INTRODUCTION:

Skin care cosmetics are significantly contributed to global economies, which is projected to grow from 460 billion in 2014 to over 800 billion by 2023, and annual growth rate of approximately 7%. The value of skin care products is more than one of third of global cosmetic market, and in Asian market particularly in China is expected to become the largest consumer of cosmetics in 2050. This growth is driven by various factors, including consumer demand, globalization, and the increasing popularity of natural products^1-8. The importance of natural products in cosmetics is due to the awareness of the risks associated with chemical use and the health benefits of natural ingredients derived from plants and other natural resources. The applications of natural products include topical care products, fragrances, moisturizers, UV protective products, and anti-wrinkle products, which emphasize their potential benefits in rejuvenation and overall skin health^2,3,5-7. The relationship between skin care cosmetics and the Thai economy is multifaceted, and influenced by cultural perceptions of beauty, globalization, and the burgeoning beauty market. The demand for skin whitening products is societal standards by favouring on skin lightening, which is significantly impacts economic activities and growth in Thailand^9,10. Thai natural products, i.e., the peel of Gac fruit is increasingly recognized for their multifunctional roles in skincare including antioxidant, melanogenesis inhibition, and collagen-stimulating properties¹¹. Aloe vera and Centella asiatica are commonly known for their anti-inflammatory and wound healing properties, which relieve skin conditions, such as acne and premature aging. In addition, herbal extract has provided the essential phytochemical nutrients for skin health, and can improve skin tone and texture^12,13.

Schumannianthus dichotomus, locally known in Thai as “Khla,” is a perennial shrub belonging to the Marantaceae family. There is growing within moist clay soils and commonly found in swampy tropical regions of South and Southeast Asia. Traditionally, rhizome from this plant has been used in folk medicine for various therapeutic purposes, particularly in the treatment of skin diseases. Phytochemical analyses have revealed that the presence of phenolic compounds with their antioxidant properties are potentially aiding skin health. The extracts of S. dichotomus rhizome had demonstrated significant anti-nociceptive effects in animal models, which had indicated its potential for pain relief, and flavonoids and phenols contained in the extracts may contribute to anti-inflammatory effects. Thus, recent studies have explored its pharmacological properties including anti-nociceptive and antipyretic effects, which may indirectly improve skin health by relieving inflammation associated with skin conditions^14,15,16. In addition, the previous study had also reported hypoglycaemic effects of S. dichotomus rhizome extracts in mice. Their extracts contained high contents of catechin hydrate, (-) epicatechin, and caffeic acid found in high concentrations¹⁷. Our previous study had also reported that the anti-diabetic activity of ethanolic extract from S. dichotomus rhizome by inhibition of α-glucosidase and α-amylase. S. dichotomus rhizome is rich in dietary fibre and iron, which is a suitable food supplement for healthy diet on weight management¹⁸. Since the ethanol extraction of S. dichotomus rhizome has chosen to mimic the common method of Thai herbal preparations with lower toxicity when compared to methanol extraction¹⁸, this study was aimed to screen the phytochemicals of S. dichotomus or “Khla” rhizome ethanol extract (KE) including total phenolic content (TPC), total flavonoid content (TFC). KE was analysed for common phenolic acids and flavonoids i.e. gallic acid, tannic acid, hydroquinin, quercetin, isoquercetin, rutin, kaempferol, apigenin, eriodictyol, and catechin. Furthermore, the biological activities of KE such as, in vitro antioxidants, anti-inflammation, anti-microbial and cytotoxicity were also screened, which were necessary to evaluated for judged it as skin care cosmetic ingredient.

