Study on Characteristic Fingerprint of Chemical Constituents in the Pholidota cantonensis Rolfe.

Peng LI Miao ZHANG Yuanping JIANG Pingfeng LI Hua ZHU

Abstract ［Objectives］ This study was conducted to establish a characteristic fingerprinting research method for the chemical constituents of the Zhuang medicine Pholidota cantonensis Rolfe. and its characteristic fingerprint of chemical constituents, in order to identify P. cantonensis.

［Methods］ Ten batches of P. cantonensis from different habitats in Guangxi and out of Guangxi (Shaoguan of Guangdong Province) were determined. The materials were extracted in 80% methanol ultrasonically for 1.5 h under the material-to-liquid ratio of 2.5 g/80 ml. The HPLC determination was carried out using an Agilent 1260 HPLP instrument equipped with an Agilent5 TC-C18 (2) column at the column temperature of 30 ℃, the wavelength of 280 nm and the injection volume of 10 μl using acetonitrile-0.4% phosphoric acid as the mobile phase and chlorogenic acid as the reference peak, and the detection took 58 min.

［Results］ The characteristic fingerprint of the chemical constituents of P. cantonensis were obtained, and the three chromatographic peaks of gallic acid, chlorogenic acid and syringaldehyde were compared.

［Conclusions］ This study can provide reference for the identification of the Zhuang medicine P. cantonensis and provide guarantee for the clinical use of Chinese medicine.

Key words Pholidota cantonensis Rolfe.; Chemical component; Fingerprint; HPLC

Pholidota cantonensis Rolfe., also known as Shuangyeyanzhu, commonly known as Shichuanpan (Guangxi) and Fushihu, is a plant in Pholidota of Orchidaceae. Its pseudobulb and whole herb are often used as a medicine［1-2］which has the effects of nourishing Yin and moistening the lungs, clearing heat to cool blood, clearing away toxic materials, and dispersing blood stasis. P. cantonensis contains chemical components such as flavonoids, phenanthrenes, phenols, terpenoids, steroids, polysaccharides, aliphatics and minerals［3-11］. At present, the research on P. cantonensis at home and abroad mainly focuses on the extraction, separation, analysis and antibacterial effect of chemical components and the identification of Chinese herbal medicines. However, there are few reports on HPLC fingerprints. Chinese medicine fingerprint, which has the characteristics of integrity and ambiguity, is a comprehensive and quantifiable identification method of quality and authenticity［12-14］. In this study, the established research method for the characteristic fingerprint of the chemical constituents in P. cantonensis were applied to compare the fingerprints of the chemical components in P. cantonensis from different regions, to obtain the characteristic common peak atlas, which was then used to indentify P. cantonensis materials according to its integrity. This study can provide reference for the identification of the Zhuang medicine P. cantonensis and provide guarantee for the safety, effectiveness and stability of the drug and further development and formulation of the quality standard of the drug.

Test materials

Instruments

1260 high performance liquid chromatograph (Agilent, USA); electronic balance (Sedolis Scientific Instrument Co., Ltd.); Milli-Q Advantage A10 pure water system (Merck Millipore); SHB-IIIA multi-use circulating-water vacuum pump (Zhengzhou Greatwall Scientific Industrial and Trade Co., Ltd.); KQ-500DA ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); H1650-W centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.); pulverizer (Tianjin City TAISITE Instrument Co. Ltd.); HWS-28 thermostat water bath (Shanghai Qixin Scientific Instrument Co., Ltd.); DHG-9240A electric blast drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.).

Reagents

Gallic acid reference substance (National Institutes for Food and Drug Control, lot number: 110831-201605, with a content of 90.8%); chlorogenic acid reference substance (National Institutes for Food and Drug Control, lot number: 110753-201415，with a content of 96.2%); syringaldehyde reference substance (HPLC, Sigma-AIdrich, with a content of 98%); methanol, ethanol, ethyl acetate and petroleum ether (grade: analytical grade, manufacturer: Sinopharm Chemical Reagent Co., Ltd.); methanol and acetonitrile (grade: chromatographic purity, manufacturer: Fisher); phosphoric acid (grade: chromatographically pure, manufacturer: Sinopharm Chemical Reagent Co., Ltd.); ultrapure water.

Medicinal materials

Due to the differences in geographical location and growth environment, the chemical constituents and contents of the medicinal materials from different habitats are not the same. According to the relevant requirements for the establishment of the fingerprints of traditional Chinese medicine, at least 10 batches are required. In this study, a total of 10 batches of P. cantonensis grown in  Guilin, Wuzhou, Hezhou, Laibin and Baise were selected (Table 1). It was identified by Professor Teng of Guangxi University of  Chinese Medicine and others as P. cantonensisin in Pholidota of Orchidaceae.

Methods and Results

Investigation of chromatographic conditions

Chromatographic conditions are important factors influencing the experimental results. The investigation was carried out from the detection wavelength, column temperature, flow rate, mobile phase, column, injection volume, elution gradient, etc., and the best chromatographic conditions were selected through visual overall chromatographic comparison and indices such as appearance time, resolution, number of peaks, and peak area.

