Georgian Scientists/ . 5 N 2, 2023 54 Georgian Scientists Vol. 5 Issue 3, 2023 https://doi.org/10.52340/gs.2023.05.03.07 Pharmacological potency of the different polarity fractions from the Rosa damascena Georgian cultivar oil waste aqueous extract. Karen Mulkijanyan 1, Marine Sulakvelidze 1, Natela Gogitidze 1, Nadezhda Mushkiashvili 1, Zhana Novikova 1, Nino Aleksidze 2, Sopio Gokadze 2, 3 and Lasha Mskhiladze 1, 2 1Department of Preclinical Pharmacological Research, Tbilisi State Medical University I. Kutateladze Institute of Pharmacochemistry, Tbilisi, Georgia 2Direction of Pharmacognosy and Pharmaceutical Botany, Tbilisi State Medical University, Tbilisi, Georgia. 3Scientific-Research and Practical Skills Laboratory of the Pharmacy Faculty, Tbilisi State Medical University, Tbilisi, Georgia. Corresponding author: Lasha Mskhiladze, l.mskhiladze@tsmu.edu ABSTRACT Damask rose (Rosa x damascena Herrm) is a well-known species within the genus Rosa L. cultivated in numerous countries due to its ornamental properties and especially for the valuable essential oil found in its flower petals. The latter has been utilized in perfumery, cosmetics, aromatherapy, and medicinal treatmentsfor centuries. Overall, the studies on R. damascena provide promising insights into its diverse effects and applications. However, it is important to note that further research is needed to validate these findings and fully understand the mechanisms underlying the observed effects. The present study aimed to assess the pharmacological features of the potency of the different polarity fractions RDF-H2O, RDF-50% and RDF-100% derived from the aqueous extract of rose oil waste from the R. damascena Georgian cultivar. In the carrageenan-induced acute inflammation model in rodents, it was established that all three fractions - RDF-H2O, RDF-50% and RDF-100% have an anti-inflammatory effect of higher-than- average strength. Among them, RDF-50% was significantly more active and, caused the 62.5% inhibition of the edema. A similar efficacy trend was revealed when evaluating the analgesic properties in mice in "Hot plate" model, but the most powerful analgesic effect was shown by fractions RDF-50% and RDF-100%, the maximum effect of which (86.0 and 99.7%, respectively) was observed 1 hour after administration. As a result of the experiment on the gastric ulcer model caused by absolute ethanol in rodents, it was revealed that the samples RDF-H2O and RDF-100% have a gastroprotective effect of medium strength (48 and 40%, respectively), whereas RDF-50% appeared less effective. The findings Georgian Scientists/ . 5 N 2, 2023 55 of current study highlight the potential of further investigation of the above-mentioned fractions to identify individual constituents responsible for the observed pharmacological effects. Keywords: Rosa damascena extract; anti-inflammatory; analgesic; gastroprotective INTRODUCTION Rosa x damascena Hermm, commonly known as Damask rose [32, 34], has been the subject of multiple studies investigating its effects in various domains, which highlight the potential benefits and applications of the plant [16, 35]. The historical significance of R. damascena as a medicinal herb is one aspect explored. For ages it has been traditionally used in different cultures for various purposes, including managing cardiovascular disorders, gastrointestinal problems, and nervous system disorders. [17, 18]. These traditional uses hint at the potential therapeutic effects of R. damascena. Another area of research focuses on the chemical composition of R. damascena essential oil and its antimicrobial and antioxidant activities. The essential oil has shown promising antimicrobial properties, inhibiting the growth of microorganisms, and it exhibits antioxidant activity, which could have implications for health and well-being [3, 8, 24, 25] Furthermore, there is scientific evidence supporting the pharmacological effects of R. damascena. In addition to aforesaid activities, studies have demonstrated that R. damascena could have broader applications in the field of pharmacology and medicine [5, 12, 13, 29, 33]. In recent years, efforts have been made to explore the waste generated during the extraction of rose oil from R. damascena flowers. Due to the relatively low oil content in the flowers, the industrial extraction process produces substantial amounts of waste, including solid residues and wastewater. In the past decade, several