Introduction

Rhododendron schlippenbachii, or the royal azalea, is a species of Rhododendron native to the Korean Peninsula and adjacent regions of Manchuria, Japan, and the Russian Far East. It is the dominant understory shrub in many Korean hillside forests, growing at an altitude of 400–1500 m. Rhododendron isone of the largest shrub genera, has been distributed through most of the northern hemisphere, and their species are well-known garden plants due to their evergreen leaves and various flower colors¹. Furthermore, the vast genus is used in the traditional medical system in China, Europe, and North America. Such application is based on a tremendous number of phytochemicals with diverse biological activities, such as anti-microbial², anti-inflammatory³, anti-diabetic⁴, and anti-oxidative properties⁵. The R.schlippenbachii Maxim. (R. schlippenbachii Maxim) species has traditionally been sought after as a garden plant because of its attractive and diverse flowers, but it also has potential as a source for natural medicines, because of its activities as a cholinesterase inhibitor, anti-hyperglycemic, and anti-oxidant^6-8 (Figure 1).

Amino acids are important for all life processes. They are essential for every metabolic process, as well as for the optimal transport and optimal storage of all nutrients (i.e., water, fat, carbohydrates, proteins, minerals, and vitamins). Many diseases such as obesity, high-cholesterol levels, diabetes, insomnia, erectile dysfunction, or arthritis can be caused by metabolic disturbances. This can also apply to hair loss and serious cases of wrinkle formation. The correct amino acid composition may be able to repair many of these metabolic deficiencies. This is confirmed by various studies that stress the importance of amino acids hair and dermal health^9-11. Additionally, the amino acid arginine can lead to considerable expansion of the blood vessels, improving blood pressure in humans^12-14. Another study showed that arginine is also important in the treatment of diabetes-related foot diseases¹⁵.

More than 60% of the protein required by humans comes from plant sources. The most important function of amino acids is that they are the building blocks of proteins. Amino acids have antioxidant effects^16-19, and free amino acids are necessary in secondary plant metabolism and the biosynthesis of compounds, such as glucosinolates and phenolics, that play important roles, either directly or indirectly, in plant–environment interactions and human health²⁰

Although many amino acids exist in nature, approximately 24 are reported to be essential to human nutrition^21,22. Several previous studies have addressed the different nutritive properties of Rhododendron; however, to our knowledge no study has shown the amino acid content in the different species of Rhododendron. The objective of the present study was to determine the profile and quantity of amino acids present in the differently colored flowers of Rhododendron.

Material and Methods

Plant material

White, violet, and red flowers of three R. schlippenbachii Maxim cultivars were maintained at the Chungnam National University Experiment Farm, Daejeon, Korea. Flowers of these three cultivars were harvested on May 10, 2015 and immediately freeze-dried at -80°C for at least 72 h before being ground using a mortar and pestle into a fine powder for amino acid analysis.

Chemicals

We obtained trichloroacetic acid (TCA, 99.0%) from Samchun Pure Chemical Co., Ltd. (Pyeongtaek, Korea). Standards for 16 amino acids and four amino acid supplements were obtained from Agilent Technologies (Waldbronn, Germany). The vitamin U (dl-methionine methylsulfonium chloride) standards and sodium phosphate monobasic monohydrate (NaH₂PO₄) were purchased from Sigma-Aldrich (St. Louis, MO, USA). High performance liquid chromatography (HPLC)-grade acetonitrile (ACN) and methanol (MeOH) were purchased from J. T. Baker (Phillipsburg, NJ, USA). Ultrapure water with a resistivity of 18.2 MΩ/cm was produced using a PureLab Option system from ELGA LabWater (Model LA 621; Marlow, UK).

Table 1: Amino acid content in white, violet and red flowers ofRhododendron schlippenbachii  Maxim

Extraction and HPLC of free amino acids

In a 2-mL Eppendorf tube, 100 mg of freeze-dried plant powder was suspended in 1.2 mL 5% (v/v) TCA solution. The mixture was vortexed and allowed to stand for at least 1 h at room temperature before being centrifuged at 15,000 ×g for 15 min at 4°C. The supernatant was filtered through a 0.45-µm hydrophilic polyvinylidene difluoride (PVDF) syringe filter (Ø13 mm, Cat. no. 6779-1304; Whatman Int. Ltd., Maidstone, UK) into an HPLC vial.

