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Abstract [Objectives] This study was conducted to establish a method for determination of chlorogenic acid in Mussaenda pubescens Ait. f.
[Methods] HPLC used SHISEIDO C18 MG II column （5 μm， 4.6 mml.D.×250 mm） as chromatographic column and acetonitrile-0.4% phosphoric acid solution （9∶91） as mobile phase. The separation was performed at a flow rate of 1.0 ml/min and a column temperature of 30 ℃， and the detection wavelength was set at 327 nm.
[Results] Chlorogenic acid had a good linear relation in the range of 0.254 7-2.547 5 μg （R 2=0.999 9）. The recovery rate was 97.8%， RSD=1.82% （n=9）.
[Conclusions] The content of chlorogenic acid in M. pubescens was determined by ultrasonic extraction and HPLC. The method was simple， stable and reliable and could be used for the quality control of M. pubescens.
Key words Chlorogenic acid; HPLC; Content determination; Mussaenda pubescens Ait. f.
Mussaenda pubescens Ait. f. is a small shrub in Mussaenda of Rubiaceae， which is widely distributed in Guangdong， Hongkong， Guangxi， Hainan and other places. Its medicinal parts are dry stems and roots， which have the effects of clearing away heat and toxic materials， and inducing diuresis and reducing edema[1]. M. pubescens can treat cold， poisoning and various inflammations， and also has the effects of contraception and abortion. At present， most M. pubescens on the market is used for clearing away heat and toxic materials and resisting inflammations. For instance， Yuye Qinghuo Tablet， Yuye Jinhua Qinghuo Tablet and Yuye Jiedu Granule are used to treat windheat syndrome， acute pharyngitis and cold and cough because of its effect of clearing away heat and toxic materials. Chlorogenic acid was found by separation of the chemical components in stems of M. pubescens. Chlorogenic acid is a phenolic acid produced by caffeic acid and quinic acid[2]. Chlorogenic acid has antibacterial， antiinflammatory， antiviral effects， etc.[3]. Chlorogenic acid has the effects similar to the active components in M. pubescens. In this study， the content of chlorogenic acid in stems of M. pubescens was determined， so as to provide reference for the correlation between the antibacterial activity of M. pubescens stems and chlorogenic acid.
Experimental materials
Instruments
Agilent 1260 infinity high performance liquid chromatograph （VWD detector， quaternay constant flow pump， online degasser， automatic sampler）， Agilent， America; electronic balance， Mettler Toledo Instruments （Shanghai） Co.， Ltd.; dualband digital ultrasonic cleaner， Kunshan Ultrasonic Instruments Co.， Ltd.; experimental hot plate， China National Analytical Center， Guangzhou; SHISEIDO C18 MG II chromatographic column （5 μmm 4.6 mml.D.×250 mm）. Agents and reagents
M. pubescens stems （identified by teacher Huang from Traditional Chinese Medicine Department of Guangxi Institute For Food and Drug Control， as dry stems of M. Pubescens Ait. f.）; chlorogenic acid reference substance （lot number： 110753201716）， for content determination; acetonitrile （chromatographically pure）; high purity water; other reagents （analytically pure）， Sinopharm Chemical Reagent Co.， Ltd.
Experimental methods
Chromatographic conditions
Chromatographic column SHISEIDO C18 MG II （5 μm， 4.6 mml.D.×250 mm）; column temperature： 30 ℃; flow rate： 1.0 ml/min; mobile phase： acetonitrile： 0.4% phosphoric acid （9∶91）[4]; detection wavelength： 327 nm. The number of theoretical plates was 31 883.
Investigation on extraction methods of chlorogenic acid in M. pubescens
Extraction method
A certain amount of M. pubescens in coarse powder （1.0 g） was accurately weighed， and 50 ml of methanol was accurately measured and weighed. The powder was extracted by reflux extraction and ultrasonic extraction methods for 30 min， respectively. The extracts were cooled， and their lost weights were complemented. The obtained liquids were then filtered with microfiltration membrane and determined according to conditions in "Chromatographic conditions".
Extraction solvent
A certain amount of M. pubescens in coarse powder （1.0 g） was accurately weighed， and 50 ml of water， 50% methanol and methanol were measured， respectively. The powder was extracted with the different extraction solvents ultrasonically for 30 min， respectively. The extracts were cooled， and their lost weights were complemented. The obtained liquids were then filtered with microfiltration membrane and determined according to conditions in "Chromatographic conditions".
Fig. 1 Contents of chlorogenic acid in M. pubescens extracted by different methods
Ultrasonic time
A certain amount of M. pubescens in coarse powder （1.0 g） was accurately weighed， and 50 ml of 50% methanol was accurately measured. The powder was ultrasonically treated for 30， 60 and 90 min， respectively. The extracts were cooled， and their lost weights were complemented. The obtained liquids were then filtered with microfiltration membrane and determined according to conditions in "Chromatographic conditions".
Solvent dosage
A certain amount of M. pubescens in coarse powder （1.0 g） was accurately weighed， and 25， 50， and 100 ml of 50% methanol were measured， respectively. The powder was ultrasonically treated with different volumes of 50% methanol for 30 min， respectively. The obtained liquids were then filtered with microfiltration membrane and determined according to "Chromatographic conditions".
