Quantification of chlorogenic acid, rosmarinic acid, and caffeic acid contents in selected Thai medicinal plants using RP-HPLC-DAD

Abstract

The chlorogenic acid, rosmarinic acid, and caffeic acid contents in 100 selected plants were determined using reversed phase high performance liquid chromatography equipped with diode array detector. The optimum condition was 0.2% phosphoric acid in water (solvent A) and methanol (solvent B) as the mobile phase, which was set at 45% B for 20 minutes at a flow rate of 1.2 mL/min. The column temperature was maintained at 30 ºC and the detection wavelength was 325 nm. Among 100 selected plants, 39.64% contained all 3 compounds, 40.54% contained 2 compounds, 14.41% contained only 1 compound, and 5.41% could not detect any of the 3 compounds. The highest contents of chlorogenic acid, rosmarinic acid, and caffeic acid were found in Lonicera japonica flowering buds, Melissa officinalis leaves, and Coffea canephora seeds at the concentration of 9.900 ± 0.004, 19.908 ± 0.171, and 1.233 ± 0.003 g/100 g of dried plant, respectively.

Keywords:
Chlorogenic acid; Rosmarinic acid; Caffeic acid; RP-HPLC-DAD; Medicinal plant

INTRODUCTION

Phenolic compounds or polyphenols, the secondary metabolites of plant, are one of the most abundant and extensively distributed groups of substances in the plant kingdom which appear in all plant organs. However, the polyphenolic profile of plants differs between varieties of the same species. For decades, polyphenols have interested many researchers for their antioxidant, antioxidative stress activities, and great abundance in food. The varieties of natural polyphenols range from simple molecules (such as phenolic acids) to highly polymerized compounds (such as tannins). Polyphenols occur primarily in a conjugated form with one or more sugar residues linked to hydroxyl groups, although direct linkages of the sugar unit to an aromatic carbon atom also exist (Bravo, 1998; Manach et al., 2004). Hydroxycinnamic acid, one of two major groups of phenolic acids, is usually found in plants. The hydroxycinnamic acid derivatives consist of a large group of simple phenolic acids, and are bountiful in fruits, seeds of fruits, vegetables, and cereals. In addition, they have been arranged into structural and functional constituents of plant cell walls and also as bioactive ingredients of diets. The derivatives of hydroxycinnamic acids are synthesized through the shikimate pathway in which phenylalanine and tyrosine are used as starting precursor molecules. The main hydroxycinnamic acid derivatives are ferulic acid, caffeic acid, p-coumaric acid, chlorogenic acid, sinapic acid, and rosmarinic acid (Lafay, Gil-Izquierdo, 2008; Manach et al., 2004; Teixeira et al., 2013). Caffeic acid (Figure 1A) is one of the most common phenolic acids that biosynthesise by hydroxylation of p-coumaric acid and is more broadly present in several food sources such as berries, coffee drinks, and dietary supplements (Magnani et al., 2014). Chlorogenic acid (Figure 1B) is an ester form of caffeic acid and quinic acid, which is widely distributed in the human diet with plants, fruits, and vegetables especially in coffee, apples, and pears (Upadhyay, Mohan Rao, 2013). Rosmarinic acid (Figure 1C), an ester of caffeic acid and 3, 4-dihydroxyphenyllactic acid, is commonly found in species of the boraginaceae, lamiaceae, and in some ferns and hornworts (Petersen, Simmonds, 2003). High performance liquid chromatography (HPLC) is a primary method for the separation and analysis of chemical compounds in many fields such as agriculture, cosmetics, pharmaceutical industries, environments, and food. It is commonly used for qualitative and quantitative analyses of chemicals in herbal extracts. The identification of compounds depends on the retention time and light spectral characteristics of each chromatographic peak (Zeng et al., 2011).

FIGURE 1
Structures of caffeic acid (A), chlorogenic acid (B) and rosmarinic acid (C).

The aim of this study was to establish a RP-HPLC-DAD condition for analysis and provide the approximate quantification of chlorogenic acid, rosmarinic acid, and caffeic acid in 100 selected Thai medicinal plants.

