1. Introduction
Semen Nelumbinis, the dry and mature seeds of nelumbo nuciferea gaertn, are produced in the northern and southern provinces of China and have important edible and medicinal value. According to the records in Compendium of Materia Medica, Semen Nelumbinis, with its sweet taste, mild and astringent nature, can nourish heart and kidney and enrich essence and blood [1], and is currently included in the China Pharmacopoeia. Alkaloid is a kind of special nitrogen-containing organic matter that exists in plants and is synthesized along with the changes of external environment [2]. Most studies have shown that plant alkaloids have special physiological activity [3]. The alkaloid active components contained in the Semen Nelumbinis and plumula nelumbinis mainly include Lotusine, Liensinine, Isoliensinine, Neferine, Nuciferine, Pronuciferine, Berberine Hydrochloride and the like [4], which have many health promotion functions and medicinal values for human body, such as anti-cancer, anti-virus, anti-bacteria, anti-arrhythmia, blood pressure reduction, blood glucose reduction, anti-oxidation, and free radical removal [5,6]. As many alkaloid components in Semen Nelumbinis are very important chemical components, which are also important indicators to evaluate the quality of and have great influence on the sensory quality and medicinal value of Semen Nelumbinis, it is of great significance to extract and quantitatively determine alkaloids in Semen Nelumbinis.
At present, the main methods for extracting alkaloids are ultrasonic extraction [7], liquid–solid extraction technique [8] and thermal reflux method [9]. Although liquid–solid extraction and thermal reflux extraction can extract most of the active components [9], their complicated and time-consuming operations may destroy the heat-sensitive components [10]. By contrast, the ultrasonic extraction of alkaloids is both economical and simple. The samples are crushed by ultrasonic in the solvent, resulting in cell wall rupture and rapid dissolution of alkaloids, improving the extraction efficiency [11]. Therefore, it has significant advantages in terms of extraction time and sample stability [12]. Chen Yuangu et al. [7], respectively, adopted six extraction methods including water decoction, ethanol reflux, acid water decoction, alkaline ethanol reflux, ethanol ultrasound and alkaline chloroform ultrasound to determine the total alkaloids and content in Radix Sophorae Flavescentis. In view of their extraction yields, production cost and safety, ethanol ultrasound extraction is considered the best extraction method.
The determination methods of alkaloids mainly include ultraviolet spectrophotometry [13], TLC scanning [14], high-performance liquid chromatography (HPLC) [15] and high-performance liquid chromatography-tandem mass spectrometry [16], among which HPLC is increasingly used for the analysis and detection of alkaloids due to its high precision, accurate results and good repeatability. In recent years, there have been reports on the determination of Liensinine, Isoliensinine and Lotusine [15] in Semen Nelumbinis by HPLC, and literature on the determination of Neferine by TLC scanning [14], but no documents on the simultaneous determination of seven representative alkaloids such as Liensinine and Isoliensinine in Semen Nelumbinis by HPLC. Therefore, in this study, a HPLC method was established to simultaneously determine the contents of seven kinds of alkaloids in Semen Nelumbinis and their products, optimize the extraction method, and determine and analyze the alkaloid contents of Semen Nelumbinis and plumula nelumbinis from different manufacturers, aiming to provide theoretical support for scientific evaluation of the quality of Semen Nelumbinis and further development and utilization of the value of alkaloids in Semen Nelumbinis.
2. Materials and Methods
2.1. Instruments and Reagents
The reference substances of Lotusine, Liensinine, Isoliensinine, Neferine, Nuciferine and Pronuciferine with purity of 98% produced by Zhuhai Anzhen Biotechnology Co., Ltd. (Zhuhai, China); reference substance of Berberine Hydrochloride with purity of 89% produced by Manhage Detection Technology Co., Ltd. (Changzhou, China); Formic acid with chromatographic purity of 98%, hydrochloric acid (analytical purity), ethanol (chromatographic purity), methanol (chromatographic purity), and acetonitrile (chromatographic purity) produced by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); Water prepared with Millipore water purifier (Burlington, MA, USA); Herbal Secret Semen Nelumbinis, Huayangmingtang Semen Nelumbinis, bulk Semen Nelumbinis, Wahaha eight-treasure porridge, and Huilong plumula nelumbinis: commercially available.
