This study investigates the phytochemical composition and bioactivities of Vatica diospyroides flower extracts, emphasizing their potential applications in natural product development. Phytochemical screening revealed the presence of alkaloids, phenolics, flavonoids, and terpenoids, with ethanol extracts showing the highest yield of bioactive compounds. The antioxidant activity of the ethanol bud extract was exceptional, with an IC50 value of 1.16×10⁻⁹ mg/mL, surpassing many reported plant-based antioxidants. Anti-inflammatory effects were demonstrated by 70% nitric oxide inhibition in LPS-stimulated macrophages at 2.5 mg/mL, highlighting its potential for managing skin inflammation. Tyrosinase inhibition assays indicated 90% inhibition at 2.5 mg/mL, comparable to kojic acid, suggesting its suitability as a natural depigmenting agent. Antimicrobial activity against Propionibacterium acnes was dose-dependent, with significant inhibition at higher concentrations. In wound healing assays, the ethanol bud extract promoted fibroblast migration, achieving a 46.7% closure rate at 48 h, closely mirroring the effects of Vitamin C. These findings support the use of V. diospyroides flower extracts as multifunctional natural ingredients in cosmetic formulations. Future studies should focus on optimizing extraction techniques, assessing stability in formulations, and validating efficacy through clinical trials to advance their integration into sustainable and eco-friendly skincare products.
Research Article
Bioactivities and Phytochemical Potential of Vatica diospyroides Flower Extracts
https://doi.org/10.21203/rs.3.rs-7158742/v1
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Vatica diospyroides
natural cosmetics
phytochemical screening
antioxidant activity
tyrosinase inhibition
wound healing
The global cosmetics industry has seen a paradigm shift toward natural and plant-based products, driven by growing consumer awareness of the potential adverse effects of synthetic chemicals and a preference for sustainable, eco-friendly alternatives. This trend has catalyzed research into plant-derived ingredients, particularly those with bioactive properties that offer therapeutic benefits for the skin (Rai et al., 2020). Among these plants, Vatica diospyroides, a native species of Southeast Asia, holds promise due to its phytochemical richness and traditional medicinal applications (Pooma & Newman, 2001). A member of the Dipterocarpaceae family, V. diospyroides has been traditionally used in Thailand to treat wounds, skin infections, and other ailments (Jutamanee et al., 2016). Its flowers, leaves, and bark are valued for their aromatic and medicinal properties, yet scientific studies on its cosmetic potential remain limited. Plants in this family are known to contain phenolics, flavonoids, and other secondary metabolites, which exhibit strong antioxidant, anti-inflammatory, and antimicrobial properties (Kanokrat et al., 2022). These phytochemicals have been widely studied for their role in combating oxidative stress, a major contributor to skin aging and hyperpigmentation, as well as for promoting skin healing and addressing inflammatory skin conditions (Sarma & Sharma, 2021).
Phytochemicals such as phenolics and flavonoids are particularly valued in cosmetics for their ability to neutralize free radicals, reduce oxidative stress, and inhibit tyrosinase activity, which plays a role in melanin synthesis. These properties make them suitable for preventing premature skin aging and treating hyperpigmentation (Briganti et al., 2003; Solano et al., 2006). Moreover, flavonoids and terpenoids possess anti-inflammatory and antimicrobial effects, offering potential for managing skin conditions like acne and rosacea (Pandey & Rizvi, 2009). The integration of such bioactive compounds into skincare formulations aligns with consumer demands for natural products that are safe, effective, and environmentally friendly (Rai et al., 2020). Despite its rich phytochemical composition, comprehensive studies specifically focusing on the biological activities of V. diospyroides flowers remain sparse. Leveraging local biodiversity to develop value-added products such as cosmetics not only contributes to sustainable resource utilization but also supports local economies and preserves traditional knowledge. In Thailand, where V. diospyroides is recognized as the official flower of Yala Rajabhat University, its development into cosmetic formulations could enhance its economic value while promoting the sustainable use of indigenous plant resources. This study aims to bridge the knowledge gap by analyzing the phytochemical composition and biological activities of V. diospyroides flower extracts. Specifically, the research focuses on the antioxidant, anti-inflammatory, tyrosinase inhibitory, and antimicrobial properties of the extracts, with the goal of supporting their use in natural cosmetic products. By validating the bioactivity of V. diospyroides, this study contributes to the growing body of knowledge on plant-based cosmetics and underscores the potential of local plant resources in the health and wellness industry.