Determination of detection wavelength

In this study, a diode array detector was used to investigate the chromatographic conditions of the samples at wavelengths of 265, 270, 275, 280, 285, 290, 295, and 315 nm. It could be seen from comparison that, the resolution, peak area, retention time, symmetry factor and number of peaks obtained at the wavelength of 280 nm were relatively better, the baseline was relatively stable, and the peak heights were relatively uniform. Therefore, the wavelength of 280 nm was selected for the determination.

Determination of column temperature for detection

In this study, the chromatographic peaks at different column temperatures (35, 30 and 25 ℃) were investigated. It could be seen from comparison that, the resolution, peak area, retention time and symmetry factor of the chromatographic peaks obtained at the temperature of 30 ℃ were better. Considering various factors, the column temperature used was 30 ℃.

Determination of flow rate

The flow rate also has a certain effect on chromatographic detection results. It is an important parameter for adjusting the resolution and appearance time of peaks. Properly reducing the flow rate under determined chromatographic column and mobile phase can improve column efficiency.   In this study, the effects of different flow rates (1, 0.8, 0.5 ml/min) on the separating effect, retention time, peak area, and symmetry factor of the chromatographic peaks of P. cantonensis were investigated. The results showed that the resolution obtained when the flow rate was 1 ml/min was better. Therefore, the flow rate chosen for this study was 1 ml/min.

Determination of mobile phase

When the stationary phase is determined, the type and proportion of the mobile phase have a significant effect on the separating effect. In this study, such six mobile phases as acetonitrile (B)-water (D), acetonitrile (B)- 0.1% phosphoric acid (D), acetonitrile (B)-0.4% phosphoric acid (D), methanol (B)-water (D), methanol (B)-0.1% phosphoric acid (D) and methanol (B)-0.4% phosphoric acid (D) had better separating effects. Comprehensively, acetonitrile-0.4% phosphoric acid was used as the mobile phase of this study.

Determination of injection volume

In this study, the injection volumes of 12, 10, and 8 μl were investigated. The results showed that the degree of separation was relatively better when the injection volume was 10 μl. Therefore, the injection volume was selected to be 10 μl.

Sample preparation

Preparation of test solutions

A certain amount of P. cantonensis powder (2.5 g) for testing dried and sieved with No. 4 sieve was accurately weighed with an electronic balance and added into a 150 ml conical flask. 80 ml of 80% methanol solution was transferred with a pipette to the conical flask. After 1.5 h of ultrasonic extraction, cooling and removal of water on the surface of the conical flask, the conical flask was weighed, and the lost weight was compensated with the 80% methanol solution, followed by shaking and filtration. The filtrate was centrifuged for 10 min, obtaining the supernatant, which was filtered with a 0.45 μm microporous membrane.

Preparation of references solutions

Gallic acid standard, chlorogenic acid standard and syringaldehyde standard were weighed accurately with a balance, respectively. Each was placed in a  10 ml volumetric flask, dissolved in methanol and diluted to constant volume, followed by shaking. The prepared solutions were  0.125, 0.058 8 and 0.112 g/ml, respectively, which were then filtered with a 0.45 μm microporous membrane, respectively.

Specificity test

In order to rule out the misjudgment of the experimental results by the presence of peaks of other interfering components and the solvent, a blank test was carried out in this study. The experimental result is shown in Fig. 1, which showed that the study was not affected by solvent peak interference.

Fingerprint analysis

A certain amount of P. cantonensis powder (2.5 g) was accurately weighed, and prepared into a test solution according to the above method. The prepared solutions were determined according to the above chromatographic conditions. It can be seen from the obtained chromatograms that peak No.2 had a  the better degree of separation and stable appearance time. The contents of various producing areas were not much different, and chlorogenic acid is one of the main components of P. cantonensis, so peak No. 2 was the main indicator component of P. cantonensis, which was used as the reference peak and labeled as S.

Establishment of characteristic fingerprints of chemical components

The ten batches of P. cantonensis samples were prepared into test solutions according to the above method, which were separately injected and analyzed with the above chromatographic conditions to obtain the chromatograms of each batch of samples. According to the chromatograms of the three reference materials, the chromatographic peaks corresponding to gallic acid, chlorogenic acid and syringaldehyde can be obtained.

Conclusions and Discussion

In this study, the characteristic fingerprint of the chemical constituents of P. cantonensis was obtained by HPLC through gradient elution with gallic acid as a reference substance. The materials were extracted in 80% methanol ultrasonically for 1.5 h under the material-to-liquid ratio of 2.5 g/80 ml. The HPLC was carried out using an Agilent 1260 HPLP instrument equipped with an Agilent5 TC-C18 (2) column at the column temperature of 30 ℃, the wavelength of 280 nm and the injection volume of 10 μl using acetonitrile-0.4% phosphoric acid as the mobile phase, and the detection took 58 min. The experimental results showed that the method established in this study can be used for the identification of the medicinal material P. cantonensis, and gallic acid, chlorogenic acid and syringaldehyde are the main effective constituents in P. cantonensis with relatively stable amounts. Therefore, gallic acid, chlorogenic acid and syringaldehyde were used as reference materials to qualitatively identify the chromatographic peaks of  P. cantonensis in this study. Through the visual comparison of the chromatographic peaks, the chemical composition of P. cantonensis of different origins can be obtained, and the medicinal materials of P. cantonensis can be identified and distinguished initially. This method can serve as an effective method for identification of the medicinal materials of P. cantonensis.

Agricultural Biotechnology2019

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