Agilent1260 HPLC: Agilent Technologies Co., Ltd. (Santa Clara, CA, USA); EOFO digital display multitubular vortex mixer: Talboys (Thorofare, NJ, USA) AS3120 ultrasonic cleaner: Tianjin Autoscience Instruments Co., Ltd. (Tianjin, China); Model ST16R high-speed refrigerated centrifuge: Thermo Fisher Scientific (China) Co., Ltd. (Shanghai, China); TurboVap LV termovap sample concentrator: Biotage, Sweden (Uppsala, Sweden) EOAA-HM-01 oscillator: Shanghai Anpel Experimental Technology Co., Ltd. (Shanghai, China); DK-98-II electric-heated thermostatic water bath: Tianjin Taisite Instruments Co., Ltd. (Tianjin, China); XS205DU electronic balance-1/100000: Mettler Toledo Group (Zurich, Switzerland) Direct-Q8R ultra-pure water machine: Millipore Company (Burlington, MA, USA) 0.85 mm sieve: Changsha Sike Instrument Sieve Factory (Changsha, China) PXC solid-phase extraction column (60 mg/3 mL): Beijing Dikema Technology Co., Ltd. (Beijing, China).
2.2. Experimental Method
2.2.1. Preparation of Standard Solution
Proper amounts of Lotusine, Liensinine, Isoliensinine, Neferine, Nuciferine, Pronuciferine and Berberine Hydrochloride reference substances were weighed and dissolved in methanol to prepare mixed standard solutions with concentrations of 1.0 mg/mL, respectively. The appropriate amount of stock solution was taken and diluted with 0.1% formic acid water + methanol solution (80 + 20) into a series of mixed working solutions with the concentration of 0.5–25 μg/mL (Lotusine), 1–50 μg/mL (Liensinine, Isoliensinine and Neferine), and 0.3–15 μg/mL (Nuciferine, Pronuciferine and Berberine Hydrochloride).
2.2.2. Sample Handling
An amount of 2 g (solid samples were pulverized to pass through a 0.85 mm sieve before sampling and homogenized before sampling of semisolid samples) of samples was weighed and put into a 50 mL centrifuge tube, and 30 mL of 1% formic acid ethanol was added into the tube. The samples were vortexed and mixed for 1 min, extracted by ultrasound for 1 h at 8000 r/min, and centrifuged for 3 min. Then, 6 mL of supernatant was collected for subsequent use and purification. Meanwhile, the supernatant was collected, filtered through a 0.2 μm microporous membrane, and analyzed by HPLC. Purification: The PXC solid-phase extraction column was sequentially activated with 3 mL of methanol, 3 mL of water, and 3 mL of 30 mmol/L hydrochloric acid. After 6 mL of the standby supernatant was taken, it passed through the column and eluted successively with 3 mL of water and 3 mL of methanol, and then with 5 mL of 5% ammonia methanol. The eluents were collected, concentrated to almost dry, and 0.1% formic acid water + methanol solution (80 + 20) was added to dilute to 1 mL, and then filtered through a 0.2 μm microporous membrane to be determined by HPLC.
2.2.3. Chromatographic Method
Welch AQ-C18(250 mm × 4.6 mm, 5 μm); Mobile phase: A (0.1% formic acid) -B (acetonitrile); 0–13 min: 0%–35% B; 13–14 min: 35%–20% B; 14–20 min: 20% B; 20–20.1 min: 20%–0% B; 20.1–25 min: 0% B; flow rate: 1.0 mL/min; temperature: 30°C; sample size: 10 μL, detection wavelength: 282 nm.