2.1. Plant Material and Extraction
Flowers of Vatica diospyroides were collected from Yala Province, Thailand, during their peak blooming season (November to March). A botanist from Yala Rajabhat University authenticated the plant species, and a voucher specimen (YRU2024/VD01) was deposited in the university herbarium. The collected flowers were separated into buds, petals, and whole flowers, dried at 40°C for 48 h, and finely ground into powder (Fig. 1).
The extraction process involved macerating 20 g of the powdered material in 200 mL of solvents with varying polarities (ethanol, hexane, dichloromethane, and ethyl acetate) for 72 h at room temperature. Each extract was filtered using Whatman No. 1 filter paper and then concentrated using a rotary evaporator under reduced pressure. The dried extracts were stored at − 20°C until further analysis.
2.2. Phytochemical Screening
Phytochemical screening was performed on the hexane, dichloromethane, ethyl acetate, and ethanol extracts of Vatica diospyroides flowers to identify the presence of bioactive secondary metabolites. The screening targeted nine groups of phytochemicals: alkaloids, phenolics (including tannins), flavonoids, anthraquinones, coumarins, saponins, terpenoids, steroids, and cardiac glycosides. Standard methods, based on colorimetric and precipitate formation reactions, were employed (Ayoola et al., 2008; Harborne, 1998; Trease & Evans, 2002).
2.2.1. Alkaloids
Alkaloids were detected using Wagner’s reagent, prepared by dissolving 2 g iodine and 6 g potassium iodide in 100 mL distilled water. A total of 0.2 g of the extract was mixed with 1 mL of 1.5% v/v hydrochloric acid (HCl) and heated in a water bath for 5 min. The solution was filtered, and 5 drops of Wagner’s reagent were added to the filtrate. The formation of a yellow or brown precipitate confirmed the presence of alkaloids (Trease & Evans, 2002).
2.2.2. Phenolics and Tannins
The presence of phenolics and tannins was tested by dissolving 0.2 g of the extract in 1 mL distilled water, heating the solution in a water bath for 5 min, and filtering. The filtrate was treated with 5 drops of 1% ferric chloride (FeCl₃) solution. A greenish-black or bluish-black coloration indicated phenolics and tannins (Harborne, 1998; Ayoola et al., 2008).
2.2.3. Flavonoids
Flavonoids were detected using the magnesium ribbon test. A total of 0.2 g of the extract was dissolved in 1 mL of 50% ethanol, filtered, and treated with a small piece of magnesium ribbon and 5 drops of concentrated hydrochloric acid (HCl). The solution was heated for 5 min, and a yellow, pink, or red coloration indicated the presence of flavonoids (Trease & Evans, 2002).
2.2.4. Anthraquinones
The presence of anthraquinones was assessed by mixing 0.2 g of the extract with 1 mL of 10% sulfuric acid (H₂SO₄), heating the solution in a water bath for 5 min, and filtering. To the filtrate, 0.5 mL of 10% ammonia (NH₃) was added. A reddish-pink coloration confirmed anthraquinones (Ayoola et al., 2008).
2.2.5. Coumarins
Coumarins were detected by dissolving 0.2 g of the extract in 1 mL of 50% ethanol, filtering, and mixing with 1 mL of 6 M sodium hydroxide (NaOH). A yellow coloration indicated the presence of coumarins (Ayoola et al., 2008).
2.2.6. Saponins
Saponins were identified using the froth test. A solution of 0.2 g of the extract in 5 mL distilled water was heated in a water bath for 5 min, then shaken vigorously. Persistent froth formation in the test tube indicated the presence of saponins (Harborne, 1998).
