Evaluation of antioxidant and anti-collagenase activity of Rosa damascena L. flower petal and receptacle extract

Sample extraction

R. damascena L. flowers were obtained from Bandung, Indonesia (-6.803662, 107.586633). In total, 1400 g and 700 g of petals and receptacles of R. damascena L. were dried and mashed, respectively. The dried samples (250 g petals and 90g receptacles) were soaked with 8000 mL and 3000 mL of 70% ethanol, respectively, and macerated. This solvent was replaced every 3 days and the extracted solution from the first to the last (15^th day) was mixed and concentrated using a rotary evaporator at 50°C for 4 h. The extracts were labeled as ethanol extract of rose petals (EERP) and ethanol extract of rose receptacles (EERR).

Phytochemical analysis

Phytochemical analysis was done to detect the presence of flavonoids, phenolics, tannins, steroids/triterpenoids, terpenoids and alkaloids. 10 mg of extracts were used for each analysis following the procedures of Widowati et al. without modification¹².

Ferric ion reducing antioxidant power (FRAP) activity

The antioxidant activity of EERP and EERR was investigated using FRAP assay following Sricharoen et al. with slight modifications¹³. Briefly, ferric chloride hexahydrate (Merck) and 2,4,6-tripyridyl-s-triazine (TPTZ) (Sigma) were mixed to form the FRAP reagent. 142.5 μL FRAP solution was added into 7.5 μL of sample extract and the mixtures were incubated at 37°C for 5 min. The absorbance of the reaction mixture was read at 539 nm. A set of concentrations 0–200 µg/mL of FeSO4.7H2O (Sigma) was used for calibration of the standard curve. The results were expressed as μM Fe(II)/μg extract.

Anti-collagenase assay

Anti-collagenase activity of EERR and EERP was assessed by evaluating the inhibition of collagenase, following the procedure of Wittenuer et al.¹⁴ 10 µL collagenase from Clostridium histolyticum (0.01 U/mL, Sigma), 60 µL of 50 mM Tricine buffer pH 7.5 (containing 10 mM CaCl₂ (Merck) and 400 mM NaCl (TCL Co., Ltd)), and 30 µL of extract (7.81 – 250 µg/mL) were mixed and incubated at 37°C for 20 min. After incubation, 20 μL of 1 mM N-[3-(2- Furyl)acryloyl]-leu-gly-Pro-Ala (FALGPA, Sigma) in Tricine buffer (Sigma) was added to the mixture. The collagenase inhibitory activities were measured at 335 nm by monitoring for 20 min after starting the reaction. The inhibition value was calculated according to Eq. (1). IC₅₀ values were determined from dose-effect curves.

Data analysis

Statistical analysis using SPSS version 23 was carried out by normality test using numeric variable with Levene test and ANOVA and continued with post-hoc honestly significant difference, and Tukey test to the confidence level of 95% (α = 0.05). All data are expressed as mean ± SEM.

Phytochemical analysis

Phytochemical investigations of R. damascena L. petal and receptacle extracts in ethanol expressed positive results of flavonoids, phenolic compounds, tannins, steroids, terpenoids, and alkaloids. The details are presented in Table 1.

The results of phytochemical screening showed steroids are detected in EERP by the bluish-green color, while in the same test, EERR gave a red color indicated it contained triterpenes. Although there was a difference between chemical compounds, both samples contained phenol and flavonoids that act as antioxidants and anti-collagenases. In previous research, Patil et al.¹⁵ found that the ethanol extract of R. damascene Mill L. contains total phenolic and flavonoid compounds at a higher level than acetone, aqueous, and chloroform extracts.

The antioxidant and anti-collagen activity of flowers depends on the compounds it contains and the solvent used. Phenolics are well known to have antioxidant capability^9–10,16, as does flavonoids and alkaloids¹⁷. Steroids, terpenoids, and triterpenoids also have been reported to have antioxidant activity¹⁸. Muccilli et al.¹⁹ reported that tannins also play a role in blocking oxidative chain reactions. The solvent extract also has a contribution to increasing antioxidant and anti-collagenase activity. Ethanol extract of R. damascena L. petals has been shown to have a lower IC₅₀ value than acetone when evaluated by DPPH¹⁵. Ethanol extracts also show the highest phenolic content and antioxidant activity than water extracts of R. spinosissima⁹.

Antioxidant activity

Total antioxidant activity of EERP and EERR were further evaluated by FRAP assay. The FRAP value was obtained by plotting the standard curve of FeSO₄ at a concentration between 0.78 and 50 μg/mL (Figure 1).

Figure 1. Standard curve of FeSO₄.

The antioxidant activity of both extracts is presented in Figure 2. In this test, the antioxidant activities were obtained based on the capacity to reduce ferric (III) to ferrous (II). The result demonstrates that EERP has higher antioxidant capacity compared to EERR.

Figure 2. Effect of various concentrations of EERP and EERR in FRAP activity.

EERP, ethanolic extraction of Rosa damascena L. petals; EERR, ethanolic extraction of R. damascena L. receptacles.

Table 2 showed FRAP values for different concentrations of EERP and EERR. The data showed normal distribution and was significantly different (p > 0.005). Both extracts showed higher antioxidant activity with higher concentrations (Figure 2). The highest antioxidant activities were observed at 50 at µg/mL (EERP: 164.23 ± 1.34 µM/mL; EERR: 12.85 ± 6.19 µM/mL).

Anti-collagenase activity

Collagen is the most abundant protein responsible for conferring skin thickness, strength, and elasticity²⁰. This metalloproteinase enzyme has a vital role in collagen degradation, which increases aging signs²¹. The percentage of collagenase inhibition of EERP and EERR found in the present study is presented in Table 3. Anti-collagenase activity showed a concentration-dependent manner, where higher concentrations of the extracts increased the anti-collagenase (and thus antiaging) activity of extracts. At the highest concentration (250 µM/mL) EERP showed the highest inhibition activity compared to EERR (91.52 ± 3.44 µg/mL and 77 ± 2.03 µg/mL, respectively).

The IC₅₀ percentage can be seen in Table 4. The IC₅₀ value of EERR is higher than EERP (141.96 ± 6.13 µM/mL and 115.48± 1.78 µM/mL, respectively). This indicates that EERR has less activity to impede collagenase activity than EERP; however, these results show that there is potential for EERR to inhibit collagenase. Park et al.²² found that R. damascena extract impacted metalloproteinase transcription by suppressing AP-1 activation. AP-1 is a protein activated by UVB penetration to the skin, and initiates changes of the extracellular matrix, including collagen, elastin, and proteoglycans degradation. Our results show that ethanol extracts of petals and receptacles of R. damascene L. possess anti-collagenase activity.

Underlying data

Figshare: Raw data Analysis of anticollagenase an IC50 value.xlsx, https://doi.org/10.6084/m9.figshare.12585626.v1²³

Figshare: Raw data FRAP Activity of Extracts of Rosa Damascene Petals and Receptacles, https://doi.org/10.6084/m9.figshare.12585671.v1²⁴

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).