3. Results and Discussion
3.1. Optimization of Chromatographic Conditions
3.1.1. Wavelength Selection
To select the optimal detection wavelength, the full wavelength scanning of seven target peaks was performed using a DAD detector at 210–800 nm, with wavelength as the abscissa and absorbance as the ordinate, as shown in Figure 1. All seven kinds of alkaloids had high absorption peaks at 210 nm. Additionally, Lotusine had a large absorption peak at 280–287 nm, Liensinine at 279–286 nm, Isoliensinine at 278–288 nm, Neferine at 275–286 nm, Nuciferine at 267–282 nm, Pronuciferine at 266–282 nm, and Berberine Hydrochloride at 265–282 nm. The appropriate wavelength [17] was selected in the wavelength band (±20 nm) before and after the maximum absorption wavelength. When the wavelength was 230 nm, the wavelength was too low to be easily interfered by impurities. When the wavelength was 282 nm, all seven target peaks had strong absorption and the sample matrix effect was weak, so the detection wavelength of 282 nm was chosen.
3.1.2. Selection of Chromatographic Column
Chromatographic columns are essential for high performance liquid chromatographic separation. Different chromatographic columns have a great impact on the appearance time, response value, peak height and tailing factor of the target substance [18], especially when the mobile phase system is a pure water phase or has high requirements on pH value [19]. Improper selection of chromatographic columns will not only cause poor separation effect, but also damage the chromatographic columns. Considering that the mobile phase in this experiment might have pure water phase during gradient elution, the AQ column that can tolerate pure water was selected for analysis. As shown in Figure 2, the analysis of the mixed standard solution by comparing Aglient AQ-C18 column with Welch AQ-C18 column revealed that Welch AQ-C18 column was superior to Aglient AQ-C18 in peak height, resolution, peak width, symmetry factor and tailing factor, and Welch AQ-C18 column required a shorter analysis time. Therefore, Welch AQ-C18 column was selected for this study.
3.1.3. Selection of Mobile Phase
In this study, seven kinds of alkaloids were analyzed, and gradient elution was selected due to the number of target compounds and large difference in polarity [20]. Since the type and ratio of organic reagent solvents determine the elution ability of the mobile phase and greatly affect the resolution and retention time [21], methanol and acetonitrile were used as the mobile phase to analyze the target substances, respectively. As shown in Figure 3, the resolution of the seven kinds of alkaloids was poor when methanol was used as the mobile phase, and the resolution of the target substances was greater than 1.5 when acetonitrile was used as the mobile phase, which met the requirements. Considering that the target was an alkaline substance and an acid was added to the mobile phase as a modifier [22], 0.01%, 0.05%, 0.1% and 0.2% formic acids were selected as the mobile phase to analyze the target at pH values of 5.40, 2.94, 2.75 and 2.65, respectively. The results showed that the retention time was longer with the lower pH value, indicating that the pH of the mobile phase had an effect on the retention time [23]. When the mobile phase was 0.01% formic acid, the degree of separation was not enough. Considering the retention time and the degree of separation, 0.1% formic acid was finally selected as the mobile phase.
3.2. Optimization of Extraction Method
The single-factor experiment was conducted to investigate the effects of solid-phase extraction column, extraction solvent, liquid-to-material ratio and extraction method on the extraction yields of seven kinds of alkaloids from Semen Nelumbinis. The results were as follows.
3.2.1. Influence of Solid Phase Extraction Column (SPE)
To reduce the interference from the sample matrix, a PXC solid-phase extraction column was selected to purify the samples [24], and the chromatograms purified with and without the solid phase extraction column were compared. As shown in Figure 4, the four substances of Lotusine, Liensinine, Isoliensinine and Neferine had a short retention time, and the samples showed good separation efficiency after purified by a solid phase extraction column. For the three substances of Nuciferine, Pronuciferine and Berberine Hydrochloride, the matrix had little effect after the appearance time, and there was a certain loss after purification by the solid phase extraction column. Therefore, the first four substances were selectively extracted, purified by a solid phase extraction column, and then analyzed and determined, and the second three substances were directly analyzed and determined after selective extraction.
