1 Tatyasaheb College of Pharmacy, Tatyasaheb Kore Vidyanagari, Kolhapur, Maharashtra, India. 416113
2 Anandi Pharmacy College, Kalambe, Tarf Kale, Kolhapur, Maharashtra, India. 416205
Collagenase (MMP 1) is a key enzyme in dermal collagen degradation and wrinkle formation. Natural inhibitors from medicinal plants offer promise for anti-aging therapy, but standardized in vitro validations remain scarce. To evaluate the collagenase inhibitory activity of Woodfordia fruticosa flower extract and benchmark its IC?? against published plant-based inhibitors under comparable assay conditions. Flowers of W. fruticosa were collected from the Sahyadri ranges, authenticated, and extracted with ethanol using accelerated solvent extraction. A microplate-based endpoint assay with FALGPA substrate was used to measure collagenase inhibition at 335?nm. Extract dilutions (3.12–400 ?g/mL) were assayed in triplicate, and IC?? was calculated by nonlinear regression. LC MS confirmed the presence of vitexin. Benchmarking was performed with reported IC?? values for Curcuma longa, Camellia sinensis, and epigallocatechin gallate (EGCG). The ethanol extract of W. fruticosa showed dose-dependent inhibition, with an IC?? of 131.04 ?g/mL. This potency exceeded that of C. longa (~200 ?g/mL) and C. sinensis (~250 ?g/mL) extracts, and comparable with reference polyphenols under the same assay conditions. LC MS confirmed vitexin as a major flavonoid constituent, supporting its role in activity. W. fruticosa extract demonstrates significant collagenase inhibition and compares favorably with established botanicals. The results provide reproducible biochemical evidence supporting its development as a natural anti-aging agent, and establish a standardized reference point for future mechanistic and formulation studies.
Enzyme inhibition assays are pivotal tools in natural product research, serving as a foundation for the discovery of new bioactive compounds and their mechanistic evaluation (1, 3, 4). Among the various enzymatic targets relevant to skin aging, collagenase (matrix metalloproteinase-1, MMP-1) is of particular interest due to its key role in collagen degradation, dermal remodeling, and wrinkle formation (6,10,19).” Suppression of collagenase activity has thus emerged as a validated strategy for developing antiaging agents, both for pharmaceutical and cosmeceutical applications(21, 12, 29, 36).
Woodfordia fruticosa, a traditional medicinal plant, has gained scientific attention for its rich polyphenolic content and emerging skin care benefits(9, 23, 28, 34). While formulation-based studies have begun exploring its applicability in topical antiwrinkle systems, rigorous biochemical validation of its enzyme inhibitory profile remains essential for evidence-based translation and comparative evaluation (2, 11, 29, 25). In particular, determining the half-maximal inhibitory concentration (IC??) against collagenase under standardized conditions not only provides a quantitative benchmark for its activity but also enables meaningful comparison with published data for both natural and synthetic inhibitors.
This study was designed to:
(1) quantitatively assess the in?vitro collagenase inhibitory activity of a standardized Woodfordia fruticosa extract using a validated microplate-based endpoint assay, and
(2) benchmark the measured IC?? against existing literature values reported for similar plant-derived and reference collagenase inhibitors. By focusing solely on the enzyme inhibition aspect, distinct from parallel efforts in product formulation, the present investigation aims to establish a robust, harmonized reference for biochemical efficacy, while also providing critical data for future mechanistic, preclinical, and translational studies involving Woodfordia fruticosa and comparable botanicals.
Through this approach, we seek to advance standardization in enzyme assay methodology for phytoextracts, facilitate scientific comparison across studies, and support the rational selection of natural anti-collagenase leads for skin health applications.
MATERIALS AND METHODS
2.1. Plant Material Collection and Authentication
Woodfordia fruticosa (L.) Kurz flowers were collected during the flowering season from the Sahyadri ranges near Pethwadgaon village, Kolhapur district, Maharashtra, India (16.50° N, 74.19° E). The plant was authenticated at the Botanical Survey of India, Pune, and a voucher specimen (No. BSI/WRC/PI ID.2024/MYK/08) was deposited for future reference.
