Different drying techniques effect on the bioactive properties of rose petals

2.1 Extraction

The rose petal samples were extracted with 80 % ethanol in a solid to liquid ratio of 1:20 on fresh weight basis using a shaker at 220 rpm for 1 h at 30 °C. The samples were then filtered using a Whatman filter paper No 2. The extracts were stored at 4 °C for further analysis.

2.2 Determination of total phenolic content (TPC)

The quantification of total phenolic contents was performed using the Folin–Ciocalteu (FC) procedure as previously reported by Alshammari et al., (2022). Methanol 4 mL was used to dissolve 1 g of sample using vortex, then the solution was filtered through Whatman No.1 filter paper. 200 µl of Folin–Ciocalteu reagent was mixed with 5 mL Milli-Q water and 100 µl sample solution, then 3 mL of sodium carbonate (Na2CO3) 20 % solution was added and incubated for 2 h at room temperature in the dark. The intensity of absorbance was recorded at 765 nm with methanol as a blank. Gallic acid standard (0–1.5 mg/mL) was used to obtain the standard curve for the calculation of total phenolic contents and results were reported as milligram gallic acid equivalent (GAE) per 100 g of sample.

2.3 Total flavonoid content (TFC)

The TFC was analyzed according to the method reported by (Salamatullah et al., 2021). In total, 250 µl of rose petal extract was mixed with 1000 µl of water and then 75 µl each of 5 % NaNO2 (w/v) and 10 % AlCl3 (w/v) was added. The prepared mixture was allowed to stand for 5 min at room temperature. After that, 600 µl of water and 500 µl of 1 M NaOH were added. Blank was prepared without extract. The absorbance was measured at 510 nm (Jasco V-630 UV–Vis spectrophotometer, Easton, MD, USA). TFC was expressed as catechin equivalent per gram dry weight of the sample (mg CE/g DW) against a catechin standard curve prepared at a concentration of 0.05–0.6 mg/ml.

2.4 DPPH radical scavenging assay (RSA)

The DPPH (1,1-diphenyl-2-picrylhydrazil, SIGMA, St. Louis, MO, USA) radical scavenging activity was determined in this study using the method of Alshammari et al., (2022). Briefly, 0.75 mL of the sample solution (0.1 g/mL) in warm water was mixed with 1.5 mL of DPPH (0.09 mg/mL) in methanol. The mixture was then incubated at 25 °C in a water bath for 5 min. The absorbance was measured at 517 nm against a blank sample consisting of sample solution with distilled water. The absorbance of a radical blank was also measured using 0.75 mL of distilled water.

The radical scavenging activity (RSA) of sample extract was expressed in terms of percentage inhibition of DPPH radical by rose petal and was calculated as follows: $$\text{RSA}\left(\text{DPPH},\text{Inhibition},\%\right)=\left[\left(\text{AB}-\text{AT}\right)/\text{AB}\right]\times 100$$

Where AB = Absorbance of radical blank (DPPH, without sample).

AT = Absorbance of test sample (DPPH, with sample).

2.5 Antibacterial and anticandidal activity

Antimicrobial activity of rose extracts using different methods was determined with agar well diffusion assay against Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans (Salamatullah et al., 2021).

2.6 Determination of minimum inhibitory concentration (MIC)

The MIC of rose extracts was obtained using the previously published microbroth dilution method. (Hussein and Joo, 2018; Alyousef, 2021).

2.7 Gas chromatography-mass spectrometry (GC–MS) analysis

Analysis was carried out using gas chromatograph coupled with mass spectrometer (Turbomass) Perkin-Elmer, Auto system XL using Elite 5MS capillary column (30 mts × 0.25 mm ID). The oven temperature program was 80 °C for 5 min, rising to 310 °C at a rate of 10 °C/min. The injector and interface temperatures were kept at 260 and 250 °C, respectively. The flow rate of the carrier gas, helium, was fixed to 1.0 mL/min. In ionization mode, mass spectral scanning was performed at 40–600 (m/z). The temperatures of the source and intake lines were set to 180 and 250 °C, respectively. To identify the compounds, the spectra were compared to the National Institute of Standards and Technology (NIST, 2005 v2.1) collection.

2.8 Statistical analysis

Data was statistically analyzed using SAS (Version 9.2, 2000–2008; SAS Institute Inc., Cary, NC, USA). The experiments were independently replicated 3 times (n = 3). The data were presented as mean standard deviation (SD). If the differences were found to be significant between the groups, a post-hoc analysis using Duncan's multiple range testing was carried out. The differences between the treatment groups were examined using one-way analysis of variance (ANOVA) at a significance threshold of p ≤ 0.05.