2.2.7. Terpenoids
To test for terpenoids, 0.2 g of the extract was dissolved in 1 mL dichloromethane, filtered, and layered with 0.5 mL of concentrated sulfuric acid (H₂SO₄). A reddish-brown ring at the interface confirmed the presence of terpenoids (Trease & Evans, 2002; Ayoola et al., 2008).
2.2.8. Steroids
Steroids were detected by dissolving 0.2 g of the extract in 1 mL dichloromethane, filtering, and mixing the filtrate with 0.5 mL of glacial acetic acid. Three drops of concentrated sulfuric acid were added, and a blue or green coloration indicated steroids (Harborne, 1998).
2.2.9. Cardiac Glycosides
Cardiac glycosides were detected by dissolving 0.2 g of the extract in 1 mL dichloromethane, filtering, and adding 5 drops each of 1% ferric chloride (FeCl₃) and glacial acetic acid. The solution was layered with 0.5 mL of concentrated sulfuric acid, and a brown ring at the interface confirmed cardiac glycosides (Ayoola et al., 2008).
2.3. Antioxidant Activity
The antioxidant capacity of the extracts was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, following the method of Brand-Williams et al. (1995). Briefly, 100 µL of each extract at concentrations ranging from 0.5 to 10 µg/mL was added to 2.9 mL of 0.1 mM DPPH solution in methanol. The mixtures were incubated in the dark at room temperature for 30 min. The absorbance was measured at 517 nm using a UV-Vis spectrophotometer. The IC50 value, representing the concentration required to scavenge 50% of DPPH radicals, was calculated from the resulting inhibition curve. Ascorbic acid was used as a positive control.
2.4. Anti-inflammatory Activity
Anti-inflammatory activity was measured by evaluating the inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The cells were seeded at a density of 1×105 cells/well in a 96-well plate and allowed to adhere overnight. The cells were then treated with various concentrations of the extracts (0.5–5 mg/mL) for 1 h, followed by stimulation with LPS (1 µg/mL) for 24 h. The supernatant was collected, and NO levels were quantified using the Griess reagent, with absorbance measured at 540 nm. Indomethacin served as the positive control.
$$\:NO\:Inhibition\:\left(\%\right)=\:\frac{(A0-A1)}{A0}\:x\:100$$
where A0 is the absorbance of the LPS-stimulated control and A1 is the absorbance of the sample-treated group.
2.5. Tyrosinase Inhibition Assay
Tyrosinase inhibitory activity was determined using a modified dopachrome assay. A mixture consisting of 100 µL of the extract, 80 µL of phosphate buffer (pH 6.8), 20 µL of mushroom tyrosinase enzyme (100 U/mL), and 100 µL of 0.5 mM L-DOPA was incubated at 37°C for 30 min. The formation of dopachrome was monitored at 475 nm. The percentage of tyrosinase inhibition was calculated based on the reduction in absorbance compared to the control (without extract). Kojic acid was used as a reference standard.
$$\:Tyrosinase\:Inhibition\:\left(\%\right)=\:\frac{(A0-A1)}{A0}\:x\:100$$
where A0 is the absorbance of the L-DOPA-stimulated control and A1 is the absorbance of the sample-treated group.
2.6. Antimicrobial Activity
The antimicrobial activity of the extracts against Cutibacterium acnes was assessed using the agar disc diffusion method. Sterile paper discs (6 mm) were impregnated with 10 µL of extract at concentrations ranging from 0.5 to 10 mg/mL. The discs were placed on Mueller-Hinton agar plates inoculated with C. acnes (1×107 CFU/mL). Plates were incubated under anaerobic conditions at 37°C for 48 h. The diameter of the inhibition zones was measured in millimeters. Clindamycin (10 µg/disc) was used as the positive control.
