Quantitative Analysis of the Flavonoid Content in the Leaves of Boehmeria nivea and Related Commercial Products

Abstract

Content analysis of flavonoids (epicatechin, epicatechin gallate, and rutin) present in the leaves of Boehmeria nivea (originating from Geumsan-myeon, Biin-myeon, Hansan-myeon, and Baeksu-eup) and their commercial products (ramie tteok, ramie songpyeon, ramie bory-tteok, and ramie tea) was conducted by HPLC. The content of epicatechin, epicatechin gallate, and rutin was highest in the leaves of B. nivea from Geumsan-myeon (0.138 mg/g), Baeksu-eup (1.654 mg/g) and Geumsan-myeon (12.205 mg/g), respectively. With respect to commercial products, the content of epicatechin and epicatechin gallate was highest in ramie tea, with concentrations of 1.879 and 1.090 mg/g, respectively. Given these flavonoid concentrations, B. nivea leaf extracts have the potential to be used as additives in natural medicinal products, health supplements, and beverages.

Introduction

Boehmeria nivea, which belongs to the family Urticaceae, is distributed throughout Asia including the Philippines, India, China, Korea, and Thailand. It is a herbaceous perennial plant with broad (6 - 12 cm) and long (7 - 15 cm) heart-shaped leaves that appear silvery due to the dense small hairs on the underside.1 B. nivea is commonly referred to as China grass, white ramie, green ramie, and rhea, and has been used as far back as 5,000 BC in Egyptian mummy cloths. Furthermore, the leaves of this plant have been used as sources of tea, cloths, and tteok (traditional Korean rice cakes).2,3

B. nivea leaves are also commonly used in folk remedies as a diuretic and anti-pyretic, and has are thought to possess hepatoprotective, anti-oxidant, and anti-inflammatory properties.4 Previous studies have indicated that these leaves contain kiwiionoside, rutin, uracil, 3-hydroxy-4-methoxy-benzoic acid, cholesterol, α-amyrin, nonacosanol, emodin, emodin-8-O-β-glucoside, physcion, polydatin, catechin, potassium nitrate, β-sitosterol, epicatechin, and epicatechin gallate.5-7 Furthermore, the leaves of B. nivea contain a large amount of phenolic compounds, which were able to inhibit angiotensin I-converting enzyme.6 In the roots of a related species, B. tricuspis contains several epicatechin dimers such as (–)-epiafzelechin-(–)-epicatechin-4,8(or 6)-dimer, and (–)-epicatechin-(–)-epicatechin-4,8-(or 6)-dimer.8 Among them, epicatechin, epicatechin gallate, and rutin are polyphenolic compounds known as flavonoids, which are known for their anti-oxidant, anti-inflammatory, anti-tumorigenic, anti-bacterial, anti-viral, and anti-allergenic properties.9-16

This study utilized HPLC to analyze the concentrations of epicatechin, epicatechin gallate, and rutin in the leaves of B. nivea originating from four different locations (Geumsan-myeon, Biin-myeon, Hansan-myeon, and Baeksu-eup), and in commercial products (ramie tteok, ramie songpyeon, ramie bory-tteok, and ramie tea).

 

Experimental

Plant material − The leaves of B. nivea were cultivated and collected by Yeonggwang Agricultural Technology & Extension Center, Korea. The collection areas for B. nivea were Geumsan-myeon, Biin-myeon, Hansan-myeon, and Baeksu-eup, Korea.

Commercial products − Commercial products (tteok, songpyeon, bory-tteok, and ramie tea) containing B. nivea leaves were purchased from Yeonggwang-gun, Korea. Among them, tteok, songpyeon, and bory-tteok were made up of B. nivea leaves and wheat flour. Ramie tea was made up of dried B. nivea leaves.

Instruments and reagents − Methanol (MeOH), n-hexane, dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and n-butanol (n-BuOH) were purchased from SamChun Pure Chemical Co. (Pyeongtaek, Korea). Epicatechin (1), epicatechin gallate (2), and DMSO were purchased from Sigma-Aldrich (St. Louis, MO, USA). Electron ionizationmass spectrometry (EI-MS) was conducted with a Jeol JMS-600W mass spectrometer (Tokyo, Japan). NMR spectra were recorded with a Bruker AVANCE 500 NMR spectrometer (Bremen, Germany) using TMS as the internal standard. Chemical shifts are reported in parts per million (δ), and coupling constants (J) are expressed in Hertz. An Eyela rotary evaporator system (Tokyo, Japan) under reflux in vacuo was used for evaporation. Thinlayer chromatography was conducted with Kiesel-gel 60 F254 plates (silica gel, 0.25 mm layer thickness; Art. 5715, Merck Co., Darmstadt, Germany), and compounds were visualized by spraying with 10% H2SO4 in MeOH, followed by heating to 100 ℃. Sephadex LH-20 (20 - 100 μm) was purchase from Sigma-Aldrich. HPLC chromatograms of flavonoids were recorded with a Waters Breeze system (Massachusetts, USA) equipped with a Waters 1525 binary HPLC pump and a 2489 system UV/VIS detector. Water and MeOH used in this study were of HPLC grade, and all other reagents were of analytical grade.