MATERIAL AND METHODS

Chemicals and material

Standard chlorogenic acid (CAS no. 327-97-9, purity ≥ 95%), rosmarinic acid (CAS no. 20283-92-5, purity 96%) and caffeic acid (CAS no. 331-39-5, purity ≥ 98%) were purchased from Sigma-Aldrich, USA. Methanol was of HPLC grade (RCI Labscan, Bangkok, Thailand). Ethanol, petroleum ether, and ortho-phosphoric acid were of analytical grade (RCI Labscan, Bangkok, Thailand). Ultra-pure water was prepared by an ultra-pure water system (NW20VF, Heal Force, China). The filters were 46 mm × 0.45 µm nylon membrane filters (National Scientific, Tennessee, USA) and 13 mm × 0.45 µm PTFE membrane syringe filters (ANPEL Laboratory Technology, Shanghai, China).

Sample collection

A selection of 100 fresh plants was obtained by randomized collection from various places in Thailand and also purchased from local markets in Thailand based on chemotaxonomy. They were authenticated by Associate Professor Dr. Nijsiri Ruangrungsi. All plant materials were dried at 45 ºC in a hot air oven, and voucher specimens were deposited at College of Public Health Sciences, Chulalongkorn University. After the removal of any foreign matter, crude drugs were grounded into coarse powders before use.

Sample extraction

Ten grams of each selected plant sample were exhaustively extracted with petroleum ether and followed by 95% ethanol using a Soxhlet apparatus. The ethanolic extract was filtered through filter-paper and evaporated to dryness under reduced pressure by a rotary evaporator. The extract yields were weighed, recorded, and stored at -20 ºC to avoid the possibility of degradation of the active compounds.

Preparation of standard solutions

One milligram of each standard was dissolved in 1 mL of methanol. The solution was filtered through a 0.45 µm PTFE membrane syringe filter.

Preparation of sample solutions

Fifty milligrams of each extract were dissolved in 1 mL of methanol and diluted to appropriate concentrations for further RP-HPLC analysis. The solution was filtered through a 0.45 µm PTFE membrane syringe filter.

Chromatographic conditions

The Shimadzu HPLC LC-20A system (Shimadzu, Japan) consists of a system controller (CMB-20A), two solvent delivery units (LC-20A), an on-line degassing unit (DGU-20A3), an auto-sample (SIL-20A), a column oven (CTO-20A), and a photo-diode array detector (SPD-M20A). System control and data analysis were processed with Shimadzu LC Solution software. The chromatographic separation was performed with an Inertsil^® ODS-3 5 µm C₁₈ column (4.6 X 250 mm) and coupled with a ReproSil^®-Pur ODS-3 C₁₈ guard column (4.0 X 10 mm). The samples were analyzed using 0.2% phosphoric acid in water, pH 1.46 (solvent A), and methanol (solvent B) as a mobile phase. The isocratic program was set at 45% B for 20 minutes at a flow rate of 1.2 mL/min. The mobile phase was filtered through 0.45 µm nylon membrane filters and degassed using an ultrasonic bath before analysis. The column temperature was maintained at 30 ºC and the injection volume of standards and sample solutions was 5 µl. The wavelength was set at 325 nm for monitoring chromatographic profile. All measurement was done in triplicate.

System suitability

The retention factor, theoretical plate number, and tailing factor were evaluated for system suitability parameters. The system performance was analyzed for five replicates of standard solution.

Method validation

According to the ICH guideline (ICH, 2005), the calibration range, specificity, accuracy, repeatability, intermediate precision, limit of detection (LOD), limit of quantitation (LOQ), and robustness were validated for analytical method. The Lonicera japonica flowering bud ethanolic extract was found to contain all 3 compounds so it was used as a sample matrix to evaluate the validity of the analytical method.

Calibration range

The calibration range was performed by plotting peak areas obtained from RP-HPLC analysis versus concentrations of standard. The stock solutions of chlorogenic acid, rosmarinic acid, and caffeic acid were dissolved in methanol and diluted together to give concentrations of 16.67, 33.33, 50.00, 66.67, and 83.33 µg/mL for evaluation of the calibration range. The calibration range of these standards was fitted by linear regression. The regression equation was calculated in the form of $\mathit{y}=a\mathit{x}+\mathit{b}$, where y is peak area and x is concentration.

Specificity

The specificity was evaluated by a peak purity test. The peak purity index of the analyte was processed by Shimadzu LC Solution software. It was determined by comparing all the spectra within the chromatographic peak to the reference spectrum at the peak apex.