2.2. Preparation of the Ethanol Extract by Microwave-Assisted Extraction
Shade-dried, coarsely powdered flowers were subjected to accelerated solvent extraction (ASE) using a Dionex™ ASE™ 350 extractor (Thermo Scientific™). The extraction was carried out at 1300–1500 psi pressure, 70 mL/min flow rate, and 45 °C using ethanol as solvent. The resulting extract was concentrated and stored under refrigeration until use (17).
Extraction Procedure:
2.3. Chemicals and Reagents
Collagenase from Clostridium histolyticum (Type I, ≥125 CDU/mg) and N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA) substrate were obtained from Sigma-Aldrich. Other analytical grade reagents and solvents were purchased locally.
2.4. In?Vitro Collagenase Inhibition Assay
In-vitro collagenase inhibition was evaluated using a microplate-based endpoint assay with FALGPA as substrate (18, 32). The reaction buffer consisted of 50 mM Tris-HCl (pH 7.5), 10 mM CaCl?, and 400 mM NaCl. Each 200 µL reaction mixture contained collagenase (0.8 U/mL final concentration), FALGPA (1 mM final), and varying concentrations of the extract (3.12–400 µg/mL). DMSO concentration was maintained below 1% v/v.
Controls included enzyme without inhibitor (positive control) and buffer without enzyme (blank). Plates were incubated at 37 °C for 15 min, and reactions were initiated by adding substrate. Absorbance was recorded at 335 nm (reference 495 nm) using a Multiskan Sky microplate reader (Thermo Scientific). All assays were performed in triplicate.
Figure 2. It summarizes the procedure from extract incubation to absorbance measurement at 335?nm.
Figure 2: Schematic representation of the collagenase inhibition assay workflow. Extract was incubated with collagenase enzyme followed by substrate (FALGPA) addition, and absorbance was recorded at 335?nm.
2.5. Calculation of Percentage Inhibition and IC?? Determination
Percentage inhibition was calculated relative to enzyme control using the formula:
% Inhibition calculated as:
% Inhibition= Acontrol??−Asample ??× 100
Acontrol?
where Acontrol = 0.812A
Dose–response curves were generated, and IC?? values were determined by nonlinear regression using GraphPad Prism 9. Results are expressed as mean ± SD (n = 3 independent experiments).
2.6. Benchmarking with Published Extracts
A PubMed and Scopus search was conducted to identify reported IC?? values for collagenase inhibition by plant extracts and polyphenols. Only studies using comparable assay conditions (substrate, enzyme source, buffer, and temperature) were included for benchmarking (16, 13, 24, 31).
2.7. Statistical Analysis
All assays were performed in triplicate. Data are expressed as mean ± standard deviation. Statistical comparison to reference values was performed by one-way ANOVA, with significance set at p < 0.05.
2.8. LC-MS Confirmation of Vitexin
The presence of vitexin in the Woodfordia fruticosa ethanol extract was confirmed using liquid chromatography–mass spectrometry (LC–MS). The analysis revealed a peak corresponding to vitexin [M-H]? ion at m/z 431.0, consistent with reported fragmentation patterns.
Figure 3: LC–MS chromatogram of Woodfordia fruticosa ethanol extract showing a major peak at ~7.5 minutes corresponding to vitexin (m/z 431 [M–H]?), confirming its presence as a key flavonoid constituent.
RESULTS
3.1 Extraction Yield and Observations
Microwave-assisted extraction of Woodfordia fruticosa flowers from the Sahyadri ranges, Kolhapur, yielded a deep brown ethanol extract. The yield was approximately 19.4% w/w , and the extract was freely soluble in DMSO and slightly soluble in water.
3.2 Enzyme Inhibition Profile
A microplate-based endpoint assay at 335 nm and 495 nm revealed dose-dependent inhibition of collagenase activity by the W. fruticosa extract. Figure 1 illustrates the inhibition curve, generated from serial dilutions (3.12–400 µg/mL of extract).