3.2.2. Influence of Liquid–Solid Ratio
Ultrasonic extraction was performed with 1% methanol hydrochloride as the extraction solvent for 1 h. The contents of seven kinds of alkaloids in Semen Nelumbinis were extracted at a liquid-solid ratio of 10:1, 15:1 and 20:1 (mL/g). The results are shown in Table 1.
Table 1 shows that the seven kinds of alkaloids had high extraction yields when the liquid–solid ratio was 15:1 and 20:1 (mL/g). The soaking of Semen Nelumbinis with the extraction reagent was not enough, which resulted in a slightly low extraction yields when the liquid–solid ratio was small. The extraction yields of the target substance increased slightly at a liquid–solid ratio of 20:1. Therefore, based on the economy of the extraction process, the appropriate liquid–solid ratio was 15: 1 (mL/g).
3.2.3. Influence of Extraction Method
The ultrasonic extraction, cold soak (6 h) ultrasonic extraction and thermal reflux extraction were performed, respectively, to extract seven kinds of alkaloids from Semen Nelumbinis with the extraction solvent of 1% methanol hydrochloride for 1 h. The ultrasonic extraction and cold soak ultrasonic extraction adopted a liquid–solid ratio of 15:1. The liquid–solid ratio of thermal reflux extraction was 30:1, considering that the extraction solvent under thermal reflux extraction should be more than one-quarter of the extraction bottle. The extraction yield results are shown in Table 2.
Table 2 shows that all the three extraction methods had good extraction yields. In view of the simplicity of the experiment and reducing the harm of organic reagents on human body during reflux [25], ultrasonic extraction was selected to achieve rapid and effective separation for the convenience and speediness of the experiment.
3.2.4. Influence of Extraction Solvent
The ultrasonic extraction was adopted to extract seven kinds of alkaloids from Semen Nelumbinis at a liquid–solid ratio of 15:1 for 1 h with the solvents of 1% methanol hydrochloride (a), 1% ethanol hydrochloride (b), 1% methanol formate (c), 1% ethanol formate (d), pure methanol (e) and pure ethanol (f). The extraction yield results are shown in Table 3.
Table 3 shows that 1% methanol hydrochloride and 1% formic acid ethanol had good separation effects and extraction yields for the seven kinds of alkaloids. For lower toxicity of ethanol [26] and less damage to human body and environment, 1% formic acid ethanol was selected as the extraction solvent.
3.3. Method Validation
3.3.1. Linearity Range and Detection Limit
An appropriate amount of stock solution was pipetted and diluted with 0.1% formic acid water+ methanol solution (80 + 20) into a series of mixed working solutions with the concentration of 0.5–25 μg/mL (Lotusine), 1–50 μg/mL (Liensinine, Isoliensinine and Neferine), and 0.3–15 μg/mL (Nuciferine, Pronuciferine and Berberine Hydrochloride). The peak area (y) was used to perform linear regression analysis on the mass concentration (x, μg/mL) of the standard. The signal-to-noise ratio S/N > 3 was taken as the limit of detection, and S/N > 10 as the limit of quantitation. The linear range, linear equation, correlation coefficient, limit of detection and limit of quantitation of the seven kinds of alkaloids are shown in Table 4.