Isolation and identification of rutin (3) − B. nivea leaves (2 kg) were dried finely powdered and immersed in MeOH for 3 h (4 L × 8) under reflux at 65 ℃-75 ℃. The solvent was evaporated in vacuo to produce the MeOH extract (294.6 g). This extract was then suspended in distilled water and partitioned with n-hexane (106.9 g), CH2Cl2 (4.1 g), EtOAc (2.4 g), and n-BuOH (23.8 g), successively. The EtOAc fraction (2.4 g) was separated on an LH-20 column (φ 2.0 × 50 cm) using MeOH/water (gradient: 1 : 3 → 1 : 0, v/v). Nine fractions were obtained by combining those with similar Rf on TLC behavior (1 → 9). Among them, compound 3 was isolated from fraction 7 by recrystallization with MeOH (Fig. 1).

Fig. 1.Structures of compounds 1 - 3.

Compound 3: FAB-MS m/z: 611 [M + H]+; 1H-NMR (500 MHz, DMSO-d6): δ 1.00 (3 H, d, J = 6.0 Hz, Rha CH3), 3.07-3.69 (12 H, m, J = 2 Hz, sugar H), 4.38 (1 H, s, Rha H-1), 5.34 (1H, d, J = 7.5 Hz, Glc H-1), 6.19 (1 H, d, J = 1.2 Hz, H-6 ), 6.38 (1 H, d, J = 1.2 Hz, H-8), 6.84 (1 H, d, J = 8.5 Hz, H-5'), 7.54 (1 H, d, J = 2.5 Hz, H-2'), 7.55 (1 H, dd, J = 2.5, 8.5 Hz, H-6'), 12.58 (1 H, s, 5-OH); 13C-NMR (125 MHz, DMSO-d6): δ 156.5 (C-2), 133.2 (C-3), 177.3 (C-4), 161.1 (C-5), 98.6 (C-6), 164.1 (C-7), 93.5 (C-8), 156.5 (C-9), 103.9 (C-10), 121.5 (C-1'), 115.2 (C-2'), 133.2 (C-3'), 148.4 (C-4'), 116.2 (C-5'), 121.1 (C-6'), 101.1 (Glc C-1), 74.0 (Glc C-2), 76.4 (Glc C-3), 70.5 (Glc C-4), 75.9 (Glc C-5), 67.0 (Glc-C6), 100.7 (Rha C-1), 70.3 (Rha C-2), 70.0 (Rha C-3), 72.0 (Rha C-4), 68.2 (Rha C-5), 17.7 (Rha C-6).

Sample preparation − To determine the content of 1, 2, and 3 in B. nivea leaves and in the commercial products (mentioned above), 50 g of either leaves or commercial products were dried finely powdered and immersed in 50% MeOH (3 × 100 mL) by reflux and evaporation in vacuo. The residue was dissolved in 1 mL of MeOH and filtered through a 0.45 μm filter. The resulting solution was used for HPLC analysis.

HPLC conditions- HPLC separation of 1, 2, and 3 for qualitative and quantitative analysis was performed using a reverse phase system. A Waters Spherisorb® ODS2 (4.6 × 250 mm, 5 μm) column was used with a mobile phase consisting of water (0.2% acetic acid) and MeOH. The elution program was a gradient solvent system of water and MeOH (80 : 20 to 50 : 50 for 30 min). UV detection was conducted at 280 nm. The injection volume was 10 μL and the flow rate was 0.5 mL/min. All injections were performed in triplicate.

Calibration curve- Stock solutions (1 mg/mL) of 1, 2, and 3 were prepared in MeOH, successively reducing the solution content to 50% to create different concentrations. The amount of 1, 2, and 3 from B. nivea leaves and commercial products were determined from the corresponding calibration curve. The calibration functions of 1, 2, and 3 were calculated using the peak area (Y), concentration (X, μg/10 μL), and mean values (n = 5) ± standard deviation.

