Major components of Rhododendron luteum leaves

Abstract β-D-Fructopyranose (1), D-Xylopyranose (2), D-Mannitol (3), and flavonoids including quercetin-3-α-L-arabinofuranoside (4) and quercetin-3-O-glucopyranoside (5) were isolated from the leaves of Rhododendron luteum for the first time. Isolated compounds were identified using 1 D- (1H-NMR, 13C-NMR) and 2 D- NMR (HMBC, HSQC, COSY) spectroscopic techniques and comparison of the literature data. In our study, a relatively high amount of Quercetin-3-O-glucopyranoside was isolated (4%). As a result, our research showed that leaves of Rhododendron luteum could be a good source of monosaccharides, sugar alcohols and bioactive flavonoid glycosides. Graphical Abstract


Introduction
Plants have been still resourcing for modern science. Rhododendron (Ericaceae) is a wide genus of plants, which consists of 80 species around the world (Carballeira et al. 2008). Some of the Rhododendron species such as Rhododendron ponticum (Ericaceae) occurs from southwestern Iberia and the southern Black Sea Basin. It is known that poisonous effects on human metabolism and livestock. Poisoning occurs from eating honey made from the nectar of some Rhododendron species. However, this honey is used in traditional medicine and it has got some interesting medicinal properties when consumed in low amounts such as lowering the blood sugar levels in the human body, inflammatory, skin disease, common cold and asthma (Jansen et al. 2012;Popescu and Kopp 2013). In previous years, few phytochemical and pharmacological studies from R. luteum were carried out. The mixture of saponin was isolated from the leaves (Dzuba and Golovko 2000). Orhan et al. reported that the CHCl 3 : MeOH extract of R. luteum inhibited acetylcholinesterase and butyrylcholinesterase activity (Orhan et al., 2004). DMSO extract of R. luteum flowers showed reducing activity and exhibited a non-selective cytotoxic effect against MCF-7 cells compared to the chemotherapeutic drug, cisplatin (Demir et al. 2016). Mahomoodally et al. reported that methanol and water extracts of aerial parts of R. luteum have the highest flavonoid and phenolic contents using HPLC-ESI-MS methods in 2020.
In the light of the above explanation, our study was aimed to define the major phytochemical profiles of the 80% ethanol extract of R. luteum leaves. Phytochemical screening of plant extracts revealed the presence of phenols, tannins, glycosides, flavonoids, steroids, sterols and terpenoids (Table S1). The five main compounds were isolated from R. luteum leaves for the first time ( Figure 1). According to the literature, isolated five compounds have significant activities (Kim et al. 2010;Falleh et al. 2011;Awuchi 2017;Alirezalu et al. 2018;Buonerba et al. 2018). As a result, we can suggest that the leaves of this plant should be evaluated in terms of the food and pharmaceutical industry as a source of the natural drug.

Results and discussion
Compound 1 was isolated as a white powder (28 mg). Its melting point measured 105 C and its specific optical rotation measured [a] 24 ¼ À95. 13 C spectrum of compound 1 displayed six-carbon signals. The signals in 13 C-NMR, d C , 98.03, 69.66, 63.34, 69.17, 67.57, and 63.89 are similar to those reported data for b-D-Fructose. Its structure was supported with literature data (Shiomi and Onodera 1990). According to 1 H and 13 C NMR spectra and literature data, compound 1 was identified as a b-D-Fructose ( Figures S1 and S2).
Compound 2 was isolated as a white powder (30 mg). Its melting point was measured as 146 C and specific optical rotation was measured as [a] 24 ¼ þ19.1 Figure 1. Structure of isolated major compounds from R. luteum leaves. (Lappalainen et al. 2006). 13 C NMR of 2 showed 10 carbon signals including 2 anomeric carbon at 92.22 (a-C) and 96.61 ppm (b-C). The presence of a and anomers of xylose was observed for anomeric protons between 4.51 (d, J ¼ 7.9, b-CH) and 5.13 (d, J: 1, a-CH) in the 1 H NMR spectrum. According to the 1 H and 13 C NMR spectra and the literature data, compound 2 was identified as a mixture of anomers of D-(þ)-Xylose. (Figures S3 and S4) (https://www.chemicalbook.com/SpectrumEN_58-86-6_1HNM.htm).
Compound 3 was isolated from the water extract as a white powder (35 mg). Its 1 H and 13 C NMR spectrums were supported with the literature data and were identified as a D-mannitol ( Figures S5 and S6; Dinda et al. 2006). Melting point measured 169 C and optical rotation measured [a] 23 ¼ þ25. Its molecular structure was differentiated from its stereoisomer D-iditol by comparing their melting points (Desphande and Upadhyay, 1967).
Compound 4 was isolated as a yellow powder (17 mg). Its chemical structure was identified with the comparison of its 1 H and 13 C NMR spectrums, HMBC, HSQC and COSY NMR spectrums and the literature data (Figures S7-S11; Zhang et al. 2005). Its melting point measured 209 C and specific optical rotation measured [a] 27 ¼ À142. Arabinose can be attached to both pyranose and furanose forms. In pyranose form methylene protons (H-5 0 ) shows a large chemical shift difference (dH ¼ 3.81/4.38 ppm) in the HSQC spectrum. On the other hand, methylene protons (H-5 0 ) in the furanose form is observed with a small difference (dH ¼ 4.10/4.15 ppm) in the HSQC spectrum. As a result, compound 4 was identified as an Avicularin (Quercetin 3-a-Larabinofuranoside).

Conclusion
In our study, five major compounds were isolated from the leaves of R. Luteum for the first time. Compound 1, fructose, is a monosaccharide that can be found widely in vegetables and fruits. Compound 2, xylose, is first isolated from the wood. It can be found naturally in the embryos of many edible plants. Xylose plays a remarkable role in the biological conversion of plant biomass to fuels and chemicals. D-Xylose is widely used as a diabetic sweetener in food and beverage. Compound 3, mannitol, is a sugar alcohol. It can be found naturally in plants and fruits (Venditti et al. 2013) It is used in the food industry as a sweetener. "Medically, D-mannitol is using as a diuretic in serums and is poorly metabolized in the intestines. It can be found as a different concentration from %5 to %20 in medicine sector" (Awuchi 2017). "On October 30, 2020, mannitol was approved by the FDA as add-on maintenance therapy for the control of pulmonary symptoms associated with cystic fibrosis in adult patients and is currently marketed for this indication under the name BRONCHITOLV R by Chiesi USA Inc" (https://go.drugbank.com/drugs/DB00742) Compound 4, Avicularin, is the secondary metabolite which biosynthesised in many plants. "It is a flavonoid glycoside which demonstrates many biological activities such as anti-oxidant activity, anti-microbial activity, anti-allergic activity, oestrogenic activity and vascular activity" (Falleh et al. 2011). Compound 5, Quercetin 3-O-glucopyranoside, is also known as isoquercetin. It possesses antioxidant activity (Alirezalu et al. 2018) as well as antiviral activity (Kim et al. 2010). "Clinically, isoquercetin has been used in studying on kidney cancer, renal cell carcinoma, thromboembolism of vein in pancreatic cancer" (Buonerba et al. 2018).
As a result, we present R. luteum leaves can be a good source for medicinal glycosides and monosaccharides as well as bioactive flavonoids glycosides. All these findings will be helpful for the food, pharmaceutical industry and/or chemotaxonomic profile of this genus for further investigations.