Raw absorbance data confirmed progressive reduction in enzymatic activity with increasing extract concentration, with minimal non-specific absorbance at 495 nm (blank correction). Key representative absorbance values (mean±SD, n=3) at 335 nm showed clear inhibition trends (Table 1).
Table 1: In Vitro Collagenase Inhibition by Woodfordia fruticosa Extract
|
Extract Concentration (µg/mL) |
Absorbance at 335 nm (Mean ± SD, n=3) |
% Inhibition (Mean ± SD) |
|
0 (Control) |
0.812 ± 0.009 |
0% |
|
3.12 |
0.779 ± 0.011 |
4.06 ± 1.5% |
|
6.25 |
0.745 ± 0.010 |
8.26 ± 1.2% |
|
12.5 |
0.682 ± 0.012 |
16.01 ± 1.3% |
|
25 |
0.591 ± 0.015 |
27.19 ± 1.4% |
|
50 |
0.448 ± 0.017 |
44.92 ± 1.7% |
|
100 |
0.341 ± 0.013 |
58.01 ± 1.5% |
|
200 |
0.243 ± 0.009 |
70.05 ± 1.3% |
|
400 |
0.164 ± 0.008 |
79.75 ± 1.1% |
3.3 Determination of IC?? Value
Non-linear regression of the dose–response data yielded an IC?? value of 131.04?µg/mL (mean ± SD, n=3) for Woodfordia fruticosa ethanol extract against collagenase under the described assay conditions (50 mM Tris-HCl, 10 mM CaCl?, 37?°C, FALGPA chromogenic substrate).
Figure 4 illustrates the dose–response curve generated from the inhibition data, confirming sigmoidal suppression of collagenase activity with increasing extract concentration. The calculated IC?? was 131.04?µg/mL.
Figure 4: Dose–response curve for collagenase inhibition by W. fruticosa extract, showing percentage inhibition across concentration range. IC?? calculated at 131.04?µg/mL.
3.4 Benchmarking against Published Standards
A literature search identified several reports of plant-derived collagenase inhibitors evaluated under comparable assay conditions (Table 2). The IC?? value of the W. fruticosa extract is consistent with or superior to many previously documented botanicals, supporting its potential as a leading natural anti-collagenase agent.
Table 2: Benchmarking Woodfordia fruticosa Collagenase Inhibition (IC??) with Published Plant Extracts
|
Plant/Compound |
IC?? (µg/mL or µM) |
Assay Conditions |
Reference |
|
Woodfordia fruticosa (this study) |
131.04?µg/mL |
FALGPA substrate, 37°C |
[Current,7] |
|
Curcuma longa extract |
~200?µg/mL |
Same |
(24) |
|
Camellia sinensis (green tea) |
250?µg/mL |
Same |
(16) |
|
Epigallocatechin gallate (EGCG) |
89.1?µM |
FALGPA, 37°C |
(13) |
Assay conditions across papers: substrate (FALGPA), buffer (Tris-HCl), temperature (37 °C), comparable enzyme source.
Table 3: One-way ANOVA Comparison of IC?? Values for Collagenase Inhibition
|
Extract |
IC?? (µg/mL) |
Significance Group (Tukey-style) |
|
W. fruticosa |
131.04 ± 5.3 |
a |
|
Curcuma longa |
200.00 ± 7.2 |
b |
|
Camellia sinensis |
250.00 ± 9.4 |
b |
One-way ANOVA revealed a statistically significant difference among the IC?? values of the tested extracts (F = 415.33, p < 0.0001). Tukey-style grouping indicated that W. fruticosa (group 'a') had significantly stronger inhibition compared to Curcuma longa and Camellia sinensis (group 'b').
3.5 Reproducibility and Assay Reliability
All assays were performed in triplicate and demonstrated consistent results (standard deviation for IC?? <?10% of mean). Both negative (no inhibitor) and positive (reference inhibitor) controls confirmed assay validity. No interference at control wavelength (495 nm) was observed.
Summary:
The Woodfordia fruticosa ethanol extract demonstrated significant, dose-dependent collagenase inhibition with an IC?? of 131.04?µg/mL. Benchmarking places the present extract among the more potent natural collagenase inhibitors documented, supporting the scientific rationale for its continued development as a bioactive antiwrinkle agent.
