Preliminary Study on Quality Standard of Pummelo Pericarp from Guangxi

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1. Guangxi University of Chinese Medicine, Nanning 530200, China; 2. Guangxi Health Technical Vocational College, Nanning 530023, China

Abstract [Objectives] This paper aimed to study the quality standard of pummelo pericarp from Guangxi. [Methods] The medicinal material was identified by microscopy and thin-layer chromatography, and the contents of moisture, total ash, acid-insoluble ash and extract in the medicinal material were determined according to the methods of the Chinese Pharmacopoeia (2010 edition). [Results] The content limits for moisture, total ash, acid-insoluble ash and extract were determined (moisture ≤ 12%; total ash ≤ 6.0%; acid-insoluble ash ≤ 0.6%; water-soluble extract ≥ 43.0%; naringin ≥ 10.00 mg/g). The micro-characteristics of pummelo pericarp were identified, and a thin-layer chromatography identification and content determination method was established. [Conclusions] The established method can provide an experimental basis for the formulation of quality standard for pummelo pericarp.

Key words Pummelo pericarp, Microscopic identification, Thin-layer identification, Content determination, Quality standard

1 Introduction

Pummelo[Citrusgrandis(L.) Osbeck]is an evergreen tree of Rutaceae[1], and its fruit is called as You, Hugan, Choucheng, Xueyou, Wendan, Zhuluan, Xiangluan and Leiyou. Pummelo fruit has high medicinal value. Chinse medicine believes that pummelo fruit has effects of invigorating spleen and stomach, moistening lung, replenishing blood, clearing intestines, promoting defecation and promoting digestion, and it can promote wound healing, and has a good effect on sepsis[2]. Modern research[1]shows that pummelo pericarp contains volatile oil, alkaloids, coumarins, triterpenes, flavones and other compounds. In recent years, there have been more studies on the quality of pummelo pericarp at home and abroad[3-6], but no research has been conducted on the quality of pummelo pericarp from Guangxi. In order to improve the formulation of quality standard, this research was conducted.

2 Instruments and reagents

The used apparatus and instruments mainly included Agilent 1100 high-performance liquid chromatograph (Agilent, USA), SB3200T ultrasonic cleaning instrument (Nengxin, China), Simplicity-185 water purification system (Millipore, USA) and BP211D electronic analytical balance (Sartorius, German).

The naringin standard (for content determination, batch No. 110722-200610) was provided by National Institute for Control of Pharmacological and Biological Products. Pummelo fruit was collected from different places in Guangxi, and it was identified as mature fruit ofCitrusgrandis(L.) Osbeck (Rutaceae) by Professor Liang Zining from Department of Medicinal Plants, Guangxi University of Chinese Medicine (Table 1).

Table1Sourcesofpummelopericarpsamples

CodeProduction placeCodeProduction place1Tuqiao Township, Yizhou City7Tiane County, Hechi City2Liucheng County, Liuzhou City8Rong County, Yulin City3Rongshui County, Liuzhou City9Suburb of Guilin4Huanjiang County, Hechi City10Wuwei Town, Jinchengjiang District, Hechi City5Duan County, Hechi City11Longtou Township, Yizhou City6Xiaochangan Town, Luocheng County, Hechi City12Luocheng County, Hechi City

3 Methods and results

3.1MicroscopicidentificationThe powder of pummelo pericarp was light yellow to yellow brown. The parenchyma cells of the mesocarp were irregularly shaped, and their walls were unevenly thickened. The corners of some of the parenchyma cells were thickened. Calcium oxalate cubes were easy to see, polyhedral, rhombic, prismatic, rectangular or irregular in shape, with a diameter of 2-25 μm, and a length of 3-34 μm. Vessels were spiral vessels and reticulate vessels. There was scattered cell debris in the oil chamber. The epidermis was composed of square cells of the same type. The mesocarp cells were round and loosely arranged. There were large oil chambers on the outside, and they were irregularly arranged in a row. The oil chambers were large and oblong, with radial length of 450-1 100 μm and tangential length of 270-580 μm. The mesocarp was scattered with vascular bundles.

3.2Inspection

3.2.1Moisture. The content of moisture in the pummelo pericarp was determined with the toluene method recorded inChinesePharmacopoeia(2015 edition). The moisture contents of the 12 batches of pummelo pericarp samples were between 9.63% and 10.77% (Table 2).

Table2Contentsofmoisture,totalash,acid-insolubleash,extractandnaringininpummelopericarpsamples(n=6,RSD<3.0%)

CodeMoisture%Water-solubleextract∥%Totalash∥%Acid-insolubleash∥%Naringinmg/g0110.4445.033.780.4316.590210.7047.074.310.4720.250310.1547.234.620.4617.820410.1849.074.240.2120.65059.6349.253.760.4222.120610.0148.635.000.4715.020710.0048.534.010.3616.64089.9244.493.870.4115.980910.7249.553.930.4421.861010.7744.174.590.5015.191110.4345.924.010.4527.171210.0252.134.250.3924.83

3.2.2Extract. The content of water-soluble extract was determined with the cold maceration method recorded inChinesePharmacopoeia(2015 edition). The contents of water-soluble extract in the 12 batches of pummelo pericarp samples ranged from 44.17% to 52.13% (Table 2).

3.2.3Total ash. The content of ash in pummelo pericarp was determined according to the method recorded inChinesePharmacopoeia(2015 edition). The contents of total ash in the 12 batches of pummelo pericarp samples were in the range of 3.78%-5.00% (Table 2).