Accuracy

The accuracy of each sample was tested by recovery method. Three different levels of standard solutions (10, 25, and 50 µg/mL) were spiked into the extract. The spiked and un-spiked samples were evaluated under the same condition in triplicate, then percent recoveries were calculated by comparing the measured amount of those standards with the amount added.

Precision

The precision was determined by repeatability (intra-day) and intermediate precision (inter-day) studies. The method was performed by analyzing three level concentrations of sample solution in triplicate on the same day for repeatability and in the five different days for intermediate precision. The precision was calculated in terms of percent relative standard deviation (% RSD) of compound content.

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

LOD and LOQ were determined from the calibration range using the following formulae:

$\begin{array}{c}\mathrm{LOD}=\frac{3.3x\sigma }{S}\hfill \\ \mathrm{LOD}=\frac{10x\sigma }{S}\hfill \end{array}$

where: σ = the residual standard deviation of the regression line; S = the slope of the regression line

Robustness

The robustness was determined for variations in flow rates (1.195, 1.200, and 1.205 mL/min), variations in column temperature (29, 30, and 31 ºC), and variations in wavelength (322, 325 and 328 nm). The robustness was calculated in terms of percent relative standard deviation (% RSD) of retention time and peak area.

RESULTS AND DISCUSSION

Optimization of chromatographic condition

The chromatographic condition optimization including mobile phase, gradient elution procedure, flow rate, column temperature, and wavelength detection were performed to provide a better separation of constituents. Numerous mobile phases and gradient programs were trialled using various proportions of different aqueous phases and organic modifiers. Formic acid, phosphoric acid, and acetic acid were usually employed to the aqueous phase to enhance the resolution, restrain the ionization, and reduce the peak tailing of compounds (Ma et al., 2011). The most suitable mobile phase that showed good resolution and symmetric peak shape were obtained using two parts as Solvent A (0.2% phosphoric acid in water) and Solvent B (methanol) with an isocratic program. The column temperature was held at 30 ºC for the duration of analysis to improve the retention time precision. Hydroxycinnamic acids have the maximum wavelength during 270 - 360 nm (Köseoglu, Kolak, 2017). The UV spectra of standard chlorogenic, rosmarinic, and caffeic acids were compared at varying wavelengths, and based on the data from the literatures. The optimal detection wavelength in this study was to be 325 nm (Haghi, Hatami, 2010; Shan et al., 2013).

Chlorogenic acid, rosmarinic acid, and caffeic acid quantification

The 100 selected plants were edible vegetables, fruits, and herbal plants in Thailand. The plant samples were exhaustively extracted with petroleum ether and followed by 95% ethanol using a Soxhlet apparatus. The percent yields of crude extracts were shown in Table I.

TABLE I
The contents of chlorogenic, rosmarinic and caffeic acids in plant samples