MATERIALS AND METHODS:

Plant identification, preparation and extraction:

The plants were collected from the small community enterprise, Udon Thani, Thailand (17°29¢06.6¢¢N 103°11¢49.6¢¢E) who use S. dichotomus stem for handicraft made. The botanical characterization of S. dichotomus was identified according to previous studies^14-18. The fresh rhizomes (5 kg) were separated from stems and roots, which were cleaned with distilled water (DW) and cut into thin pieces. The pieces of rhizome were dried with sunlight (2-3 days) and hot air oven at 60 °C for overnight. Dried rhizome pieces (800 g) were ground in fine powder form (500 g) for extraction with ethanol. The remaining of dried rhizome pieces (~300 g) were sent to the laboratory service, the Central Laboratory Co., Ltd., Bangkok, Thailand who responsible for the analysis of common ten phenolic acids and flavonoids by liquid chromatography-mass spectrophotometry (LC-MS), the operating condition was later explained. The ground rhizome powder was macerated in one litter of 95% ethanol (RCI Labscan, Thailand), and continuous sonicated at 25 °C for 72 h. The KE was prepared from the pooling of three-repeated time extraction. The ethanol was removed from KE by rotary evaporator and steam of nitrogen gas. The constant weight of KE was calculated for extraction yield¹⁸.

Phytochemical screening:

TPC measurement:

The TPC was determined using Folin-Ciocalteu colorimetric method. Briefly, KE (5.0 mg/ml) was dissolved in dimethyl sulfoxide, DMSO (RCI Labscan, Thailand). 20 mL of dissolved KE was added with 100 mL of 10% Folin-Ciocalteu reagent following with 80 mL of 7.5% Na₂CO₃ (RCI Labscan, Thailand), and mixed thoroughly. The mixture stood in dark room at 25 °C for 30 min. The mixture was absorbed at 765 nm using a microplate spectrophotometric reader. The range of gallic acid (Sigma-Aldrich, USA) was diluted, similarly analyzed, and plotted as standard curve. Standard curve equation was y(Abs.) = 80.742x(Conc.) + 0.0031 with r² = 0.9994. TPC was expressed as gallic acid equivalents (GAE) per g of KE dry weight¹⁹.

TFC measurement:

The TFC was determined using an aluminum chloride (AlCl₃) colorimetric method. Briefly, 20 ml of dissolved SDRE (2.0 mg/ml) was added with 80 ml of DW, 6 μl of 15% NaNO2 (RCI Labscan, Thailand) and 6 μl of 10% AlCl3 (Loba Chemie, India). The mixture was filled with 80 μl of 4% (w/v) NaOH (RCI Labscan, Thailand) and the total volume was adjusted to 200 μl by DW. Then, amalgam (LABCHEM, Australia) was adequately mixed thoroughly and shake for 15 mins at room temperature. The absorbance of reaction mixture was measured at 510 nm by employing a microplate spectrophotometric reader. The range of quercetin (HWI Analytik GmbH, Germany) dilutions were similarly analyzed and prepared standard curve. Standard curve equation was y(Abs.) = 3.7999(Conc.) + 0.0031 with r² = 0.9994. The TFC within KE was represented as mg of quercetin equivalents (QE)/ per g of dry weight²⁰.

Measurement of phenolics and flavonoids:

The dried rhizome pieces (300 g) were sent to the laboratory service, the Central Laboratory Co., Ltd., Bangkok, Thailand who responsible for the analysis of phenolic acids and flavonoids including gallic acid, tannic acid, hydroquinin, quercetin, isoquercetin, rutin, kaempferol, apigenin, eriodictyol, and catechin by liquid chromatography-mass spectrophotometry (LC-MS). This extraction protocol and measurement were referenced and modified according to Ti et al., 2014²¹. Briefly, 0.5 g of rhizome powder was filled with 50 mL of chilled acidified methanol (95% methanol and 1 M HCl 85:15, v/v). The mixture was homogenized and centrifuged at 10,000 rpm for 5 min using a homogenizer with an ice-bath. The supernatants were separated by centrifugation at 2500g for 10 min and concentrated under a vacuum at 45 °C. Extracted sample was reconstituted to chilled acidified methanol (final volume = 10 ml) with. The extracts were stored at -20 °C before LC-MS analysis. The 250 mm ´ 4.6 mm, 5.0 mm Zorbox SB-C18 column was equipped with HPLC system. The mobile phase was a 0.4% aqueous solution of acetic acid (solution A) and acetonitrile (solution B). The gradient of two mobile phases was 5–25%, 25–35% and 35–50% of solution B at 0–40 min, 40–45 min, and 45–50 min, respectively. The flow rate was constant at 1.0 mL/min. Injection volume was 20 ml. Total running time was 50 min. The phenolics and flavonoids in KE were detected at 280 nm, which were identified by comparison the retention time of each peak referenced to retention time from standard phenolics and flavonoids²¹. Both extracted ion chromatogram (EICs) extraction and chromatographic peak detection are important represented results from LC-MS. Atmospheric pressure ionization-electrospray (API-ES) with selected ion monitoring (SIM) was used in a mass spectrometry scanning mode in which only a limited mass-to-charge ratio (m/z) range is detected, which is significantly increased sensitivity.