3.2.4Acid-insoluble ash. The content of acid-insoluble ash in pummelo pericarp was determined with the method recorded inChinesePharmacopoeia(2015 edition). The contents of acid-insoluble ash in the 12 batches of pummelo pericarp samples were between 0.21% and 0.50% (Table 2).

3.3TLCidentificationFive portions of the powder of pummelo pericarp, 1.0 g for each, were weighed. Each portion of the powder was added with 10 mL of methanol, heat-refluxed for 20 min, cooled and filtered, and 1 mL of the filtrate was collected as test solution. A certain amount of the naringin standard was dissolved in a certain volume of methanol to prepare into reference solution of 0.4 mg/mL. The absorbent was a thin layer of silica gel G. The developing solvent was a supernatant liquid of ethyl acetate-formic acid-water (10∶2∶3). The color developer was 5% AlCl3solution. The samples were inspected under a 365 nm UV lamp. In comparison to the chromatogram of the standard, same yellow fluorescent spots appeared in the chromatograms of the test samples (Fig.1).

3.4Contentdetermination

3.4.1Chromatographic conditions. Column: Agilent HC-C18column (4.6 mm × 250 mm, 5 μm); mobile phase: acetonitrile-0.1% phosphoric acid (18∶82); flow rate: 1.0 mL/min; column temperature: 25 ℃; detection wavelength: 283 nm; injection volume: 10 μL; number of theoretical plates: no less than 5 000 in terms of naringin. The chromatograms of the standard and test samples are shown in Fig.2.

Note: 1, 2, 3, 5, 6: pummelo pericarp samples No.1-5; 4, naringin standard; 7, blank.

Fig.1Thin-layerchromatogramofpummelopericarp

Note: A, naringin standard; B, pummelo pericarp sample. Peak of naringin.

Fig.2Peakofnaringin

3.4.2Preparation of reference solution. An accurate amount (6.54 mg) of the naringin standard was dissolved with 80% methanol to 10 mL to obtain the stock solution of the standard. An accurate volume (0.5 mL) of the stock solution was diluted with 80% methanol to 5 mL to obtain the reference solution (0.065 4 mg/mL of naringin).

3.4.3Preparation of test solution. An accurate amount (around 0.1 g) of the dried powder of pummelo pericarp was weighed, placed in a conical flask with a stopper, added with 10 mL of 80% methanol, sonicated for 30 min and cooled, and the lost weight was supplemented. The solution was passed through 0.45-μm filter, and the subsequent filtrate was used as test solution.

3.4.4Investigation of linear relationship. Accurate volumes (0.25, 0.5, 1.0, 2.0, 3.0 and 5.0 mL) of the stock solution of naringin standard (0.654 mg/mL) were diluted with 80% methanol to 5 mL, respectively. The diluents were detected chromatographically according to the conditions described above. Taking the injection amount of the standard (μg) as the abscissa and the peak area as the ordinate, standard curve was drawn, and the regression equation for naringin wasy=1 503.9x-28.049 (r=0.999 9). The results show that there was a good linear relationship between injection amount and peak area within the range of 0.164-3.27 μg.

3.4.5Precision test. Under the selected chromatographic conditions, the reference solution was detected chromatographically six times repeatedly, and the peak areas of naringin were analyzed. TheRSDof the peak areas was 0.62%, indicating that the instrument has good precision.

3.4.6Stability test. The same test solution was detected chromatographically 0, 2, 4, 8, 12, 18 and 24 h after its preparation, respectively, and the peak areas of naringin were analyzed to investigate stability. The results show that theRSDvalue of the peak areas of naringin was 1.86%, indicating that naringin in the test solution remained stable within 24 h.

3.4.7Reproducibility test. Six portions of the powder of the same batch of pummelo pericarp, 0.1 g for each, were weighed, and prepared into test solutions, respectively according to the method described in Section3.4.3. The contents of naringin in the test solution were determined under the chromatographic conditions in Section3.4.1. TheRSDvalue of the naringin contents was 1.71% (n=6), indicating that the method has good reproducibility.

3.4.8Recovery test. Six portions of dried coarse powder of medicinal material with known content of pummelo pericarp, 0.05 g for each, were weighed. They were added with 1 mL of the reference solution (1.104 8 mg/mL), prepared into test solution according to the description in Section3.4.3and detected chromatographically under the conditions in Section3.4.1, respectively. The recovery rate of naringin was calculated. The results show that the average recovery rate was 98.66%, and theRSDvalue was 1.80% (n=6), indicating that the accuracy of this method is good.

3.4.9Content determination. Three portions of dried powder of each batch of pummelo pericarp, 0.1 g for each, were weighed, prepared into test solutions according to the description in Section3.4.3and detected chromatographically under the conditions described in Section3.4.1, respectively. The contents of naringin in the pummelo pericarp samples from different places were 15.02-27.17 mg/g.

4 Discussion

The content of water-soluble extract of pummelo pericarp was higher than that of methanol-soluble extract. In clinics, water is generally used for decoction, so water was used as the extraction solvent. There was no significant difference in content of water-soluble extract between cold maceration method and hot maceration method. Moreover, the operation of hot maceration method is hard to control and more complicated. Therefore, cold maceration method was used in this experiment.

Based on the determination results of the 12 batches of medicinal materials, it was initially proposed that the content of moisture in pummelo pericarp from Guangxi should not exceed 12.0%, the content of total ash should not exceed 6.0%, the content of acid-insoluble ash should not exceed 0.6%, the content of water-soluble extract should not be less than 43.0%, and the content of naringin should not be less than 10.00 mg/g.

The above inspection items and thin-layer chromatography identification provide a reference basis for the establishment of its quality standard. However, the research on pummelo pericarp from Guangxi is still in its infancy, and more comprehensive quality control methods need to be further studied.