Voucher specimen Number	Scientific plant name	Plant parts used	% yield (g/100 g)	Content (g/100 g of dried plant)
				Chlorogenic acid	Rosmarinic acid	Caffeic acid
	Family: Acanthaceae
CCPh001	Andrographis paniculata (Burm.f.) Wall. ex Nees	Leaves	25.595	0.625 ± 0.001	0.338 ± 0.000	0.045 ± 0.000
	Family: Alliaceae
CCPh002	Allium sativum L.	Bulbs	10.393	-	-	0.005 ± 0.000
CCPh003	Allium cepa L.	Bulbs	66.467	0.034 ± 0.000	-	-
	Family: Amaranthaceae
CCPh004	Spinacia oleracea L.	Leaves	21.103	-	0.039 ± 0.000	0.037 ± 0.000
	Family: Anacardiaceae
CCPh005	Mangifera indica L. cv. Okrong	Leaves	28.698	0.354 ± 0.003	-	1.008 ± 0.008
	Family: Apiaceae
CCPh006	Anethum graveolens L.	Aerial part	28.714	7.361 ± 0.038	-	0.184 ± 0.002
CCPh007	Apium graveolens L.	Aerial part	36.739	0.913 ± 0.003	2.880 ± 0.018	0.088 ± 0.001
CCPh008	Apium graveolens L. var. secalinum	Aerial part	36.066	2.574 ± 0.011	2.556 ± 0.014	0.112 ± 0.000
CCPh009	Centella asiatica (L.) Urb.	Aerial part	31.917	0.848 ± 0.003	0.910 ± 0.003	0.086 ± 0.002
CCPh010	Coriandrum sativum L.	Seeds	4.770	0.028 ± 0.000	0.061 ± 0.001	0.024 ± 0.000
CCPh011	Daucus carota L. subsp. sativus (Hoffm.) Arcang.	Roots	52.063	0.326 ± 0.003	-	0.085 ± 0.000
CCPh012	Eryngium foetidum L.	Leaves	30.451	4.979 ± 0.006	4.302 ± 0.100	0.160 ± 0.007
CCPh013	Petroselinum crispum (Mill.) Nyman ex A.W. Hill	Aerial part	31.826	0.058 ± 0.004	9.153 ± 0.602	0.048 ± 0.001
	Family: Apocynaceae
CCPh014	Telosma cordata (Burm. f.) Merr.	Flowers	34.584	0.350 ± 0.000	-	0.184 ± 0.002
	Family: Asteraceae
CCPh015	Artemisia dracunculus L.	Aerial part	28.885	5.253 ± 0.047	5.359 ± 0.037	0.062 ± 0.008
CCPh016	Artemisia pallens Wall. ex DC.	Aerial part	8.141	0.042 ± 0.000	-	0.011 ± 0.000
CCPh017	Chromolaena odorata (L.) R. M. King & H. Rob.	Leaves	28.239	4.137 ± 0.035	0.279 ± 0.009	0.621 ± 0.018
CCPh018	Gnaphalium polycaulon Pers.	Aerial part	23.409	0.699 ± 0.009	1.207 ± 0.012	0.236 ± 0.009
CCPh019	Helianthus annuus L.	Pericarps	2.880	0.038 ± 0.000	0.004 ± 0.000	0.003 ± 0.000
CCPh020	Helianthus annuus L.	Seeds	11.523	2.144 ± 0.006	0.008 ± 0.000	0.110 ± 0.000
CCPh021	Helianthus annuus L.	Sprouts	30.726	2.199 ± 0.005	3.117 ± 0.029	0.093 ± 0.002
CCPh022	Lactuca sativa L.	Leaves	24.088	1.396 ± 0.036	0.343 ± 0.001	0.258 ± 0.002
	Family: Brassicaceae
CCPh023	Brassica juncea (L.) Czern.	Leaves	28.414	0.351 ± 0.002	-	0.083 ± 0.001
CCPh024	Brassica oleracea L. Group Capitata	Aerial part	38.510	0.172 ± 0.002	-	-
CCPh025	Brassica rapa L. Group Pekinensis	Aerial part	41.573	0.154 ± 0.001	-	-
CCPh026	Raphanus sativus L.	Roots	52.566	0.072 ± 0.002	-	0.033 ± 0.001
	Family: Caprifoliaceae
CCPh027	Lonicera japonica Thunb.	Flowering bud	32.734	9.896 ± 0.004	2.543 ± 0.007	0.195 ± 0.002
	Family: Caricaceae
CCPh028	Carica papaya L.	Leaves	17.759	0.049 ± 0.000	0.023 ± 0.001	0.508 ± 0.000
	Family: Convolvulaceae
CCPh029	Ipomoea aquatica Forssk.	Aerial part	30.503	2.541 ± 0.014	8.303 ± 0.042	0.113 ± 0.002
	Family: Cucurbitaceae
CCPh030	Momordica charantia L. (Thai varieties)	Fruits	28.359	0.042 ± 0.000	-	<LOQ
CCPh031	Momordica charantia L. (Chinese varieties)	Fruits	35.728	0.085 ± 0.001	-	<LOQ
	Family: Eucommiaceae
CCPh032	Eucommia ulmoides Oliv.	Stem barks	10.441	0.039 ± 0.000	-	0.019 ± 0.000
	Family: Euphorbiaceae
CCPh033	Euphorbia hirta L.	Aerial part	11.150	0.234 ± 0.000	-	0.021 ± 0.000
CCPh034	Phyllanthus emblica L.	Fruits	28.671	0.300 ± 0.001	0.077 ± 0.000	0.029 ± 0.000