methods for valorisation of rose waste have been developed [30, 31]. Studies have revealed that the waste contains a significant amount of biologically active compounds, particularly polyphenols, flavonoids, and polysaccharides [2, 4, 6, 14, 11, 15, 20, 23]. These compounds hold potential for various applications in the pharmaceutical, nutraceutical, and cosmetic industries. By utilizing the waste, researchers aim to maximize the utilization of the R. damascena plant and minimize environmental impact. Research on the polar and non-polar extracts obtained from R. damascena flower petals has provided valuable insights into their chemical composition, bioactive compounds, and potential applications. These extracts have attracted significant attention due to the presence of various bioactive components that contribute to their pharmacological properties and industrial significance. [27]. Non-polar extracts, such as essential oils obtained through processes like hydro distillation, have been a subject of interest due to their aromatic properties and commercial applications. R. damascena essential oil is highly valued in the perfume and cosmetic industries for its exquisite fragrance. The chemical composition of essential oils from R. damascena can vary significantly depending on factors such as climatic conditions, soil composition, and extraction techniques [10]. The major constituents of these oils typically include various terpenes and aromatic compounds. The unique composition of essential oils contributes to their pleasant scent and therapeutic effects [9]. Furthermore, the essential oil has been utilized in traditional Georgian Scientists/ . 5 N 2, 2023 56 medicine for its medicinal properties, including its use as a base material in aromatherapy and for the treatment of certain diseases. Recently we reported about biological potency of the aqueous extract of rose oil waste from the R. damascena Georgian cultivar [22] and the present study aimed to assess the pharmacological features of the potency of the different polarity fractions RDF-H2O, RDF-50% and RDF-100% derived from the above-mentioned extract. MATERIALS AND METHODS Materials, chemicals and reagents The plant material was collected from Kakheti floristical region of Georgia during the active flowering season in May, 2020. The identification of the plant was carried out in the Department of Botany at Iovel Kutateladze Institute of Pharmacochemistry, Tbilisi State Medical University. A voucher specimen (TBPH–21167) was deposited in the herbarium of the named institute. Aqueous extract of R. damascena petals (AERD) was obteined in accordance with the European Pharmacopoeia (Ph. Eur. 2008) guideline. All reagents, and solvents, and chemicals used in the experiments were of analytical grade and obtained from Sigma Aldrich (USA). Fractionation of AERD AERD was processed as following: a 20 g dry extract was chromatographed on a Diaion HP-20 column. H2O-MeOH gradient (100:0, 50:50, and 0:100 v/v) was used as the mobile phase. The fractions were concentrated using rotary vacuum evaporator below 40ºC and were frozen in a layer of 10 mm in the Petri dishes at -20 °C in freezer for 12 h, then vacuum dried at -90 °C under 3.33 Pa absolute pressure to constant weight. The dried material was powder-grinded and stored in vacuum desiccator until further use. Biological assays Animals: Inbred white mice weighing 28 ± 2 g (n = 40) was housed under standard conditions: including a temperature of 20 ± 2°C, humidity of 55-65%, a 12/12-hour light/darkness cycle, and provided with granulated food (4 g/animal/day) and water ad libitum. All experimental procedures adhered to the requirements of the EU Directive 2010/63 [7] and were approved by the Ethics Committee on Animal Research at Tbilisi State Medical University (registration number AP-61-2023). Analgesic Activity (Hot plate assay) The analgesic activity was evaluated using a setup comprising an open cylindrical space with transparent vertical walls and a metal floor heated to 52 ± 2°C. The mice were administered the test compounds (50 mg/kg intraperitoneally), and the reaction time, either hind paw licking or jumping, was recorded at regular 30 min intervals over a period of one hour. The analgesic effect was calculated using the formula: E% = ((To-Tn) /T0) × 100, where to represented the reaction time before the extract injection, and Tn represented the reaction time after the specified period (30 or 60 min) following injection [26]. Georgian Scientists/ . 