In vitro biological assay:

Assays on antioxidant activity:

DPPH radical scavenging assay: 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of KE were determined by a modified DPPH assay. Briefly, 50 µl of the serial KE concentrations (0.01-100 mg/ml) dissolved in ethanol, and 50 µl of DPPH in ethanol were transferred into each well of a 96-well microplate and incubated for 30 min at room temperature. The absorbance was determined at 515 nm by microplate reader, which was against negative control. Positive control was vitamin C in same as sample concentrations. All samples or controls were conducted in three-time repeated experiments. The 50% DPPH scavenging concentration (SC₅₀) was represented from the calibration curve between the scavenging activity (%) and KE concentration (mg/ml)^22-25.

NO radical scavenging assay: Nitric oxide (NO) is formed by the natural degradation of sodium nitroprusside in 20 mM phosphate buffer (pH 7.4). Once nitric oxide is formed, it interacts with oxygen to produce nitrite ions, which are measured by the Griess reaction. The experimental mixture consisting of 10 mM of sodium nitroprusside in phosphate buffer and KE in different concentrations, which was stood for 1 hour at 37°C. The aliquot of sample or control was taken and homogenized with Griess reagent (1:1). The absorbance of chemical reaction was measured at 540 nm. The nitric oxide inhibition was measured and calculated by comparing the absorbance values of the negative control, 10 mM sodium nitroprusside (without sample) and with the experimental preparation. The results were expressed as the SC₅₀ of KE on nitric oxide inhibition^22-25.

Metal chelating assay: The metal ion chelating activity of the KE was modified from the ferrous ion chelating method. Briefly, 100 µl of the serial KE concentrations (0.01-100 mg/ml) dissolved in ethanol were transferred into each well of a 96-well microplate and 50 µl of 2 mM FeCl₂ in distilled water was added. The adding of 5 mM ferrozine (50 µl) started the reaction, and the mixture volume (300 µl) was adjusted by DW. The reaction mixture was incubated for 15 min at room temperature. The absorbance was determined at 570 nm by the microplate reader. Positive control was EDTA (0.001-10 mg/ml). Negative control was FeCl₂-ferrozine complex solution. All samples or controls were conducted in three-time repeated experiments. The 50% metal chelating concentration (MC₅₀) was represented from calibration curve between the metal chelating activity (%) and KE concentration (mg/ml)^22-25.

Assay on inhibition of lipid peroxidation: The inhibition of lipid peroxidation of KE was modified from the ferric-thiocyanate method. Briefly, 50 µl of the serial KE concentrations (0.01-100 mg/ml) dissolved in ethanol was added into each well of microplate and added with 50 µl of linoleic acid in 50% (v/v) DMSO. The adding of 5 mM NH₄SCN (50 µl) and 2 mM FeCl₂ (50 µl) were started the reaction. The reaction mixture was incubated at 37±2ºC for 1 h, and the absorbance was determined at 490 nm. Negative control was reaction mixture without KE sample. Positive control was α-tocopherol (0.001-10 mg/ml). All samples or controls were conducted in three-time repeated experiments. The concentration for 50% inhibition of lipid peroxidation (IPC₅₀) was represented from calibration curve between the activity on inhibition of lipid peroxidation (%) and KE concentration (mg/ml)^22-25.