CCPh035	Ricinus communis L.	Leaves	22.599	0.039 ± 0.000	-	0.352 ± 0.004
	Family: Fabaceae
CCPh036	Pisum sativum L.	Fruits	45.838	-	-	-
CCPh037	Pisum sativum L. var. macrocarpon	Fruits	43.713	-	-	-
CCPh038	Sesbania grandiflora (L.) Poir.	Flowers	42.093	0.016 ± 0.000	0.204 ± 0.007	<LOQ
CCPh039	Sesbania grandiflora (L.) Poir.	Stem barks	3.960	0.009 ± 0.000	-	0.006 ± 0.000
	Family: Gnetaceae
CCPh040	Gnetum gnemon L. var. tenerum Markgr.	Leaves	22.424	0.092 ± 0.001	-	-
	Family: Labiatae
CCPh041	Hyptis suaveolens (L.) Poit.	Aerial part	11.194	0.078 ± 0.001	3.961 ± 0.006	0.111 ± 0.000
CCPh042	Leonotis nepetifolia (L.) R. Br.	Leaves	22.287	0.171 ± 0.001	-	0.418 ± 0.010
CCPh043	Leonurus sibiricus L.	Aerial part	14.098	0.045 ± 0.003	-	0.012 ± 0.000
CCPh044	Melissa officinalis L.	Leaves	20.371	0.048 ± 0.004	19.908 ± 0.171	0.174 ± 0.006
CCPh045	Mentha arvensis L. var. piperascens Malinv.	Leaves	19.968	1.038 ± 0.002	6.809 ± 0.086	0.108 ± 0.001
CCPh046	Mentha cordifolia Opiz ex Fresen	Leaves	21.461	0.117 ± 0.000	7.537 ± 0.010	0.100 ± 0.001
CCPh047	Ocimum africanum Lour.	Leaves	15.466	0.033 ± 0.000	1.691 ± 0.002	0.092 ± 0.000
CCPh048	Ocimum basilicum L.	Leaves	15.756	0.243 ± 0.001	0.597 ± 0.003	0.311 ± 0.001
CCPh049	Ocimum gratissimum L. var. macrophyllum Briq.	Leaves	19.372	0.189 ± 0.002	1.756 ± 0.001	0.240 ± 0.003
CCPh050	Ocimum tenuiflorum L.	Leaves	17.317	0.215 ± 0.001	2.292 ± 0.003	0.112 ± 0.002
CCPh051	Origanum majorana L.	Leaves	24.119	-	7.954 ± 0.028	0.065 ± 0.003
CCPh052	Origanum vulgare L.	Leaves	31.593	0.123 ± 0.005	9.902 ± 0.091	0.410 ± 0.009
CCPh053	Orthosiphon aristatus (Blume) Miq.	Leaves	11.332	0.072 ± 0.002	2.101 ± 0.003	0.115 ± 0.001
CCPh054	Perilla frutescens (L.) Britton	Leaves	21.621	0.042 ± 0.001	13.185 ± 0.021	0.189 ± 0.001
	Family: Labiatae
CCPh055	Plectranthus amboinicus (Lour.) Spreng.	Leaves	12.369	0.049 ± 0.001	0.279 ± 0.003	0.121 ± 0.001
CCPh056	Plectranthus rotundifolius (Poir.) Spreng.	Leaves	22.849	-	0.334 ± 0.003	0.086 ± 0.001
CCPh057	Plectranthus rotundifolius (Poir.) Spreng.	Tubers	6.628	-	0.669 ± 0.005	0.270 ± 0.004
CCPh058	Plectranthus scutellarioides (L.) R. Br.	Leaves	26.274	-	2.594 ±0.016	0.217 ± 0.001
CCPh059	Rosmarinus officinalis L.	Aerial part	17.989	0.124 ± 0.005	2.611 ± 0.023	0.146 ± 0.010
CCPh060	Salvia hispanica L.	Seeds	5.427	0.029 ± 0.000	0.576 ± 0.025	0.007 ± 0.000
CCPh061	Salvia officinalis L.	Aerial part	20.719	-	6.829 ± 0.070	0.166 ± 0.009
CCPh062	Thymus citriodorus (Pers.) Schreb.	Aerial part	19.963	0.165 ± 0.001	10.176 ± 0.417	0.128 ± 0.010
CCPh063	Thymus vulgaris L.	Aerial part	21.499	0.093 ± 0.005	4.349 ± 0.025	0.155 ± 0.002
	Family: Lauraceae
CCPh064	Persea americana Mill.	Flesh	16.142	0.029 ± 0.000	-	-
CCPh065	Persea americana Mill.	Peels	15.259	0.539 ± 0.001	-	0.008 ± 0.000
CCPh066	Persea americana Mill.	Seeds	22.143	1.381 ± 0.005	-	-
	Family: Malvaceae
CCPh067	Hibiscus sabdariffa L.	Leaves	31.829	1.117 ± 0.002	-	0.351 ± 0.002
	Family: Meliaceae
CCPh068	Azadirachta indica A. Juss.	Leaves	16.665	-	-	-
	Family: Moraceae
CCPh069	Morus alba L.	Leaves	20.045	3.028 ± 0.005	0.050 ± 0.001	0.025 ± 0.001
	Family: Moringaceae
CCPh070	Moringa oleifera Lam.	Leaves	16.509	0.525 ± 0.002	-	0.030 ± 0.000
CCPh071	Moringa oleifera Lam.	Seeds	8.414	-	-	0.004 ± 0.000
	Family: Myrtaceae
CCPh072	Psidium guajava L.	Fruits	50.035	-	-	0.022 ± 0.000
CCPh073	Syzygium antisepticum (Blume) Merr. & L. M. Perry	Leaves	23.821	-	-	-