5 N 2, 2023 57 Anti-inflammatory Activity (Carrageenan-induced edema assay) The anti-inflammatory activity was assessed using the carrageenan-induced paw edema model [21]. A 50 l injection of 1% carrageenan solution in normal saline was administered into the aponeurosis of the right hind paw of each mouse. One hour prior to the carrageenan injection, the control group received 0.5 ml of normal saline, while the experimental groups received 0.5 ml of the test fractions at a dose of 50 mg/kg, both administered intraperitoneally. The thickness of the paw was measured using a digital micrometer before (baseline) and 2 hours after the carrageenan injection. The anti- inflammatory efficacy was calculated using the formula: E%=(1-( Texp/ Tcon)) x 100, where Tcon and Texp represented the mean differences in paw thickness before and 2 hours after carrageenan administration in the control and experimental groups, respectively. Gastroprotective Activity (Ethanol-induced ulcer model): The gastroprotective activity experiment followed the method described by Adinortey et al. [1]. A total of 24 outbred mice were randomly divided into four groups, with six mice in each group. Prior to the experiment, food intake was restricted for 24 hours, and to prevent coprophagy the mice were placed in cages with a raised wire mesh floor. To prevent excessive dehydration during fasting, all mice had free access to a nutritive solution of 8% sucrose in 0.2% NaCl. On the second day, the test group received test fractions (at a dose of 50 mg/kg) intraperitoneally, while the control group - normal saline (0.4 ml/animal). Subsequently, all animals were orally administered absolute ethanol (1 ml/100 g). After 1 hour, the mice were euthanized using CO2 inhalation. The stomachs were promptly removed, opened along the greater curvature, and rinsed with water and a 10% formalin solution (containing approximately 4% formaldehyde w/v). The stomachs were then fixed on a white polystyrene board and digitally photographed. The macroscopic ulcer index (MUI) was calculated for each stomach based on the following scale, determined by three independent observers: 1 - no lesions, 2 - single petechial lesions, 2.5 - multiple petechial or short linear haemorrhagic lesions, 3 - long linear haemorrhagic lesions, 4 - continuous linear haemorrhagic lesions along the entire length of the glandular part of the stomach. The efficacy of test compounds was calculated using the following formula: % I = (MUIcontrol - MUItest) / MUIcontrol × 100, where MUIcontrol and MUItest represent the macroscopic ulcer indexes in the control and test groups, respectively. Statistical Analysis. Statistical analysis of the experimental data was conducted using Student's t-test [19]. A significance level of p 0.05 was considered statistically significant. RESULTS AND DISCUSSION Hot plate assay. When evaluating the analgesic properties in "Hot plate" model in mice it was discovered that maximal analgesic effect for all tested fractions was observed 1 hour after the administration. RDF-50% and RDF-100% fractions exhibited nearly similar efficacy: 86.0% and 99.7%, respectively (p<0.05). Fraction RDF-H2O with 19.5% analgesic effect appeared 4.5-fold less active. (Fig. 1, Fig.4, A) Georgian Scientists/ . 5 N 2, 2023 58 Figure 1. Analgesic efficacy of tested fractions in “Hot plate” model. Each value represents mean±SEM of 6 animals; * - p<0.05; ** - p<0.01 vs control. Carrageenan-induced edema assay. In the model of acute inflammation (carrageenan-induced swelling) in rodents, it was established that 2 hours after the carrageenan administration all three fractions - RDF-H2O, RDF-50% and RDF-100% have an anti-inflammatory effect of higher-than-average strength. An increase in the thickness of the paw varied from 49 to 72 m, whereas in control animals it was 131 m (Fig. 2). Among fractions, RDF- 50% appeared the most active one causing the 62.5% (p<0.01) inhibition of the edema. (Fig.2, Fig. 4, B) Figure 2. Dynamics of carrageenan-induced edema development in animals from control and experimental groups. Each value represents mean±SEM of 6 animals; * - p<0.05; ** - p<0.01 vs control. Ethanol-induced ulceration assay Georgian Scientists/ . 