Anti-microbial test:

This agar-disc diffusion was tested for anti-microbial activity of KE according to Kirby-Bauer method. The skin pathogens include Staphylococcus aureus, Cutibacterium acnes, Candida albicans and Malassezia furfur were used in this test. Brain heart infusion, BHI (HiMedia Laboratories, India) was applied for S. aureus and Cu. acnes culture, and Sabaurad dextrose agar (HiMedia Laboratories, India) was applied for C. albicans and M. furfur culture. Each KE concentration (0.05, 0.5 and 5.0 mg) was applied to a 6 mm filtered-paper disc (MachereyNagel, Germany), while ethanol was negative control. Antimicrobial discs (Oxoid, UK): erythromycin (0.015 mg), clindamycin (0.002 mg), fluconazole (0.025 mg), and ketoconazole (0.2 mg) discs were the positive controls for S. aureus, Cu. acnes, C. albicans and M. furfur, respectively. The anti-microbial activity was represented as the diameter (mm) of clearing zone surrounded by testing and control discs. Anti-microbial test for each pathogen was three-time repeated measurement²⁶.

Anti-inflammation test:

The generation of the mediators and cytokines from inflammatory cells are common role on monitoring of inflammatory process. NO is the important mediators produced from macrophage cells. When NO production is reduced, the inflammatory process will cessation or slow down. The anti-inflammatory activity was monitored NO production, which was released from induced macrophage cell. Briefly, mouse macrophage cell RAW264.7 was maintained in Dulbecco ¢s modified Eagle ¢s medium, DMEM (Invitrogen, USA), containing foetal bovine serum, FBS (10%) and penicillin/streptomycin (1%) at appropriate condition, which was deposited to a 24-well plate and cell density was controlled to 1 × 10⁵ cells with 500 μl of medium per well. The cell suspension was incubated with KE or control for 1 h and activated with lipopolysaccharide, LPS (Sigma, USA) for 24 h. 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 was determined by microplate reader at 540 nm. Result was represented as IC₅₀ of KE compared with triamcinolone acetonide as positive control^27,28.

Cytotoxicity test:

Normal human skin fibroblast passage 80 was cultivated in DMEM and deposited in a 75-cm² flask (Nunc, Denmark) and culture condition was like macrophage cultivation in anti-inflammation test with appropriate modification. The harvesting cells were maintained and resuspended in 0.25% (w/v) trypsin, 0.06 mM EDTA in phosphate buffer saline (PBS), and DMEM medium. The cell counting was using a hemacytometer. The determination of KE cytotoxicity against human skin fibroblasts was conducted by the sulforhodamine B colorimetric (SRB) assay. The cell culture was adjusted to 1.0×10⁴ cells per well in 96-well plate. The facilitation of cellular adhesion was conducted by incubated in 5% CO₂ at 37 °C for 24 h. After that suspension of the incubating cell was treated with KE (0.001 to 10 mg/ml) for 24 h. Each procedure was run in four-time repeated. The result was represented as cell viability (%) of vital staining cells after treating them with KE²⁹.

Statistical analysis:

The yield of extraction and phytochemical contents of S. dichotomus rhizome were analysed and represented as descriptive statistics. The biological activities of KE were also used descriptive statistics and compared with controls. In addition, antioxidant and anti-microbial activities were calculated from three-time repeated experiments. Whereas cell culture-based tests including anti-inflammatory activity and cytotoxicity were calculated from four-time repeated experiments.