	Family: Oxalidaceae
CCPh074	Averrhoa carambola L.	Fruits	63.412	0.045 ± 0.001	-	0.009 ± 0.000
	Family: Piperaceae
CCPh075	Piper betle L.	Leaves	21.431	0.136 ± 0.000	-	0.041 ± 0.000
CCPh076	Piper nigrum L. (Black pepper)	Fruits	7.985	-	-	-
CCPh077	Piper nigrum L. (White pepper)	Seeds	5.619	-	-	-
	Family: Poaceae
CCPh078	Cymbopogon citratus (DC.) Stapf	Rhizomes	26.886	0.132 ± 0.002	-	0.073 ± 0.003
	Family: Polygonaceae
CCPh079	Persicaria odorata (Lour.) Soják	Leaves	17.391	0.289 ± 0.017	-	0.091 ± 0.000
	Family: Punicaceae
CCPh080	Punica granatum L. var. granatum	Leaves	43.525	0.194 ± 0.009	-	0.305 ± 0.006
CCPh081	Punica granatum L. var. granatum	Peels	40.172	-	0.049 ± 0.000	0.363 ± 0.003
	Family: Rosaceae
CCPh082	Fragaria vesca L.	Fruits	70.506	0.046 ± 0.001	-	-
CCPh083	Malus domestica Borkh.	Fruits	85.332	0.277 ± 0.001	-	-
CCPh084	Pyrus communis L.	Fruits	74.522	0.407 ± 0.001	-	-
	Family: Rubiaceae
CCPh085	Coffea arabica L.	Seeds	8.228	5.967 ± 0.007	0.845 ± 0.002	0.380 ± 0.001
CCPh086	Coffea canephora Pierre ex A. Froehner	Seeds	12.262	7.843 ± 0.037	1.663 ± 0.007	1.233 ± 0.003
CCPh087	Morinda citrifolia L.	Fruits	23.686	0.046 ± 0.001	-	0.010 ± 0.000
CCPh088	Morinda citrifolia L.	Leaves	28.441	0.081 ± 0.000	-	-
	Family: Scrophulariaceae
CCPh089	Limnophila aromatica (Lam.) Merr.	Aerial part	16.237	0.649 ± 0.001	-	0.058 ± 0.001
	Family: Solanaceae
CCPh090	Capsicum annuum L. (Green bell pepper)	Fruits	51.863	0.148 ± 0.000	-	0.025 ± 0.000
CCPh091	Capsicum annuum L. (Orange bell pepper)	Fruits	55.366	0.169 ± 0.001	-	0.026 ± 0.001
CCPh092	Capsicum annuum L. (Red bell pepper)	Fruits	58.196	0.164 ± 0.002	-	0.085 ± 0.002
CCPh093	Capsicum annuum L. (Yellow bell pepper)	Fruits	58.237	0.175 ± 0.003	-	0.026 ± 0.001
CCPh094	Nicotiana tabacum L.	Leaves	30.838	3.317 ± 0.001	-	0.057 ± 0.000
CCPh095	Physalis angulata L.	Aerial part	12.733	0.196 ± 0.002	-	0.012 ± 0.000
CCPh096	Physalis peruviana L.	Fruits	61.376	0.097 ± 0.001	-	<LOQ
CCPh097	Physalis peruviana L.	Calyx	13.857	0.474 ± 0.001	-	0.086 ± 0.001
CCPh098	Solanum lycopersicum L. var. cerasiforme	Fruits	45.374	0.225 ± 0.010	-	0.037 ± 0.000
CCPh099	Solanum lycopersicum L. var. lycopersicum	Fruits	60.356	0.280 ± 0.009	-	0.035 ± 0.000
	Family: Strychnaceae
CCPh100	Strychnos nux-vomica L.	Seeds	3.228	0.389 ± 0.000	-	0.004 ± 0.000
	Family: Theaceae
CCPh 101	Camellia sinensis (L.) Kuntze var. assamica (Mast.) Kitam.	Leaves	46.363	0.719 ± 0.003	-	0.051 ± 0.001
	Family: Thunbergiaceae
CCPh 102	Thunbergia laurifolia Lindl.	Leaves	14.292	0.082 ± 0.004	11.487 ± 0.019	0.218 ± 0.001
	Family: Verbenaceae
CCPh 103	Clerodendrum calamitosum L.	Leaves	20.401	0.023 ± 0.001	-	0.010 ± 0.000
CCPh 104	Clerodendrum indicum (L.) Kuntze	Leaves	24.400	-	-	0.011 ± 0.001
CCPh 105	Clerodendrum quadriloculare (Blanco) Merr.	Leaves	21.407	0.066 ± 0.001	-	-
CCPh 106	Clerodendrum serratum (L.) Moon	Leaves	33.686	1.804 ± 0.006	-	-
CCPh 107	Clerodendrum thomsoniae Balf. f.	Leaves	19.390	0.018 ± 0.002	0.903 ± 0.004	0.077 ± 0.002
CCPh 108	Vitex agnus-castus L.	Leaves	32.946	5.557 ± 0.068	3.083 ± 0.037	0.084 ± 0.001
CCPh 109	Vitex negundo L.	Leaves	28.441	1.238 ± 0.002	1.135 ± 0.005	0.054 ± 0.004
CCPh 110	Vitex trifolia L. subsp. litoralis Steenis	Leaves	28.545	0.379 ± 0.003	1.497 ± 0.008	0.167 ± 0.003
CCPh 111	Vitex trifolia L. subsp. trifolia	Leaves	31.403	2.180 ± 0.056	2.745 ± 0.098	0.177 ± 0.006