3.3.2. Recovery and Precision Experiment
The recovery was determined by the spike recovery method. Amounts of 2g (pulverized through a 0.85 mm sieve before sampling) of each sample of Herbal Secret Semen Nelumbinis were weighed (contents of Lotusine, Liensinine, Isoliensinine, Neferine, Nuciferine, Pronuciferine, and Berberine Hydrochloride were 16.97 mg/kg, 5.39 mg/kg, 3.17 mg/kg, 13.21 mg/kg, 2.27 mg/kg, 0.76 mg/kg, and 0 mg/kg, respectively) and put in a 50 mL centrifuge tube; the mixed standards of seven kinds of alkaloids at low, medium and high levels were added for the experiment and determined seven times in parallel. The recovery of spiked alkaloids was 83.33%-116.04%, which was good. The relative standard deviation was 1.06%-5.25%. The accuracy of the method was high. The recovery and precision results are shown in Table 5.
3.4. Sample Analysis
Three parallel samples of each of the five commercially available samples were weighed, extracted and purified according to the sample treatment method in 1.2.2, and the contents of the seven kinds of alkaloids were determined by HPLC. The measurement results are shown in Table 6.
The contents of Lotusine, Liensinine, Isoliensinine and Neferine in Semen Nelumbinis from three different manufacturers were determined to be within 8–56 mg/kg, and the contents of Nuciferine, Pronuciferine and Berberine Hydrochloride were all lower than the limit of quantitation. The contents of the Lotusine, Liensinine, Isoliensinine and Neferine in the plumula nelumbinis were 330–2400 mg/kg, those of Nuciferine and Pronuciferine were 8–52 mg/kg, and the content of the Berberine Hydrochloride was lower than the limit of quantitation. The contents of Lotusine and Neferine were the highest among the five kinds of Semen Nelumbinis and their products, while those of Nuciferine, Pronuciferine and Berberine Hydrochloride were relatively low. The contents of seven kinds of alkaloids in plumula nelumbinis were all much higher than that in pulp, and those of Lotusine and Neferine could be as high as 0.2%, indicating that the alkaloid content in plumula nelumbinis was mainly concentrated in plumula nelumbinis [27].
4. Conclusions
In this study, the ultrasonic method was used to extract seven kinds of alkaloids from Semen Nelumbinis and its products. With the recovery of spiked samples as the index, the extraction process was optimized by single factor experiment and the chromatographic detection conditions were optimized. Experimental results showed that after ultrasonic extraction with the extraction solvent of 1% ethanol formate at a liquid-solid ratio of 15:1 (mg/L), it was purified by the solid phase extraction column, and then analyzed with the Welch AQ-C18 column, the mobile phase of acetonitrile +0.1% formic acid and a detection wavelength of 282 nm. The method had the advantages of simple operation, economy and environmental protection in the pretreatment process, as well as a wide detection linear range, correlation coefficients above 0.999, high recovery and good repeatability, and provided a stable and effective detection method for the quantitative analysis of seven kinds of alkaloids in Semen Nelumbinis and its products. The determination revealed that the contents of Lotusine and Neferine were the highest in Semen Nelumbinis and its products, while those of Nuciferine, Pronuciferine and Berberine Hydrochloride were lower, and the alkaloid content in plumula nelumbinis was much higher than that in pulp. Therefore, the method can be used to determine the content of seven kinds of alkaloids, which will provide a theoretical basis for the comprehensive evaluation of the quality of Semen Nelumbinis and its products.
Conceptualization, F.Z.; methodology, N.D., F.Y., J.H. and L.W.; software, N.D., L.O. and H.Z. (Hui Zeng); validation, L.Y., J.H., Z.W. and J.G.; data curation, N.D. and L.O.; writing-original draft preparation, F.Z. and N.D.; writing-review and editing, H.Z. (Hui Zhou), N.D. and L.O.; visualization, F.Z., D.L. and H.Z. (Hui Zhou); supervision, F.Z. and H.Z. (Hui Zhou); project administration, F.Z.; funding acquisition, F.Z. and H.Z. (Hui Zhou). All authors have read and agreed to the published version of the manuscript.
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The authors declare no conflict of interest.