RESULT:

The KE appearance was solid material with dark-brownish colored, its yield of extraction was about 6.5-7.2%. The standard curves of TPC and TFC with their formulae were shown on Fig. 1. The graphs represented the relationships between antioxidant activities and sample concentrations on triplicate experiments, which were depicted in Fig. 2. According to the standard curves, TPC and TFC of KE were 299.83±6.23 mg GAE/g and 81.10±4.10 mg QE/g, respectively. The chromatographic indication of common phenolic acids and flavonoids in dried S. dichotomus rhizome were found catechin and rutin (22.76 and 3.67 mg/kg, respectively), which were in Fig 3. The KE exhibited DPPH and NO radical scavenging activity, and inhibited lipid peroxidation (SC₅₀ = 0.01±0.0 and 6.49±1.59 mg/ml; IPC₅₀ = 0.01±0.0 mg/ml, respectively), while it was lack of metal chelating activity (Table 1). The KE exhibited anti-bacterial activity against Cu. acnes, the major acne-associated bacteria, which was comparable to clindamycin (Fig. 4). Therefore, it was unable to inhibit S. aureus, another bacterial skin and yeasts including C. albicans and M. furfur (data not shown). KE (0.1 mg/ml) was exerted anti-inflammatory activity by reduced NO production (24.15±1.45%) from LPS-induced macrophages (Table 2), which was comparable with triamcinolone acetonide (25.18±1.17% at 0.1 mg/ml). KE (0.0001 to 1.0 mg/ml) was a lack of cytotoxicity against mouse macrophages (Table 3). However, KE (1.0 mg/ml) was cytotoxic against human skin fibroblast (Table 3), while lower concentrations (0.0001 to 0.1 mg/ml) were safe (Fig. 5).

Table 1: Phytochemical contents and biological activities of KE

Sample	Assay
Sample	TPC	TFC	DPPH*	NO*	MC*	LPI*
(Units)	(mg GAE/g)	(mg QE/g)	(mg/ml)	(mg/ml)	(mg/ml)	(mg/ml)
KE	299.83±6.23	81.10±4.10	0.01±0.0	6.49±1.59	ND	0.01±0.0
Ascorbic acid	-	-	0.01±0.0	0.13±0.03	-	-
EDTA	-	-	-	-	0.02±0.01	-
α-Tocopherol	-	-	-	-	-	0.003±0.0

*Biological activities are given as 50% scavenging concentration, SC₅₀ (mg/ml) for 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH) and nitric oxide scavenging activity (NO), 50% metal chelating concentration, MC₅₀ (mg/ml) for metal chelating activity (MC); the concentration for 50% inhibition of lipid peroxidation, IPC₅₀ (mg/ml) for assay on inhibition of lipid peroxidation (LPI). TPC = total phenolic content; TFC = total flavonoid content; KE = S. dichotomus (Khla) rhizome extract; EDTA = ethylenediaminetetraacetic acid; ND = Not determined

Table 2: Reduction of NO production released from LPS-induced macrophages affected from KE and control

Sample (mg/ml)	Inhibition of NO production (%)
Sample (mg/ml)	0.0001	0.001	0.01	0.1	1.0
KE	19.25±0.87	22.26±2.61	23.02±2.76	24.15±1.45	20.75±0.87*
Triamcinolone acetonide	23.38±1.38	24.10±2.16	24.46±2.49	25.18±1.17	28.06±1.17

* The interference from sample color

Table 3: Cell viability (%) of mouse macrophage cells and human skin fibroblasts after treatment with KE and controls

Cells	Sample (mg/ml)	Cell viability (%)
Cells	Sample (mg/ml)	0.0001	0.001	0.01	0.1	1.0
Mouse macrophage cells	KE	96.88±3.76	110.34±4.34	104.76±3.14	94.10±5.52	92.31±2.82
Mouse macrophage cells	Triamcinolone acetonide	99.25±3.27	90.17±2.20	96.57±2.42	95.43±2.55	101.83±2.33
Human skin fibroblasts	KE	95.65±4.73	94.98±2.88	92.66±2.88	87.05±5.09	55.70±1.42
Human skin fibroblasts	Sodium lauryl sulphate	94.42±8.14	93.56±6.09	88.85±6.95	10.26±2.04	9.43±0.85