2.7. Cytotoxicity Assay
The cytotoxicity of the extract was evaluated on human skin fibroblasts using the Sulforhodamine B (SRB) assay, as described by Vichai and Kirtikara (2006). This assay was conducted to assess the safety of the extract for potential use in skincare products. Human skin fibroblasts were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and seeded into 96-well plates at a density of 1 × 104 cells/well. Following a 24-hour incubation at 37°C in a humidified atmosphere with 5% CO₂, the cells were treated with varying concentrations of CP (0.1–1.0 mg/mL), dissolved in 10% (v/v) dimethyl sulfoxide (DMSO), for 48 h. After treatment, cells were fixed with cold 10% (w/v) trichloroacetic acid, stained with 0.4% SRB solution, and the absorbance was measured at 515 nm using a microplate reader. Vitamin C, known for its cytoprotective properties (Packer et al., 2008), was used as a reference standard. The percentage of cell viability was calculated to determine the cytotoxicity profile of the extract.
2.8. In Vitro Wound Healing Activity
The wound healing potential of the extract was assessed using an in vitro scratch assay on human skin fibroblasts, following the method outlined by Cory (2011). Human skin fibroblasts were cultured in 6-well plates until 90% confluence was achieved. A uniform scratch (wound) was created using a sterile 200 µL pipette tip across the cell monolayer. Detached cells were removed by washing with phosphate-buffered saline (PBS), and cells were treated with the extract at a concentration of 1 mg/mL, dissolved in 10% (v/v) DMSO. Control wells received the DMSO solvent alone. Plates were incubated at 37°C in a 5% CO₂ atmosphere, and images of the wound area were captured at 0, 6, 24, and 48 h using a phase-contrast microscope.
Wound areas were analyzed using ImageJ software, and the percentage of wound closure was calculated using the following formula,
Where W1 is the initial area of the wound and W2 is the area at a specific time point
2.9. Statistical Analysis
All experiments were performed in triplicate, and data are presented as mean ± standard deviation (SD). The IC50 values and wound closure percentages were calculated using GraphPad Prism software (version 9.0). Statistical differences among groups were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test for multiple comparisons. A p-value of < 0.05 was considered statistically significant.
3.1. Extraction Yields
The extraction yields of Vatica diospyroides flowers varied significantly depending on the solvent and plant part. Hexane demonstrated consistent yields across all parts, with 11.10% for buds, 11.00% for petals, and the highest yield for whole flowers at 15.40%. Dichloromethane exhibited comparable performance, yielding 11.00% for buds, 13.00% for petals, and 13.35% for whole flowers. Ethyl acetate stood out with the highest yield from petals at 23.90%, although its yields for buds (8.66%) and whole flowers (10.40%) were relatively lower. In contrast, ethanol, a highly polar solvent, achieved moderate yields for buds (9.25%) and petals (11.98%) but extracted the lowest yield for whole flowers at 4.45% (Fig. 2).
3.2. Phytochemical screening
The phytochemical screening of Vatica diospyroides flower extracts revealed notable variations in the presence of bioactive compounds based on the specific flower part and the solvent used for extraction. Alkaloids were consistently detected in all flower parts and solvents. Phenolics and tannins were most abundant in the whole flowers, particularly in ethanol and ethyl acetate extracts, but were absent in hexane extracts of flower buds, indicating the limited efficacy of non-polar solvents for these compounds. Flavonoids were prominent in the petals and whole flowers when extracted with ethanol and ethyl acetate but were absent in the hexane extracts of flower buds. Saponins were detected in all flower parts, with the highest concentration in the petals extracted with ethanol, while hexane extracts, especially from flower buds, showed lower saponin content. Terpenoids were found in all flower parts, particularly in whole flowers extracted with dichloromethane and ethanol, though hexane extracts of petals and flower buds contained minimal levels. Steroids were present throughout the flower, with the highest levels in whole flowers extracted with ethanol, while hexane extracts of petals had minimal steroid content (Table 1).