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Effect of liquid–solid ratio on the extraction yields of seven kinds of alkaloids.
| Liquid-Solid Ratio | Lotusine/(%) | Liensinine/(%) | Isoliensinine/(%) | Neferine/(%)) | Nuciferine/(%) | Pronuciferine/(%) | Berberine Hydrochloride/(%)) |
|---|---|---|---|---|---|---|---|
| 10:1 | 12.72 | 68.95 | 75.97 | 72.48 | 78.43 | 77.50 | 87.91 |
| 15:1 | 66.98 | 87.04 | 79.96 | 93.13 | 85.61 | 85.16 | 92.16 |
| 20:1 | 78.49 | 88.42 | 82.38 | 87.59 | 88.72 | 89.84 | 94.04 |
Effects of different extraction methods on the extraction yields of seven kinds of alkaloids.
| Extraction Methods | Lotusine/(%) | Liensinine/(%) | Isoliensinine/(%) | Neferine/(%) | Nuciferine/(%) | Pronuciferine/ |
Berberine |
|---|---|---|---|---|---|---|---|
| Ultrasonic extraction | 66.98 | 87.04 | 79.96 | 93.13 | 85.61 | 85.16 | 92.16 |
| Cold soaking ultrasonic extraction | 70.61 | 84.95 | 83.16 | 90.16 | 98.83 | 89.90 | 90.31 |
| Thermal reflux | 73.13 | 96.03 | 84.33 | 83.25 | 85.25 | 98.65 | 116.75 |
Effects of different extraction solvents on extraction yields of seven kinds of alkaloids.
| Extraction |
Lotusine/(%) | Liensinine/(%) | Isoliensinine/(%) | Neferine/(%) | Nuciferine/(%) | Pronuciferine/(%) | Berberine Hydrochloride/(%) |
|---|---|---|---|---|---|---|---|
| a | 66.98 | 87.04 | 79.96 | 93.13 | 85.61 | 85.16 | 92.16 |
| b | 64.26 | 61.49 | 71.25 | 66.93 | 87.10 | 82.19 | 86.34 |
| c | /* | 73.38 | 81.80 | 88.77 | 93.96 | 88.72 | 95.52 |
| d | 80.29 | 66.53 | 71.86 | 91.72 | 93.82 | 89.25 | 91.06 |
| e | 81.07 | 62.00 | 72.32 | 69.58 | 93.93 | 89.56 | 86.94 |
| f | 54.25 | 56.66 | 66.91 | 62.89 | 87.30 | 87.38 | 80.41 |
*: Lotusine was not separated from the matrix under this extraction condition.
Linear range, linear equation, correlation coefficient, limit of detection and limit of quantitation of seven kinds of alkaloids.
| Name of Alkaloids | Linear Range |
Linear Equation | Correlation Coefficient | Limit of |
Limit of |
|---|---|---|---|---|---|
| Lotusine | 0.5–25 | Y = 6.87000x − 0.24991 | 0.999896 | 0.5 | 1.25 |
| Liensinine | 0.5–25 | Y = 8.50444x − 1.11677 | 0.999819 | 0.5 | 1.25 |
| Isoliensinine | 1–50 | Y = 9.35156x − 1.21131 | 0.999828 | 1.25 | 2.5 |
| Neferine | 1–50 | Y = 9.63994x − 1.47243 | 0.999775 | 1.25 | 2.5 |
| Nuciferine | 0.3–15 | Y = 18.79125x − 0.45259 | 0.999881 | 1.5 | 4.5 |
| Pronuciferine | 0.3–15 | Y = 30.26949x − 0.73528 | 0.999889 | 1.5 | 4.5 |
| Berberine Hydrochloride | 0.3–15 | Y = 32.72460x − 1.08856 | 0.999856 | 1.5 | 4.5 |
Recovery and precision of 7 kinds of alkaloids (n = 7).
