Simultaneous Determination of Four Compounds in a Nelumbo nucifera Seed Embryo by HPLC-DAD

Nelumbo nucifera has a variety of biological activities. So it was importantly used as various herbal medicines since traditional times. A simple, fast, and sensitive high-performance liquid chromatographic (HPLC) method was developed in this study for efficient quality control of N. nucifera. Four different compounds, including neferine, 1,2,3,4-tetrahydro-1-[(4-hydroxyphenyl) methyl]-2-methyl-7-isoquinolinol, 1-hydroxy-2-methylpropene, and 3-(prop-1-enyl)benzene-1,2,4,5-tetrol, were simultaneously determined. The four compounds were isolated through a Dionex C18 column by gradient elution with 0.1% TFA-water and methanol. The flow rate was 1.0mL/min, and the wavelength was detected at 205, 254, 280, and 330 nm. The chromatograms were acquired at 205 nm. The four compounds showed good linear relationships (r2 > 0 96) over five different concentrations, and an average recovery of the method ranged from 96.27% to 108.78%. Through the analysis validation test and application of the method, the optimized conditions verified that it is efficient to isolate the compounds of N. nucifera seed embryos.


Introduction
Lotus (Nelumbo nucifera) is an aquatic plant, which is widely distributed in East Asia, Australia, and North America [1].It is evaluated as one of the important Chinese herbal medicines.Nearly all of the parts of the lotus, including leaves, leaf stalks, flowers, seeds, and rhizomes, are edible and used as food products and medicines [2].Different parts possess diverse activities such as anti-ischaemia, antioxidant, anticancer, hepatoprotection, antidiarrhoeal, and anti-inflammation.
The lotus seed is also known as "kamal gatta" in Indian markets, and its embryos are mainly used to treat nervous disorders, cardiovascular diseases, inflammation, and cancer [3].N. nucifera is composed of many phenolic compounds.The major bioactive compounds of an N. nucifera seed are phenolic compounds, for example, lotusine, nuciferine, neferine, isoliensinine, and liensinie [4].Phenolic compounds show high anti-inflammatory and antioxidant properties, which are determined by the presence of a hydroxyl group bonded to an aromatic ring to give the molecular formula, C 6 H 5 OH [5,6].Phenols have been isolated from various plants [7,8], and recent studies have shown that the embryos of the lotus seed also have abundant phenolic compounds [9,10].It has also been reported that seed embryos of the lotus have antioxidant activities [4].Oxidative stress and immoderate inflammatory responses can be critical etiological causes of neurodegenerative diseases, for example, Alzheimer's disease, Parkinson's disease, and dementia [11].Based on the relationship of the structure and the activity of antioxidant and anti-inflammation, a previous study that focused on the neuroprotective effect of lotus embryo had been accomplished before this report [12].The study about an effective isolation method is also accomplished.
The purpose of this report is to establish a simple, sensitive, and fast HPLC analytical method for compounds of N. nucifera embryos.There are already some papers about the analysis of N. nucifera seed embryo compounds [9,10], but in this report, the three compounds that have not been analyzed are simultaneously determined, including neferine.
The described method will be useful for the quality control of this plant and the development of health food or medicine.

Materials and Methods
2.1.Plant Material.The N. nucifera seed embryos were supplied by the Wildlife Genetic Resources Center, National Institute of Biological Resources (Incheon, Korea).The materials were validated by Dr. Young Bae Seo, a professor at the College of Oriental Medicine, Daejeon University (Daejeon, Korea).The extracted and evaporated specimens (number CJ129M) were stored at Kangwon National University (Chuncheon, Korea).

HPLC Analysis.
All the solvents were HPLC grade and purchased from J.T. Baker.N. nucifera solutions were analyzed by HPLC-DAD.The high-performance liquid chromatography (HPLC) (Dionex) was composed of an LPG 3X00 pump, an ACC-3000 autosampler, a DAD-3000(RS) diode array UV/VIS detector, and a column oven.Each sample was injected and isolated through a Dionex C 18 column (5 μm, 120 Å, 4.6 mm × 150 mm) at 25 °C.The mobile phase consisted of 0.1% TFA-water and methanol.The injection volume of samples was 20 μL.The output signal of the detector was transcribed by a Dionex Chromeleon™ Chromatography Data System.The UV wavelength was 205, 254, 280, and 330 nm, respectively, and the chromatograms were acquired at 205 nm.2.5.2.Limit of Detection (LOD) and Limit of Quantification (LOQ).The limit of detection (LOD) was determined as the lowest concentration of sample, and the limit of detection quantification (LOQ) was determined as the lowest concentration of compounds by injecting the diluted standard solution when the signal-to-noise ratio reached 3.3~10.