• *cannot be detected

A quantitative analysis of chlorogenic acid, rosmarinic acid, and caffeic acid in selected plants was performed by RP-HPLC analysis. The standard markers to quantify in this study are chlorogenic acid, rosmarinic acid, and caffeic acid which are hydroxycinnamic acid derivatives. Hydroxycinnamic acid derivatives, a subgroup of phenylpropanoids, are synthesised by the shikimate pathway where the starter precursor molecules are phenylalanine and tyrosine. Chlorogenic acid, rosmarinic acid, and caffeic acid in extracts were identified by comparing the retention time and UV spectrum of each peak with a reference of standard compounds (Figure 2). The contents of chlorogenic acid, rosmarinic acid, and caffeic acid in the 100 selected plants were shown in Table I. The results of RP-HPLC analysis demonstrated that the distribution of these 3 phenolic compounds varied in many samples. Among 100 selected plants, 39.64% contained all 3 compounds, 40.54% contained 2 compounds, 14.41% contained only 1 compound, and 5.41% could not detect these 3 compounds. Lonicera japonica flowering buds were found to be the richest source of chlorogenic acid content at 9.90 g/100 g of dried crude drug, and Melissa officinalis leaves showed the most rosmarinic acid content at 19.91 g/100 g of dried crude drug. The most caffeic acid content was found in Coffea canephora seeds at 1.23 g/100 g of dried crude drug. Chlorogenic acid was found in many families and is the main active constituent in L. japonica flowering bud (Chaowuttikul, Palanuvej, Ruangrungsi, 2017). It is also the main phenolic compound in coffee (Coffea spp.) that supported this study (Ayelign, Sabally, 2013). Rosmarinic acid was mostly found in the Labiatae family, relating to a previous report of high rosmarinic acid content in plants of this family, especially in Mentha spicata, Salvia officicalis, and Melissa officinalis (Shekarchi et al., 2012).

FIGURE 2
HPLC chromatogram of standard chlorogenic acid, rosmarinic acid and caffeic acid at 325 nm by RP-HPLC-DAD.

System suitability

The retention factor, theoretical plate number, and tailing factor were found to be 4.30 ± 0.01, 2745.17 ± 158.17, and 1.027 ± 0.07, respectively (Table II). These parameters confirmed that the condition is appropriate for analysis according to the FDA criteria.