 

Results and Discussion

In this study, B. nivea leaves were extracted with MeOH, and partitioned successively with n-hexane, CH2Cl2, EtOAc and n-BuOH. The n-hexane, CH2Cl2, EtOAc, and n-BuOH fractions were applied sequentially to open column chromatography over silica gel to yield compound 3. Compound 3 was obtained as a white powder from the EtOAc fraction. The typical flavonoid signals of 3 were observed in the 1H- and 13C-NMR spectra. The 1H-NMR spectra revealed that it had an ABX system (H-2', -5', and -6'), as demonstrated by the coupling constant signal at δ 7.54 (d, H-2'), 7.55 (dd, H-6'), and 6.84 (d, H-5') in the B-ring structure. Compound 3 is a flavonoid glycoside. The 13C-NMR of 3 can be correlated with the flavonoid moiety of quercetin with rutinose, with the latter comprising glucose and rhamnose units. The FAB-MS spectrum of 3 showed a quasimolecular ion peak at m/z 611, corresponding to a molecular formula of C27H30O16. Accordingly, the structure of 3 was elucidated as rutin (5,7,3',4'-tetrahydroxyflavone-3-rutinose).14

Simultaneous determination of 1, 2, and 3 in the leaves of B. nivea was conducted by HPLC. The HPLC separation of analytes was conducted using a reverse phase system with a mobile phase consisting of water and MeOH (80 : 20 to 50 : 50 for 30 min) and all identified flavonoids were detected at 280 nm (Table 1). Using an optimized analytical method, compounds 1, 2, and 3 in the leaves of B. nivea were determined simultaneously (Fig. 2). The concentrations of 1, 2, and 3 were detected in B. nivea leaves from Geumsan-myeon (0.138, 0.233, and 12.205 mg/g, respectively), Biin-myeon (0.025, 1.227, and 5.510 mg/g, respectively), Hansan-myeon (0.022, 1.230, and 4.969 mg/g, respectively), and Baeksu-eup (0.025, 1.654, and 11.638 mg/g, respectively) (Table 2). The content of 3 was consistently higher than 1 and 2 in B. nivea leaves originating from the four areas investigated. Additionally, the concentrations of 1 and 2 were detected in the commercial products ramie tteok (0.0100 and 0.0423 mg/g, respectively), ramie songpyeon (0.055 and 0.039 mg/g, respectively), ramie bory-tteok (0.655 and 0.842 mg/g, respectively), and ramie tea (1.879 and 1.090 mg/g, respectively) (Table 3).

Table 1.aY = peak area, X = concentration of standard (μg/ml). br2 = correlation coefficient for three data points in the calibration curve (n = 5).

Fig. 2.HPLC chromatograms of 1 (A), 2 (B), and 3 (C) and the MeOH extract of B. nivea from Hansan-myeon (D)

Table 2.Data are represented as the mean ± S.D. (n = 4) in mg/g of the dried samples.

Table 3.Data are represented as the mean ± S.D. (n = 3) in mg/g of the dried samples. Tteok, Songpyeon: half-moon-shaped rice cake, and Bory-tteok: traditional Korean rice cakes Tea: dried leaf of tea

From our analyses, the concentrations of 1 and 3 were highest in the leaves of B. nivea from Geumsan-myeon (0.138 and 12.205mg/g, respectively), and the concentration of 2 was highest in the leaves from Baeksu-eup (1.654 mg/g). The concentration of 1 in B. nivea leaves was similar when taken from either Biin-myeon, Hansan-myeon, or Baeksu-eup. The concentration of 2 in B. nivea leaves originating from Biin-myeon was similar to that seen in leaves from Hansan-myeon, however the leaves from Geumsan-myeon contained a significantly smaller amount of 2 than that found in the other locations. Lastly, the concentration of 3 in the leaves of B. nivea from Geumsan-myeon and Baeksu-eup was similar, and likewise, the concentration of 3 in leaves from Biin-myeon and Hansan-myeon was similar. In the total content of B. nivea from Geumsan-myeon, Biin-myeon, Hansan-myeon, and Baeksu-eup, the content of Baeksu-eup was higher than that of Geumsan-myeon, Biin-myeon, and Hansan-myeon because of the different geological features. Baeksu-eup is similar in climate and precipitation. Also, in the total content of commercial products, ramie tteok, ramie songpyeon, ramie bory-tteok, and ramie tea in B. nivea, the content of ramie tea was higher than that of ramie tteok, ramie songpyeon, and ramie bory-tteok because of production process. Therefore, ingesting ramie tea is likely one of the best ways to obtain large amounts of 1 and 2. This may be important medicinally as increased consumption of green tea is associated with lower risks of obesity and obesity-related disorders, including inflammation, cardiovascular disease, and nonalcoholic fatty liver disease.17

These results demonstrate that B. nivea contains large amounts of 1, 2, and 3, and has the potential to be a new additive for natural medicinal products, health supplements, and beverages.