Figure 1: The standard curves: a) TPC, equivalent with gallic acid b) TFC, equivalent with quercetin (mg/ml) and its absorbance

DISCUSSION:

Previously, we had focused on the anti-diabetic activity of S. dichotomus rhizome extract by in vitro inhibiting of α-glucosidase and α-amylase with high phenolic contents¹⁸; and this finding correlated with the hypoglycaemic activities of S. dichotomus rhizome extract in dextrose-induced hyperglycaemic mice¹⁷. Most active constituents in the methanol extract from rhizome are catechin hydrate, (-) epicatechin and caffeic acid, however when we were prepared dried-rhizome by simple methods and used ethanol for extraction that like Traditional Thai herbal preparation, the major active constituents were changed. According to our study, KE extract was high content of TPC and TFC, and catechin and rutin were major active compounds, this finding corresponded to previous study^17,18. In addition, the analysis of common plant active compounds including gallic acid, tannic acid, hydroquinin, quercetin, isoquercetin, rutin, kaempferol, apigenin, eriodictyol, and catechin, which can be service from the Central Lab Thai who responsible for agricultural analysis as nationally laboratory support and the methods are also modified that qualified to international standard^21,30. Thus, it was implied that we would use catechin and rutin as the markers of active compounds for quality control of this herbal ingredient in nutraceutical or cosmetic products. As our results. The antioxidant activity of KE was exhibited DPPH and NO radical scavenging activity, and inhibited lipid peroxidation. In addition, KE was also the anti-inflammatory activity by reduced NO production from induced macrophages. This finding was well explained about the folk medicinal use of S. dichotomus rhizome on treatment of fever and skin diseases, which are through antinociceptive and antipyretic effects by anti-inflammation^14-16. The phytochemicals contained in plants extracts are significantly modulating the inflammatory process by their free radical scavengers and lipid peroxidation inhibitors. Various studies have reported the antioxidant properties of plant extracts, which can mitigate oxidative stress and inflammation on treatment of related diseases^31-36. Based on our study, the modulation of inflammatory process may be due to antioxidant activity and anti-inflammation of S. dichotomus rhizome extract. In addition, rhizome extract had anti-bacterial activity against Cu. acnes, the major acne-associated bacteria^37,38. Thus, there can prevent skin acne with relief of inflammation as traditional use. However, this extract had cytotoxic against human skin fibroblast at 1.0 mg/ml, whereas there was safe at concentration up to 0.1 mg/ml. This our finding was provided with information on the maximum concentration of S. dichotomus rhizome that can be used for cosmetic purposes. Natural cosmetic products are usually preferred in markets, while the synthetic compounds are concerned with adverse effects. Many herbal extracts have proved their effectiveness, and applied in skin care, anti-aging and protection of cosmetics by preclinical studies^39-42. This study explained only the preclinical effectiveness, therefore clinical research on S. dichotomus rhizome is needed to extend.

CONCLUSION:

The ethanol extract of S. dichotomus rhizome (KE) contained high phenolic and flavonoid contents; and catechin and rutin were active compounds, which were suitable for dose monitoring. The in vitro biological activity of KE for skincare included antioxidants, DPPH and NO scavenging, and inhibition of lipid peroxidation); anti-inflammation, reduction of NO releasing from LPS-induced macrophages; anti-bacterial caused acne, inhibited Cu. acnes. At maximum concentration 0.1 mg/ml, KE was safe for use of cosmetic ingredients by lack of cytotoxicity against human skin fibroblasts.

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 Faculty of Science and Technology for partial technical assistance. Our appreciation also extends to the Samut Songkhram Campus, Suan Sunandha Rajabhat University, provided invaluable support in the identification of herbal specimen and the providing of application data in Thai Traditional Medicine.

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Received on 01.05.2025 Revised on 20.12.2025

Accepted on 22.04.2026 Published on 20.05.2026

Available online from May 25, 2026

Research J. Pharmacy and Technology. 2026;19(5):2236-2244.

DOI: 10.52711/0974-360X.2026.00322

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