5 N 2, 2023 59 As a result of the experiment on the gastric ulcer model caused by absolute ethanol in rodents, it was revealed that the samples RDF-H2O and RDF-100% have a gastroprotective effect of medium strength (48 and 40%, respectively), whereas RDF-50% appeared less effective (Fig. 3, Fig 4, C). The findings of current study highlight the potential of further investigation of the above-mentioned fractions to identify individual constituents responsible for the observed pharmacological effects. Figure 3. Gastroprotective activity of tested fractions in ethanol-induced gastric ulcer model in mice. MUI - Macroscopic ulcer index. Each value represents mean of 6 animals; * - p<0.05 vs control DISCUSSION Polar extracts, such as hydroalcoholic and aqueous extracts, rich in biologically active compounds, including polyphenols, flavonoids, and polysaccharides [4, 15] have been extensively studied for their diverse pharmacological activities [2, 27]. Studies have shown that polar extracts of R. damascena exhibit antioxidant properties, which can help protect against oxidative stress-related diseases. The presence of polyphenols and flavonoids in these extracts contributes to their antioxidant effects by scavenging free radicals and inhibiting oxidative damage [3]. Additionally, the hydroalcoholic and aqueous extracts have demonstrated analgesic, anti-inflammatory [8, 12], hypnotic, memory- enhancing, and anticonvulsant activities [28, 29] Thus, the results of our investigation well correlate with literary data. On the other hand, based on the results obtained (Fig. 4), it is obvious that further research is needed both to identify the compounds responsible for the revealed activities and to explore the full potential of these extracts in various industries and therapeutic applications. Georgian Scientists/ . 5 N 2, 2023 60 Figure 4. Pharmacological efficacy of tested fractions (consolidated data). * - p<0.05; ** - p<0.01 vs corresponding controls. CONCLUSION The present study provided additional data on biological potency of the rose oil waste products from the R. gallica Georgian cultivar. It can be concluded that these products contain valuable amounts of bioactive substances that can be used in the pharmaceutical and cosmetic industry as main or auxiliary components. The recovery of such substances additionally carries an ecological impact because it aids in the complete utilization and hence reduction the overall amount of waste produced during the production of rose oil. COMPLIANCE WITH ETHICAL STANDARDS Acknowledgments The authors are thankful to Pirose LTD for supplying the raw plant material for this study. Disclosure of conflict of interest The authors declare no financial or any other conflicts of interest in this work. Statement of ethical approval The animal research protocol was approved by the Tbilisi State Medical University Ethics Committee on Animal Research (registration #AP-61-2023). Georgian Scientists/ . 5 N 2, 2023 61 REFERENCES [1] Adinortey MB, Ansah C, Galyuon I, Nyarko A. (2013). In Vivo Models Used for Evaluation of Potential Antigastroduodenal Ulcer Agents. Ulcers. [2] Akram M, Riaz M, Munir N, et al. (2019) Chemical constituents, experimental and clinical pharmacology of Rosa damascena: a literature review. J Pharm Pharmacol. 2020 Feb;72(2):161- 174. https://doi.org/10.1111/jphp.13185 [3] Basim E, Basim H (2003) Antibacterial activity of Rosa damascena essential oil. Fitoterapia. 74(4), 394-6. 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Chinese Herbal Medicines, 12(3), 336-341. https://doi.org/10.1016/j.chmed.2020.05.005 [34] World Flora Online (WFO) http://www.worldfloraonline.org/ [35] Yousefi B., Jaimand K. (2018) Chemical Variation in the Essential Oil of Iranian Rosa damascena Landraces under Semi-arid and Cool Conditions. International Journal of Horticultural Science and Technology 5(1); 81-92. https://doi.org/10.22059/ijhst.2018.256329.234 Rosa x damascena 1, 1, 1, 1, 1, 2, 2, 3, 1,2 * 1 . , , , 2 , , , 3 , , , (Rosa x damascena Herrm) Rosa L.- , . , , , . . , , Georgian Scientists/ . 5 N 2, 2023 64 . R. damascena- RDF-H2O, RDF-50% RDF-100% in vivo . , - RDF-H2O, RDF-50% RDF-100% . , RDF-50% 62.5% . " " , RDF-50% RDF-100% , (86.0 99.7%, ) 1 . , RDF-H2O RDF- 100% (48 40%, ), RDF-50% . , . : Rosa damascena ; ; ; Georgian Scientists/ . 5 N 2, 2023 65 Graphical Abstract Aqueous Extract of Rosa damascena - AERD RDF-H2O RDF-50% MeOH RDF-100% MeOH PHARMACOLOGICAL ASSESSMENT “Hot Plate” assay Carrageenan induced inflammation assay AERD CHROMATOGRAPY On DIAION HP-20 Fractions Ethanol-induced gastric ulceration