3.1 Effect of drying method on the total polyphenol content of rose

Fig. 1 shows the effect of drying methods on the total polyphenol content (TPC) of rose petals. The drying methods showed a significant effect on the TPC of rose samples (P < 0.05). Fresh sample showed the lowest TPC (15.6 mg gallic acid equivalent (GAE)/g fresh weight (FW)), while the highest TPC (34.24 mg GAE/g FW) was exhibited by the oven-dried sample. Statistically, there was no differences (P ≥ 0.05) between the TPC of shade-dried and sun-dried samples. The results of our study are in contrary to the findings of Barimah et al. (2017) who reported that the fresh leaves of Taraxacum officinale exhibited the highest TPC (7.78 mg GAE/g) as compared to the solar dried (4.19 mg GAE/g), hot-air oven dried (2.95 mg GAE/g), and freeze dried (4.31 mg GAE/g). In another study, the total polyphenol contents of Magnolia liliflora extracts were high after shade drying, microwave drying, and hot air drying. While the sun drying caused damage and showed a lower content (Feng-Ying et al., 2015). The total polyphenol content of rose petals ranged from 7.61−9.65 g GAE/100 g dry weight collected from 7 industrial scale plantations from Bulgaria for two consecutive growing seasons (Ginova et al., 2013). The TPC of the methanolic extract of fresh Rosa damascena flower from Turkey was reported as 276.02 mg GAE/g. Alizadeh and Fattahi (2021) studied 24 accessions of cultivated Damask rose and found that the TPC of rose petals ranged from 64.92 to 165.16 mg GAE/g dry weight. In previous work, the amount of TPC (GAE/g DW) for Damask rose petals was reported as 211.92–268.71 mg (Baydar and Baydar, 2013). The divergence in results can be attributed to the location, variety, and the extraction method (Özkan et al., 2004). Convection and vacuum-microwave drying method was studied by Matłok et al. (2020). Polyphenol content are high (208gm 100 g-1 dry matter) in the samples of pink rose. Stępień et al. (2019) reported that the convection drying at 40 °C and vacuum-microwave drying results in highest bioactive total polyphenol content and antioxidant activity.

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Fig. 1

Drying methods effect on the total polyphenol content of rose.

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3.2 Effect of drying methods on the total flavonoid content of rose

The effect of drying methods on the total flavonoid content (TFC) is shown in Fig. 2. The drying methods almost exhibited a similar trend for TFC as it was shown for the TPC of the rose samples. The TFC of the samples were in the following order: fresh < sun-dried < shade-dried < oven-dried, which were significantly different from each other (P < 0.05). In a recent study, the TFC of 24 accessions of cultivated Damask rose petals was reported between 28.59 and 81.35 mg Qu/g dry weight (Alizadeh and Fattahi, 2021). In another study, the TFC of Damask rose petals was reported as 28.1–98 (mg catechin equivalents/ g dry weight) (Baydar and Baydar, 2013). The total flavonoid content of the mint leaves was increased on drying compared to the fresh one (Hayat, 2020). The vacuum drying method of Bletilla striata flowers exhibited a higher TFC content than the hot-air drying, microwave drying, infrared drying natural shade drying, and freeze-drying methods (Lu et al., 2021). The hot-air drying, microwave drying, infrared drying, natural shade drying, and freeze-drying methods (Lu et al., 2021). In an earlier study it was found that the total flavonoid contents of Magnolia liliflora extracts were high after shade drying, microwave drying, and hot air drying, while the sun drying showed a lower flavonoid content (Feng-Ying et al., 2015). There are contradicting reports in the literature about the drying methods impact on plant samples. On one hand, the cell wall of plant materials may rupture during the drying process to facilitate the release of bioactive compounds into the extracting solvent (Hayat et al., 2010; Bernard et al., 2014; Hayat et al., 2019; Hayat, 2020). On the other hand, drying could cause the changes in the chemical structure of the phenolic compounds or cause them to adhere to each other components like proteins, which makes their extraction difficult (Youssef and Mokhtar, 2014). Moreover, the degradation enzymes such as polyphenol oxidase could play an important role in certain moisture content of plant materials (Orphanides et al., 2013). However, the plant species and cell wall stability have been reported to influence the effect of drying on the phenolic content of plants (Youssef and Mokhtar, 2014). Italian apple Mela Rosa dei Monti Sibillini was subjected to dehydrated by drying and freez-lyohilization methods (López et al., 2020). Flavan-3-ols, flavonol glycosides and dihyrochalcones are high in the extracts obtained by freez-lyophilization.

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Fig. 2

Drying methods on the total flavonoid content of rose.

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3.3 Effect of drying methods on the DPPH scavenging of rose

The 2,2-diphenyl-1-picrylhydazyl scavenging activity of the rose samples under different drying methods is depicted in Fig. 3. The oven dried sample displayed the high DPPH scavenging activity, reflecting the results of TPC and TFC. (60.30 %) followed by the sample dried under shade (52.24 %). While, the lowest activity (26.66 %) was obtained for the fresh rose sample. Özkan et al. (2004) reported the DPPH antiradical activity of 74.51 % for the fresh Rosa damascena flower collected from Turkey. In a previous study, which assessed the effect of drying methods on Taraxacum officinale leaves, it was reported that fresh leaves had DPPH scavenging effect, which was significantly higher 83.71 % (freeze), 74.34 % (solar), and 69.51 % for oven dried samples (Barimah et al., 2017). Lu et al. (2021) recently studied the vacuum drying and shade drying showed higher DPPH scavenging for the Bletilla striata flowers as compared to the hot-air drying, microwave drying, infrared drying, and freeze-drying methods. The bioactive components of the fresh bitter water rose flower were better preserved by freeze-drying treatment than the hot air drying, and the resulting DPPH scavenging capacity was strong (Gao et al., 2014). Hendrysiak et al. (2023) reported the significant influence on the antioxidant capacity of rosehip (Rosa canina L.) with the addition of carrier substances.