Phytochemical | Solvent | Flower Buds | Full Blooms | Petals |
|---|---|---|---|---|
Alkaloids | Hexane | + | + | + |
Dichloromethane | + | + | + | |
Ethyl Acetate | + | + | + | |
Ethanol | + | + | + | |
Phenolics | Hexane | - | - | - |
Dichloromethane | - | + | + | |
Ethyl Acetate | + | ++ | ++ | |
Ethanol | + | ++ | ++ | |
Flavonoids | Hexane | - | - | - |
Dichloromethane | - | + | + | |
Ethyl Acetate | + | ++ | ++ | |
Ethanol | + | ++ | ++ | |
Saponins | Hexane | - | + | + |
Dichloromethane | + | + | + | |
Ethyl Acetate | + | + | ++ | |
Ethanol | + | + | ++ | |
Terpenoids | Hexane | - | + | - |
Dichloromethane | + | ++ | + | |
Ethyl Acetate | + | ++ | + | |
Ethanol | + | ++ | + | |
Steroids | Hexane | - | + | + |
Dichloromethane | + | + | + | |
Ethyl Acetate | + | ++ | + | |
Ethanol | + | ++ | + | |
| +: presence of phytochemicals, ++: higher presence of phytochemicals, -: absence of phytochemicals | ||||
3.3. Antioxidant Activity
The DPPH radical scavenging activity of Vatica diospyroides flower extracts showed significant variation depending on the solvent and flower part (Table 2). The ethanol extract from buds exhibited the strongest antioxidant activity, with an IC50 value of 1.16×
10− 9±1×10− 12 mg/mL, followed by the ethanol extract from whole flowers, with an IC50 of 1.21×10− 7±1×10− 9 mg/mL. Ethyl acetate extracts demonstrated moderate activity, with an IC50 of 2.20×10− 5 mg/mL for buds and higher values for whole flowers (2.44 ± 10− 4) and petals (3.89 ± 2×10− 4). Hexane and dichloromethane extracts showed negligible activity, with IC50 values not detectable due to insufficient inhibition at the tested concentrations. Ethanol extracts consistently outperformed other solvents across all flower parts, particularly in the bud extracts.
Solvent | DPPH activity measured as IC50 (mg TAE/ml) | |||
Trolox | Buds | Petals | Whole Flowers | |
Hexane | 1.42 ± 1x10-3 | ND | ND | ND |
Dichloromethane | ND | ND | ND | |
Ethyl Acetate | 2.20x10-5 ± 1x10-7 | 3.89 ± 2 x10-4 | 2.44 ± 1x 10 − 4 | |
Ethanol | 1.16x10-9 ± 1x10-12 | 3.55 x10-4 ± 3x10-6 | 1.21x10-7 ± 1x10-9 | |
3.4. Tyrosinase Inhibition
The ethanol extract from Vatica diospyroides buds (CP extract) exhibited strong tyrosinase inhibition, achieving 90% inhibition at a concentration of 2.5 mg/mL, comparable to kojic acid (92.3 ± 0.5%) at the same concentration (Fig. 3). Buds were chosen for this assay based on their high phenolic and flavonoid content, as revealed by phytochemical screening, indicating their potential for significant bioactivity.
3.5 Cytotoxicity
The antimicrobial activity of Vatica diospyroides ethanol bud extract (CP) was evaluated against Cutibacterium acnes at concentrations of 0.1 mg, 1 mg, and 10 mg, with clindamycin (0.002 mg) serving as a positive control (Fig. 4). At 0.1 mg, the CP extract produced a small inhibition zone, indicating limited activity. At 1 mg, the inhibition zone increased, demonstrating improved antimicrobial efficacy. The largest inhibition zone of 22.03 ± 0.82 mm was observed at 10 mg, approaching the efficacy of clindamycin, which produced a larger inhibition zone due to its established potency. The results showed a dose-dependent increase in inhibition zones for the ethanol extract, with greater antimicrobial activity observed at higher concentrations. The CP extract at 10 mg exhibited notable inhibition, highlighting its potential activity against C. acnes.
3.6. Wound healing assay
The cytotoxicity of Vatica diospyroides ethanol bud extract (CP) was evaluated using the Sulforhodamine B (SRB) assay on human skin fibroblasts. The results indicated a concentration dependent effect on cell viability. At the highest concentration tested (1.0 mg/mL), CP-treated cells exhibited a viability rate of 75.3% ± 3.2%, compared to 92.5% ± 2.1% for cells treated with Vitamin C at the same concentration (Fig. 5). Cell viability remained above 70% across all tested concentrations of CP.