| Alkaloids | Addition Level |
Average Recovery/(%) | Relative Standard Deviation/(%) |
|---|---|---|---|
| Lotusine | 1.25 | 87.43 | 3.27 |
| 2.5 | 99.29 | 2.46 | |
| 12.5 | 116.04 | 2.89 | |
| Liensinine | 2.5 | 77.44 | 3.20 |
| 5.0 | 92.59 | 3.38 | |
| 25 | 95.78 | 1.15 | |
| Isoliensinine | 2.5 | 85.18 | 2.18 |
| 5.0 | 92.90 | 5.25 | |
| 25 | 88.52 | 1.15 | |
| Neferine | 2.5 | 83.33 | 3.59 |
| 5.0 | 112.83 | 4.11 | |
| 25 | 107.46 | 1.54 | |
| Nuciferine | 4.5 | 95.36 | 4.00 |
| 9.0 | 105.48 | 1.69 | |
| 45 | 106.55 | 1.29 | |
| Pronuciferine | 4.5 | 87.16 | 2.46 |
| 9.0 | 94.18 | 1.86 | |
| 45 | 98.69 | 1.06 | |
| Berberine Hydrochloride | 4.5 | 103.58 | 2.66 |
| 9.0 | 104.72 | 1.23 | |
| 45 | 100.22 | 1.59 |
Determination of 7 kinds of alkaloids in different samples.
| Samples | Lotusine/ |
Liensinine/ |
Isoliensinine/ |
Neferine/ |
Nuciferine/ |
Pronuciferine/ |
Berberine |
|---|---|---|---|---|---|---|---|
| Herbal Secret Semen Nelumbinis-1 | 16.98 | 5.37 | 3.16 | 13.25 | 2.27 | ND* | ND* |
| Herbal Secret Semen Nelumbinis-2 | 16.97 | 5.41 | 3.18 | 13.24 | 2.25 | ND* | ND* |
| Herbal Secret Semen Nelumbinis-3 | 16.96 | 5.38 | 3.16 | 13.15 | 2.28 | ND* | ND* |
| Huayangmingtang Semen Nelumbinis-1 | 56.24 | 9.47 | 13.11 | 55.05 | ND* | ND* | ND* |
| Huayangmingtang Semen Nelumbinis-2 | 56.44 | 8.83 | 12.67 | 57.04 | ND* | ND* | ND* |
| Huayangmingtang Semen Nelumbinis-3 | 56.15 | 8.76 | 12.29 | 56.79 | ND* | ND* | ND* |
| Bulk Semen Nelumbinis-1 | 19.37 | 1.35 | 6.02 | 9.61 | 2.31 | ND* | ND* |
| Bulk Semen Nelumbinis-2 | 23.82 | 1.65 | 7.93 | 14.00 | 2.36 | ND* | ND* |
| Bulk Semen Nelumbinis-3 | 19.25 | 1.43 | 5.74 | 10.60 | 2.92 | ND* | ND* |
| Wahaha eight-treasure porridge -1 | 9.64 | 0.50 | ND* | ND* | 2.67 | ND* | ND* |
| Wahaha eight-treasure porridge -2 | 9.44 | 0.53 | ND* | ND* | 3.07 | ND* | ND* |
| Wahaha eight-treasure porridge -3 | 9.94 | 0.52 | ND* | ND* | 2.97 | ND* | ND* |
| Huilong plumula nelumbinis-1 | 1843.44 | 288.89 | 674.70 | 1923.79 | 52.58 | 8.78 | ND* |
| Huilong plumula nelumbinis-2 | 2173.45 | 336.14 | 756.89 | 2042.93 | 52.72 | 8.53 | ND* |
| Huilong plumula nelumbinis-3 | 2412.04 | 394.31 | 923.64 | 2421.55 | 52.79 | 9.09 | ND* |
ND*: Not detected (below detectable limit).
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