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Fig. 3

Drying methods effect on the DPPH scavenging of rose.

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3.4 Effect of drying methods on the reducing power of rose

Fig. 4 represent the influence of drying procedures on the reducing power of rose samples. The reducing power followed the same pattern as the TPC activity. The highest reducing power was found for the dried rose sample by oven (absorbance 1.138), while the lowest activity (abs 0.886) was noted for the fresh sample, respectively. There was no significant difference (P > 0.05) between the reducing power of shade- (1.032) and sun-dried (0.999) samples. These results showed that the reducing power was due to at least a part of the TPC and TFC of rose sample. The antioxidant capacity of shade dried, microwave dried, and hot air-dried Magnolia liliflora was good, while sun drying caused great damage to polyphenols and flavonoids, and the antioxidant activity was low (Feng-Ying et al., 2015). The roses retained strong antioxidant activity after oven drying, but low temperature refrigeration or frozen storage accelerated the decomposition and loss of antioxidant components.

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Fig. 4

Drying methods effect on the reducing power of rose.

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3.5 Antimicrobial activity of the rose extracts

Extracts of rose were also assessed for their antibacterial and antifungal activity against 4 bacterial strains and 1 Candida sp. We observed that highest antibacterial and anticandidal activity was demonstrated Fig. 5, by the extracts that were prepared from the oven dried sample followed by the extract of shade dried sample, sun dried and lowest activity was recorded for the fresh samples. After treatment with extract prepared from the oven dried sample as depicted in the figure, growth suppression zones of 16, 15, 17, 15, 14 mm were obtained against S. aureus, L. monocytogenes, E. coli, P. aeruginosa, and C. albicans, respectively. Fluconazole and doxycycline were employed as positive controls drugs, since they are both antifungal and antibacterial respectively. Microorganism are responsible for the spoilage of food leading to food-borne disease that cause severe health issues to the consumers and incur huge economic losses (Uthpala et al., 2021). Extract prepared from the oven dried rose samples demonstrated highest inhibition against the test bacteria and Candida sp. and thus could prove effective against food associated pathogens.

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Fig. 5

Antimicrobial activity of rose extracts subjected to different drying techniques.

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The lowest concentration at which the extracts demonstrates complete growth inhibition of the bacteria is termed as its minimum inhibitory concentration (MIC) Fig. 6. We observed that lowest MICs were demonstrated by extract prepared from oven-dried sample against all test pathogens (Figure). MIC values were recorded to be 1 mg/mL against S. aureus and E. coli while 2 mg/ml against L. monocytogenes, P. aeruginosa and C. albicans. MICs ranged from 4 −16 mg/ml, 2–8 mg/ml and 4–8 mg/ml for the fresh, shade dried and sun-dried samples, respectively. Similar MIC values have been reported previously with the extract of Acmella flowers that were subjected to different drying techniques (Uthpala et al., 2021).

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Fig. 6

MIC of rose extracts subjected to different drying techniques against test pathogens.

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3.6 Gas chromatography-mass spectrometry (GC–MS) analysis

Results of GC–MS analysis is reported in Table 1. Fresh sample shows the aliphatic hydrocarbons are the dominant compounds 64.73 %. Whereas aliphatic hydrocarbon for sun and oven dried samples are 11.69 and 8.93 %, respectively. Generally the representative aliphatic hydrocarbons for rose samples was tetracosane, heptacosane and 11-decyl-docosane in all rose samples. Major compound in sun and oven dried rose samples were Phenylethyl alcohol, 30.41 % and 40.21 % respectively.

Fresh Rose sample GC–MS analysis revealed a high ratio of heptacosane 64.56 % and citronellyl propionate 28.35 %. Pentadecyl 2-phenylethyl ester oxalic acid was the second dominant compound in sun and oven dried rose samples, 18.5 % and 14.79 %, respectively. Fresh rose samples have three major compounds and other compounds are found as traces comparing with sun and oven dried samples. Higher monoterpene alcohols and lower quantity of alkanes indicate good quality of rose oil (Baser, 1992). Therefore, this agrees to our analysis for sun and oven dried rose samples, this corelates with the TPC, TFC, and DPPH scavenging activity results. However, extraction of fresh Rose samples can be considered as a good source of aliphatic hydrocarbon. Halawani (2014) reported citronellol (15.9–33.3 %) nonadecane (5.5–16.0 %) in rose oil. Ulusoy et al. (2009) reported that citronellol and geraniol are the major compounds in Turkish rose oils. El-Sharnouby et al. (2021) reported the concentrations of geraniol, phenyl ethyl alcohol, and citronellol were 8.67, 9.87, and 16.56 % respectively for rose samples grown under 500 PPM salinity water.