Analysis of the Volatile Components of Polygonum chinense L. Extracted with Different Solvents by GC-MS

Anda WEI Hua ZHU Fengfeng XIE Miao ZHANG Piaoling HUANG Wenqi YANG

Abstract [Objectives] This study was conducted to investigate the volatile components of Polygonum chinense L.

[Methods]The volatile components of the methanol extract， ethyl acetate extract， chloroform extract and petroleum ether extract of P. chinense were analyzed and determined by gas chromatography.

[Results] The volatile components of the methanol extract from P. chinense were the most， and phenolic acids and ketones accounted for a relatively high proportion， among which pyrogallic acid had the highest content. The ethyl acetate extract contained the second most volatile components， mostly acids and esters， of which β-sitosterol had the highest content; the chloroform and petroleum ether extracts had relatively few types of volatile components， most of which were alkanes， and the content of γ-sitosterol shared by the two was the highest. The common substances of the four extracts were palmitic acid and phytol.

[Conclusions]This study provides a theoretical basis for the quality evaluation of P. chinense and a scientific basis for its resource development.

Key words Polygonum chinense L.; Volatile components; Different solvents; Gas chromatography-mass spectrometry （GC-MS）

Received： January 23， 2021  Accepted： March 26， 2021

Supported by Development of Huotanmu Capsules， a New Drug for Hepatitis B Treatment （20183046-1）; Collaborative Innovation Center for Zhuang and Yao Medicines （GJKY [2013] 20）; Guangxi Key Laboratory of Zhuang and Yao Medicines （GKJZ [2014] 32）; Guangxi Key Discipline： Zhuang Medicine （GJKY [2013] 16）; First-class Discipline in Guangxi of Traditional Chinese Pharmacology （GJKY [2018] 12）.

Anda WEI （1995-）， male， P. R. China， master， devoted to research about identification and development of traditional Chinese medicine and ethnic medicine.

*Corresponding author. E-mail： zhuhuagx@163.com.

The medicinal materials of Polygonum chinense L refer to dried whole plant of P. chinense of Polygonum in Polygonaceae. It was originally recorded in "Illustrated Classics of Materia Medica" to have the effects of clearing away heat and removing dampness， removing pathogenic heat from the blood and toxic material from the body， benefiting liver and improving eyesight， and promoting blood circulation. P. chinense medicinal materials are commonly used folk medicines， especially commonly used in the Lingnan area. The curative effect has been well received， and it has development value in health care and clinical treatment[1]. Modern research shows that the medicinal materials of P. chinense mainly contain flavonoids， phenolic acids and other substances. In view of relatively few research reports on volatile chemical components from P. chinense， in this study， the volatile components of P. chinense extracted with different solvents were detected， analyzed， and compared by gas chromatography-mass spectrometry （GC-MS）. The study on the volatile components of P. chinense provides a theoretical basis for the quality evaluation of P. chinense and provides a scientific basis for its resource development.

Materials and Methods

Experimental samples

The tested medicinal material of P. chinense： It was collected from Baise City， Guangxi Zhuang Autonomous Region， naturally dried in the shade， and identified as the whole plant of P. chinense L. by Professor Teng from College of Pharmacy of Guangxi University of Chinese Medicine.

Experimental instruments and reagents

Instruments： Agilent 7697A autosampler; gas chromatograph-mass spectrometer （7890B GC-5977B MSD， equipped with 30 m×250 μm×0.25 μm HP-5MS quartz capillary column）; 5 ml sample bottles， sealing bottle caps and capping device （Agilent company in the United States）.

Reagents： methanol， ethyl acetate， petroleum ether and chloroform （Sinopharm Chemical Reagent Co.， Ltd.）.

Methods

Preparation of sample solution

The dried medicinal material was cut， pulverized and sieved through a 20-mesh sieve， for later use. After pre-experiment for the extraction of the product， it was decided to choose ultrasound for extraction. Four parts of the medicinal material （1 g each） were weighed and ultrasonically treated， respectively， with 15 ml of methanol， ethyl acetate， petroleum ether and chloroform for 30 min. After filtration， the extracts obtained were filtered with 0.22 μm microporous membranes， and 1 ml of each P. chinense extract was taken as a GC-MS sample[2].

Gas chromatography-mass spectrometry （GC-MS）

The chromatographic column was an HP-5MS quartz capillary column （30 m×250 μm×0.25 μm）. The heating program was started with the column temperature of 50 ℃， which was kept for 1 min， and increased at 6 ℃/min to 220 ℃， which was kept for 5 min， and increased at 6 ℃/min to 310 ℃， which was kept for 1 min. The carrier gas was high He which flowed at a rate of 1.0 ml/min; the injection port temperature was 250 ℃; the injection volume was 1 μl; and the split ratio was 5∶1.

Mass spectrometry conditions： Ion source EI source， with electron energy 70 eV; ion source temperature 230 ℃; quadrupole temperature 150 ℃; solvent delay 3 min; mass range 50-550 amu; scan mode： full scan; NIST17.L library.

Results and Analysis

The prepared sample solutions were measured by GC-MS according to the above method， and the total ion current diagram of each part （Fig. 1-Fig. 4） was obtained. After the ChemStation data processing， the area normalization method was used to calculate the relative percentage of each component from each total ion current diagram， and each component was determined by searching the mass spectrum of each peak in the mass spectrum database， combining with the relative retention time， with the matching degree SI greater than 80% as the screening basis.