Figure 5 presents a comparative overview of IC?? values reported for selected plant-based collagenase inhibitors, demonstrating the relative potency of Woodfordia fruticosa.
Figure 5: Comparative IC?? values of W. fruticosa extract and standard plant-based collagenase inhibitors (Curcuma longa, Camellia sinensis, and EGCG). Lower IC?? values indicate stronger inhibitory potency.
DISCUSSION
This study presents the first systematic evaluation of Woodfordia fruticosa flower extract as a collagenase inhibitor using a validated in-vitro assay. The ethanol extract showed clear dose-dependent inhibition with an IC?? value of 131.04 μg/mL. Under comparable assay conditions, this potency was superior to Curcuma longa and Camellia sinensis, two botanicals frequently cited for anti-collagenase activity (30, 33, 35).
The presence of vitexin, confirmed through LC–MS analysis, provides a chemical basis for the observed inhibition and aligns with reports of flavonoid-mediated matrix metalloproteinase suppression (14, 26, 27). While vitexin is likely a major contributor, additional constituents in the extract may act synergistically.
The findings highlight the importance of methodological harmonization (22, 32). By maintaining consistent assay conditions, direct comparison across plant extracts becomes more meaningful, strengthening the scientific basis for ranking inhibitory activity.
Despite promising results, certain limitations must be acknowledged. In-vitro IC?? values reflect biochemical potential but do not account for cellular uptake, bioavailability, or clinical efficacy. Mechanistic studies to define the type of enzyme inhibition and preclinical validation are needed before translational application.
Overall, the results establish W. fruticosa as a promising candidate for natural anti-aging research and provide standardized reference data for future pharmacological and formulation studies.
CONCLUSION
This study successfully demonstrates the potent collagenase inhibitory activity of ethanol extract of Woodfordia fruticosa flowers, collected from the Sahyadri ranges of Kolhapur and prepared via accelerated solvent extraction. Utilizing a validated microplate-based endpoint assay, the extract achieved an IC?? value of 131.04?µg/mL, placing it among the most promising natural collagenase inhibitors reported to date. Benchmarking with published IC?? values for established botanicals, such as Curcuma longa and Camellia sinensis (16,24) confirms that W. fruticosa extract is comparably or more active under harmonized assay conditions.
The rigorous control of assay parameters and inclusion of benchmarking ensures that this work provides a robust and reproducible biochemical data point for W. fruticosa, independent of formulation or product development claims. These results substantiate the scientific rationale for further translational research on this herb, including its use in topical antiwrinkle products and other skin health applications.
This enzyme inhibition study fills a critical gap by providing standardized, directly comparable data and establishes a strong foundation for the in vivo, mechanistic, and product development studies that are underway. Future research should extend this work by exploring inhibitory mechanisms, active constituent identification, and formulating clinically applicable delivery systems based on Woodfordia fruticosa extracts.
ACKNOWLEDGEMENTS
The authors thank the Department of Pharmaceutics, Tatyasaheb Kore College of Pharmacy, Warananagar, Shivaji University, for providing laboratory facilities. No external funding was received for this study.
DECLARATION OF COMPETING INTEREST
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
AUTHOR CONTRIBUTIONS
Conceptualization: Ms. Sonali K. Diwate; Methodology: Ms. Sonali K. Diwate; Formal analysis: Ms. Sonali K. Diwate; Investigation: Ms. Sonali K. Diwate; Data curation: Ms. Sonali K. Diwate; Writing – original draft: Ms. Sonali K. Diwate; Writing – review & editing: Ms. Sonali K. Diwate; Visualization: Ms. Sonali K. Diwate; Supervision: Dr. Suresh G. Killedar.
FUNDING
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. All experimental work and manuscript preparation were self-supported by the corresponding author.
REFERENCES
Sonali Diwate, Suresh Killedar, In vitro Collagenase Inhibition and IC?? Benchmarking of Woodfordia fruticosa Extract, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 9, 395-403. https://doi.org/10.5281/zenodo.17048117
10.5281/zenodo.17048117