Repeatability and Precision.
The precisions of the method were evaluated by intraday and interday injections.
The intraday experiment was performed by three injections for a day, and the interday was performed by three injections for 3 days.Both tests were examined in three different concentrations which were confirmed in calibration curves.The reproducibility and precision were expressed as relative standard deviations (RSD, %).2.6.Sample Analysis Using Established Method.The efficiency of optimized simultaneous determination method was verified by analyzing the total N. nucifera sample.Each content of four target compounds was detected and evaluated.

Optimization of HPLC-DAD Conditions.
The reason for an optimized experiment using HPLC was to gain higher separation efficiency and peak resolution of target compounds in a shorter run time.To achieve this goal in Nelumbo nucifera, the effective HPLC-DAD conditions including suitable column, mobile phase, and wavelength of UV spectrum were chosen by performing many preliminary tests.The selected column was a Dionex C 18 column, one of reversed-phase columns.The multistep gradient solution system consisted of 0.1% TFA-water (A) and methanol (B) was used as a mobile phase.The most appropriate condition for the separation of seed embryo was 0-10 min, 10% B; 10-20 min, 10-20% B; 20-25 min, 20-30% B; and 25-30 min, 30% B at a flow rate of 1.0 mL/min.This gradient led to the separation of four peaks within 30 min.The wavelength of the DAD detector was tested at 205, 254, 280, and 330 nm and the chromatograms were selected at 205 nm by UV spectrum.The column temperature had no significant influence on the separation, so room temperature was used.The peak of each compound was assured by comparing retention time and UV spectrum of their corresponding marker solution.The chromatograms of the N. nucifera compounds are shown in Figure 2.

Analysis Method Validation.
To approve the validation of this method, the experiments for verifying linearity, detection and quantification limits, precision and accuracy, and recovery were performed.The regression equations were established by five concentration trials of each standard in triplicate.In linear regression, equation Y = ax + b, where x is a concentration of marker components, and Y is the peak area.The slope and intercept of the calibration curve were determined by this equation.The high correlation coefficients (r 2 > 0 96) showed that all calibration curves had good linearities within the test ranges as shown in Table 1.According to this linear regression, the results of the limits of detection (LOD) ranged from 1.31 to 4.4 μg/mL and limits of quantification (LOQ) were in the range of 3.98-13.24μg/mL, respectively.This means that a few compounds can be detected and quantified.
To evaluate the reproducibility and precision of the method, within-day test (intraday analysis, n = 3) and intermediate-day test (interday analysis, n = 3) were performed.The intraday test was examined three times during a single day, and the interday test was examined on three different days (1, 3, and 5 days).The RSD values of the intraday test were 0.23%-1.35%and interday were 0.05%-6.08%,respectively (Table 2).These results showed that the method was very reproducible and precise.

Sample Analysis
Using an Established Method.The method described above was successfully applied to investigate the four target content in N. nucifera seed embryos.As summarized in Table 4, the amounts of the tested compounds varied significantly.The dominant content of the compound was neferine which was 79.39 mg/g in the whole sample.Other compounds were relatively small in quantity.The content of 1,2,3,4-tetrahydro-1-[(4-hydroxyphenyl) methyl]-2-methyl-7-isoquinolinol, 1-hydroxy-2methylpropene, and 3-(prop-1-enyl)benzene-1,2,4,5-tetrol were 9.56 mg, 13.83 mg, and 6.75 mg per 1 g of N. nucifera sample, respectively.A total of four compounds were able to be detected simultaneously by HPLC-DAD in optimized conditions.

Conclusion
In this study, the developed HPLC-DAD method was used for the qualitative analysis of compounds in Nelumbo nucifera seed embryos.Four compounds, including neferine, 1,2,3,4-tetrahydro-1-[(4-hydroxyphenyl) methyl]-2-methyl-7-isoquinolinol, 1-hydroxy-2-methylpropene, and 3-(prop-1-enyl)benzene-1,2,4,5-tetrol, were simultaneously detected within 30 min for the first time.Also, the optimized method was selective and accurate which was verified by the validation test including linearity, limits of detection and quantification, repeatability, precision, and accuracy test.These qualitative and quantitative analytical results can help with the quality control of N. nucifera seed embryos.According to a previous study, neferine showed significant neuroprotective effects, among the four compounds [12,13].Based on the activity of the compounds, this method is useful to develop the healthy food or medicine for neuroprotection.It is also expected to be used in research and development of activities and biological mechanisms.

Figure 1 :
Figure 1: Chemical structures of compounds in N. nucifera embryos.

2. 5 . 4 .
Recovery Test.The accuracy of the method was evaluated by a recovery study.It was conducted by adding a measured amount of compound solutions to the whole N.

Table 2 :
Intra-and interday precision of four compounds in the extract of lotus embryo by HPLC-DAD.
a RSD: relative standard deviation.

Table 1 :
Calibration curves, LOD, and LOQ of four compounds in N. nucifera embryos.