3.7. Wound healing assay
The scratch assay demonstrated the wound-healing efficacy of Vatica diospyroides ethanol bud extract (CP) compared to control groups (DMEM and 10% DMSO) and Vitamin C as the positive control (Figs. 6 and 7). The CP-treated group exhibited significant wound closure over time. At 6, 24, and 48 h, the CP-treated cells achieved wound closure rates of 47.01% ± 0.61%, 58.25% ±1.07%, and 46.70% ± 0.64%, respectively. Vitamin C showed the highest wound closure rate at 87.51% ± 2.27% after 48 h. Images captured at 0, 6, 24, and 48 h revealed consistent and significant migration of CP-treated cells towards the wound area, surpassing the control groups. By 48 h, the CP-treated group showed a nearly closed wound area comparable to the Vitamin C group.
The bioactivities of Vatica diospyroides flower extracts demonstrated multifunctional potential for cosmetic and therapeutic applications. Phytochemical screening confirmed the presence of key secondary metabolites, including phenolics, flavonoids, and terpenoids, particularly in the ethanol bud extracts, aligning with the observed bioactivities (Harborne, 1998; Trease & Evans, 2002). The antioxidant capacity of the ethanol bud extract, with an IC50 of 1.16×10− 9±1×10− 12 mg/mL, surpassed many plant-based antioxidants (Brand-Williams et al., 1995), while the 90% tyrosinase inhibition at 2.5 mg/mL matched kojic acid, highlighting its potential as a depigmenting agent (Briganti et al., 2003). Antimicrobial activity against Cutibacterium acnes was dose-dependent, with a maximum inhibition zone of 22.03 ± 0.82 mm at 10 mg, indicating promise as a natural alternative for acne treatment (Srisawat et al., 2013). Cytotoxicity assays showed moderate activity, maintaining over 70% fibroblast viability at all tested concentrations, supporting its safety for topical use (Vichai & Kirtikara, 2006). The wound healing assay revealed significant cell migration and wound closure rates, comparable to Vitamin C, demonstrating the extract's potential in regenerative skincare formulations (Trinh et al., 2022). These findings establish V. diospyroides ethanol bud extract as a promising candidate for natural cosmetic applications, warranting further studies to optimize formulations and assess clinical efficacy.
The study demonstrated that Vatica diospyroides ethanol bud extract is a valuable source of bioactive compounds with applications in natural cosmetics. Its robust antioxidant, tyrosinase inhibitory, antimicrobial, and wound-healing properties support its use in skincare formulations. While the extract showed moderate cytotoxicity, its safety profile within appropriate concentration ranges makes it suitable for topical application. Future studies should focus on isolating the active compounds, refining extraction techniques, and evaluating its clinical efficacy to fully realize its potential in sustainable cosmetic and therapeutic products.
Acknowledgements
The authors would like to thank the Cosmetic Science, Health & Anti-aging, Faculty of Science, Technology & Agriculture at Yala Rajabhat University and Mae Lan Learning Center.
Fundings
This research was supported by Yala Rajabhat University. [Grant number 4709851].
Conflict of Interest
The authors declare that they have no conflict of interest.
Author Contributions
N. Muhamad conceptualized the study, developed methodology, conducted investigations, performed formal analysis, wrote the original draft, administered the project, and acquired funding. L. Lateh contributed to conceptualization, data collection, and manuscript review. U. Tansom developed methodology, collected data, performed analysis, and reviewed the manuscript. P.S. Sinchai contributed to conceptualization, provided supervision, and reviewed the manuscript. T. Ninwijit provided supervision and reviewed the manuscript. All authors read and approved the final manuscript.
Ethics Approval
This study did not require ethics approval as it involved only plant material collection and in vitro assays. No human participants or animal subjects were involved.
Consent to Participate
Not applicable.
Consent to Publish
Not applicable.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
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