[Methods] HPLC used SHISEIDO C18 MG II column （5 μm， 4.6 mml.D.×250 mm） as chromatographic column and acetonitrile-0.4% phosphoric acid solution （9∶91） as mobile phase. The separation was performed at a flow rate of 1.0 ml/min and a column temperature of 30 ℃， and the detection wavelength was set at 327 nm.
[Results] Chlorogenic acid had a good linear relation in the range of 0.254 7-2.547 5 μg （R 2=0.999 9）. The recovery rate was 97.8%， RSD=1.82% （n=9）.
[Conclusions] The content of chlorogenic acid in M. pubescens was determined by ultrasonic extraction and HPLC. The method was simple， stable and reliable and could be used for the quality control of M. pubescens.
Key words Chlorogenic acid; HPLC; Content determination; Mussaenda pubescens Ait. f.
Mussaenda pubescens Ait. f. is a small shrub in Mussaenda of Rubiaceae， which is widely distributed in Guangdong， Hongkong， Guangxi， Hainan and other places. Its medicinal parts are dry stems and roots， which have the effects of clearing away heat and toxic materials， and inducing diuresis and reducing edema[1]. M. pubescens can treat cold， poisoning and various inflammations， and also has the effects of contraception and abortion. At present， most M. pubescens on the market is used for clearing away heat and toxic materials and resisting inflammations. For instance， Yuye Qinghuo Tablet， Yuye Jinhua Qinghuo Tablet and Yuye Jiedu Granule are used to treat windheat syndrome， acute pharyngitis and cold and cough because of its effect of clearing away heat and toxic materials. Chlorogenic acid was found by separation of the chemical components in stems of M. pubescens. Chlorogenic acid is a phenolic acid produced by caffeic acid and quinic acid[2]. Chlorogenic acid has antibacterial， antiinflammatory， antiviral effects， etc.[3]. Chlorogenic acid has the effects similar to the active components in M. pubescens. In this study， the content of chlorogenic acid in stems of M. pubescens was determined， so as to provide reference for the correlation between the antibacterial activity of M. pubescens stems and chlorogenic acid.
Experimental materials
Instruments
Agilent 1260 infinity high performance liquid chromatograph （VWD detector， quaternay constant flow pump， online degasser， automatic sampler）， Agilent， America; electronic balance， Mettler Toledo Instruments （Shanghai） Co.， Ltd.; dualband digital ultrasonic cleaner， Kunshan Ultrasonic Instruments Co.， Ltd.; experimental hot plate， China National Analytical Center， Guangzhou; SHISEIDO C18 MG II chromatographic column （5 μmm 4.6 mml.D.×250 mm）. Agents and reagents
M. pubescens stems （identified by teacher Huang from Traditional Chinese Medicine Department of Guangxi Institute For Food and Drug Control， as dry stems of M. Pubescens Ait. f.）; chlorogenic acid reference substance （lot number： 110753201716）， for content determination; acetonitrile （chromatographically pure）; high purity water; other reagents （analytically pure）， Sinopharm Chemical Reagent Co.， Ltd.
Experimental methods
Chromatographic conditions
Chromatographic column SHISEIDO C18 MG II （5 μm， 4.6 mml.D.×250 mm）; column temperature： 30 ℃; flow rate： 1.0 ml/min; mobile phase： acetonitrile： 0.4% phosphoric acid （9∶91）[4]; detection wavelength： 327 nm. The number of theoretical plates was 31 883.
Investigation on extraction methods of chlorogenic acid in M. pubescens
Extraction method
A certain amount of M. pubescens in coarse powder （1.0 g） was accurately weighed， and 50 ml of methanol was accurately measured and weighed. The powder was extracted by reflux extraction and ultrasonic extraction methods for 30 min， respectively. The extracts were cooled， and their lost weights were complemented. The obtained liquids were then filtered with microfiltration membrane and determined according to conditions in "Chromatographic conditions".
Extraction solvent
A certain amount of M. pubescens in coarse powder （1.0 g） was accurately weighed， and 50 ml of water， 50% methanol and methanol were measured， respectively. The powder was extracted with the different extraction solvents ultrasonically for 30 min， respectively. The extracts were cooled， and their lost weights were complemented. The obtained liquids were then filtered with microfiltration membrane and determined according to conditions in "Chromatographic conditions".
Fig. 1 Contents of chlorogenic acid in M. pubescens extracted by different methods
Ultrasonic time
A certain amount of M. pubescens in coarse powder （1.0 g） was accurately weighed， and 50 ml of 50% methanol was accurately measured. The powder was ultrasonically treated for 30， 60 and 90 min， respectively. The extracts were cooled， and their lost weights were complemented. The obtained liquids were then filtered with microfiltration membrane and determined according to conditions in "Chromatographic conditions".
Solvent dosage
A certain amount of M. pubescens in coarse powder （1.0 g） was accurately weighed， and 25， 50， and 100 ml of 50% methanol were measured， respectively. The powder was ultrasonically treated with different volumes of 50% methanol for 30 min， respectively. The obtained liquids were then filtered with microfiltration membrane and determined according to "Chromatographic conditions".