HPLC analysis of the free amino acids was performed as described previously²³. Briefly, 20 different free amino acids were identified using an Agilent 1200 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with Zorbax Eclipse Amino Acid Analysis (AAA) columns (150 × 4.6 mm i.d., particle size 5 μm) and Zorbax Eclipse AAA Guard columns (12.5 × 4.6 mm i.d., particle size 5 μm, 4-pack). A wavelength of 338 nm, 40f, and flow rate of 2.0 mL/min were used in the HPLC conditions. The mobile phase consisted of 40 mM NaH₂PO₄ (pH 7.8, solvent A) and ACN:MeOH:H₂O (45:45:10, v/v/v) (solvent B). The HPLC gradient protocol was as follows: a linear step from 0% to 57% of solvent B from 1.9 to 21.1 min; 57% to 100% of solvent B from 21.1 to 21.6 min; isocratic conditions with 100% solvent B from 21.6 to 25.0 min; followed by a rapid drop to 0% solvent B at 25.1 min; and then isocratic conditions with 0% solvent B until completion (total 30 min). Standards were prepared as individual solutions (50 pmol/µL [0.05 mM]) of 20 amino acids. The free amino acids quantification was based on HPLC peak areas calculated as equivalents of the standard compounds. All quantities were expressed as milligrams per 100 grams fresh weight (FW). All samples were run in triplicate.

Results and Discussion

Amino acid content in white, violet, and red flowers of R. schlippenbachii

Analysis of the different Rhododendron flowers revealed 22 types of amino acids that differed in content based on flower color (Table 1). Arginine was not detected in any of the flowers and among the 21 amino acids, vitamin U and glycine were not detected in white Rhododendron flowers; and vitamin U, glycine, and leucine were not detected in violet Rhododendron flowers. In addition, no methionine, norvaline, or phenylalanine was detected in red flowers. Among the amino acids that were found in Rhododendron, the levels of histidine and glutamine were much higher in both violet and white flowers than in red flowers.

Violet Rhododendron flowers contained the highest total quantity of amino acids (1051.55 mg/100 g dry wt.), which was 2.24 and 1.31 times higher than that in red and white Rhododendron, respectively. Violet Rhododendron flowers had the highest quantities of aspartate, glutamate, glutamine, histidine, γ-aminobutyric acid (GABA), and methionine. The contents of these amino acids ranged 38.96–59.68, 8.16–15.21, 35.42–158.91, 2.37–555.41, 18.48–27.34, and 2.77–3.45 mg/100 g the dry weight, respectively; among the different Rhododendron flowers. Violet flowers contained 1.53, 1.86, 4.49, 234.35, and 1.48 times higher amounts of aspartate, glutamate, glutamine, histidine, and GABA, respectively, and red Rhododendron flowers had the lowest amino acid content. We observed the highest quantities of serine, threonine, alanine, valine, norvaline, tryptophan, phenylalanine, isoleucine, and leucine in white flowers. The ranges of these amino acids were 24.09–29.90, 11.65–21.82, 19.59–49.35, 2.97–9.83, 3.47–4.45, 3.33–4.48, 1.93–4.51, 2.13–5.23, 1.46–2.17, and 2.87–4.01 mg/100 g the dry weight, respectively, among the different Rhododendron flowers. White flowers contained 1.87, 2.52, and 3.31 times higher amounts of threonine, alanine, and valine, respectively, than did red Rhododendron flowers. Further, we observed 1.24, 1.35, 2.34, and 2.46 times higher amounts of Serine, tryptophan, phenylalanine, isoleucine, and leucine, respectively, in white flowers than in violet flowers. Although the total amino acid content was the lowest in red flowers, they contained the highest quantities asparagine, vitamin U, glutamine, glycine, tyrosine, cysteine, and lysine. The asparagine content was much higher in red flowers than in any other Rhododendron flower. Additionally, red flowers contained 3.70 and 2.48 times higher levels of asparagine than that in white and violet flowers, respectively. Red flowersalso contained 2.83 and 1.51 times higher levels of tyrosine and cysteine than did white flowers. The lysine level was 1.4 times higher in red flowers than in violet Rhododendron flowers.

Among the cultivars of Momordica charantia variation due to amino acid was noticed by  Kim et al.²³, who showed that among all the amino acids isolated from M. charantia, arginine was present in remarkably high quantities, whereas cysteine and methionine were present at the lowest concentrations. Variations in amino acid content have also been observed in different organs of Scutellaria baicalensis²⁴, green and red mustard²⁵, and in different species of aloe²⁶. Previously, Li et al.²⁷ reported that amino acid and GABA content varied in cultivars of Liriope platyphylla, a finding supported by the results of the present study.

Acknowledgements

This research was supported by Korea Institute of energy Technology Evaluation and Planning (KETEP) (Project number: 2015-048402).

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