Analysis of volatile components in methanol extract of P. chinense extract

The GC-MS gas chromatography peak substance information of the volatile components in the methanol extract of P. chinense is shown in Table 1.

There are 23 substances in Table  among which pyrogallic acid and 4H-pyran-4-one， 2，3-dihydro-3，5-dihydroxy-6-methyl showed higher contents of 21.52% and 4.22% respectively. Among the volatile components of the methanol extract of P. chinense， there were many types of organic compounds such as hydrocarbons， alcohols， ketones， aldehydes， amines， phenols， and esters[3-4]. The volatile components of the methanol extract of P. chinense were more complex and diverse， and it is recommended to use methanol as the extraction solvent for the research and development of the volatile components of P. chinense. In the substances with high contents in the methanol extract， pyrogallic acid has the pharmacological effect of protecting red cell membrane from peroxidation damage， etc.， and is widely used in antiperoxidation experiments[5]; and 4H-pyran-4-one， 2，3-dihydro-3，5-dihydroxy-6-methyl is produced during the firing of certain foods， and the mixture obtained after heating has the aroma of burnt sweet and melted butter.

Analysis of volatile components in ethyl acetate extract of P. chinense

The GC-MS gas chromatographic peak substance information of the volatile components of the ethyl acetate extract of P. chinense is shown in Table 2.

There are a total of 21 substances in Table  among which the substances with higher contents were beta-sitosterol and octadecane， the contents of which were 24.58% and 8.30% respectively. Among the volatile components of the ethyl acetate extract of P. chinense， there were many types of organic compounds such as hydrocarbons， alcohols， aldehydes， phenols， and esters.  In the substances with high contents in the ethyl acetate extract， beta-sitosterol has the effect of protecting the liver， and studies have shown that it exerts an anti-hepatic fibrosis effect by inhibiting the TNF-α-NF-κB and TβR1-Smad2/3 signaling pathways[6]; and octacodecane exists in the form of a mixture of paraffin components， etc.， and is mainly used as a fuel.

Analysis of volatile components of chloroform extract from P. chinense

The GC-MS gas chromatographic peak substance information of the volatile components of the chloroform extract of P. chinense is shown in Table 3.

There are 12 kinds of substances in Table 3， among which substances with higher contents were γ-sitosterol and toluene， the contents of which were 12.48% and 6.96%， respectively. Among the volatile components of the chloroform extract of P. chinense， there were many types of organic compounds such as hydrocarbons， alcohols， and esters. In substances with higher contents in the chloroform extract， γ-sitosterol can improve the pathological symptoms of diabetes， and studies have shown that γ-sitosterol can significantly improve the pathological symptoms of type 1 diabetes in experimental rats[7]; and in industrial production， toluene is widely used as a solvent and high-octane gasoline additive， and it is an important raw material for organic chemical industry.

Analysis of volatile components of petroleum ether extract from P. chinense

The GC-MS gas chromatographic peak substance information of the volatile components of the petroleum ether extract of P. chinense is shown in Table 4.

There are 18 kinds of substances in Table 4， among which the substances with higher contents were γ-sitosterol and n-eicosane， the contents of which were 28.80% and 11.25%， respectively. Among the volatile components of the petroleum ether extract of P. chinense， there were many types of organic compounds such as hydrocarbons， alcohols， acids， esters and so on. The petroleum ether extract and chloroform extract contained similar substances， among which the common compounds were cyclooctatetraene， neophytadiene， palmitic acid， phytol， and γ-sitosterol.

After testing， in the four extracts， palmitic acid and phytol were the common compounds. Palmitic acid is a fatty acid， which accounts for a high proportion of total fatty acids in plasma. It plays a key role in the development of lipid metabolism-related diseases， such as diabetes and cardiovascular diseases. According to reports， palmitic acid has pharmacological effects such as improving the fatty degeneration of HepG2 cells[8]. Phytol， also known as plant alcohol， is a branched chain of plant chlorophyll and belongs to chain diterpenoids. It is a fatty alcohol containing multiple branches. Studies have reported that phytol has a regulatory effect on white adipocyte differentiation， brown adipocyte differentiation， and liver glucose and lipid metabolism[9-10].

The polarity of methanol， ethyl acetate， chloroform， and petroleum ether decrease in turn. Methanol has a strong polarity， while petroleum ether is non-polar. From this study， we know that due to the different polarity， the dissolution and extraction results of different substances in P. chinense were also different， that is， the components of the obtained P. chinense extracts were also different， which led to different volatile composition of the extracts. In a comprehensive comparison， the volatile components of the methanol extract of P. chinense were the most diverse， followed by the ethyl acetate extract， while the chloroform and petroleum ether extracts had relatively few volatile components， most of which were similar in types， and there were many common substances. Among the volatile components of the methanol extract of P. chinense， there were many types of organic compounds such as hydrocarbons， alcohols， ketones， aldehydes， amines， phenols， and esters. The types of volatile components in the ethyl acetate extract of P. chinense were the second. Among the volatile components of the ethyl acetate extract of P. chinense， there were many types of organic compounds such as hydrocarbons， alcohols， aldehydes， phenols， and esters. There were few types of volatile components in the chloroform and petroleum ether extracts of P. chinense， and organic compounds included hydrocarbons， alcohols， and esters. Because there were many types of volatile components in the methanol extract of P. chinense， and it contained catechol， pyrogallic acid， palmitic acid， phytol and other substances with higher medicinal value， it is recommended to use methanol as the extraction solvent for the further study of the volatile components in P. chinense.

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