TABLE II
System suitability of standard solution (n = 5) compared to criteria of the U.S. Food and Drug Administration (FDA, 1994)

Parameter	Result	Acceptance criteria
Retention factor (k)	4.30 ± 0.01	k ˃ 2
Theoretical plate number (N)	2745.17 ± 158.17	N ˃ 2000
Tailing factor (Tf)	1.027 ± 0.07	Tf ≤ 2

Method validation

The analytical method validation is the process that confirms precise, accurate, and reliable quantitative data. According to the ICH guideline, calibration range, specificity, accuracy, repeatability, intermediate precision, limit of detection, limit of quantitation, and robustness should be validated for analytical analysis.

Standard chlorogenic acid, rosmarinic acid, and caffeic acid at 5 concentrations were investigated for linearity by RP-HPLC method. The calibration curves of standard compounds were linear in the range of 16.67 - 83.33 µg/mL. The regression equation of chlorogenic acid, rosmarinic acid, and caffeic acid were $\mathit{y}=2874.5\mathit{x}+813.03,\mathit{y}=2833.8\mathit{x}-1858.3$, and $\mathit{y}=5202.2\mathit{x}+673.32$, respectively (Figures 3 - 5). The linearity showed good correlation (R² ≥ 0.999). An analytical technique is acceptable when the correlation of method (R²) value achieved is 0.99 or better.

FIGURE 3
The calibration curve of chlorogenic acid.

FIGURE 4
The calibration curve of rosmarinic acid

FIGURE 5
The calibration curve of caffeic acid.

The specificity was evaluated by peak purity test and confirmed that analyte chromatographic peak is not attributable with another compound. This test is based on the absorbance spectrum, which is detected by diode array detectors. If all of the individual spectra recorded during the elution of a peak are identical, even if detected at any periods of a peak, the peak is considered pure (Hansen, Pedersen-Bjergaard, Rasmussen, 2011). An identical peak resulted in a peak purity index of 100% or peak purity index of 1.0, indicating that all spectra are similar. The results showed the peak purity index of the three compounds was more than 0.999 (Figures 6-8), thus no impurity was detected in these peaks.

FIGURE 6
The peak purity of chlorogenic acid in L. japonica flowering bud ethanolic extract (Peak purity index: 1.000000).

FIGURE 7
The peak purity of rosmarinic acid in L. japonica flowering bud ethanolic extract (Peak purity index: 0.999952).

FIGURE 8
The peak purity of caffeic acid in L. japonica flowering bud ethanolic extract (Peak purity index: 0.999999).

The accuracy was evaluated by the recovery method. Three concentrations of standard compounds were spiked into the sample. The accuracy of chlorogenic acid, rosmarinic acid, and caffeic acid quantitative analysis in L. japonica flowering bud ethanolic extract ranged from 103.98-108.63, 97.23-99.09, and 99.41-100.85% recoveries, respectively (Table III). The repeatability and intermediate precision were performed on samples with three different concentrations of standard compounds at the same day and five different days of experiments, respectively. The values were shown as %RSD which meant the error of the method. The repeatability and intermediate precision were shown in Table III. The acceptable range of recovery is 80 - 120% of the test concentration (ICH, 2005) and the criteria of repeatability and intermediate precision was not more than 15% RSD (U.S. Food and Drug Administration, 2001). Thus, the results indicated that this RP-HPLC analysis was accurate and precise for the quantification of the three compounds in plant samples.

TABLE III
Accuracy and precision of chlorogenic acid, rosmarinic acid and caffeic acid in L. japonica flowering bud

Compounds	Spike concentration (µg/mL)	% recovery (n = 3)	%RSD
			Repeatability precision (n = 3)	Intermediate precision (n = 5)
Chlorogenic acid	10	108.632	0.130	0.989
	25	103.976	0.077	0.699
	50	107.396	0.054	1.770
Rosmarinic acid	10	97.230	0.259	1.522
	25	99.089	0.234	1.039
	50	98.328	0.135	1.415
Caffeic acid	10	100.447	0.169	6.468
	25	99.407	0.046	5.795
	50	100.851	0.074	3.119

LOD and LOQ analysis were calculated by the residual standard deviation of a regression line and the slope of the calibration curve. The LOD of chlorogenic acid, rosmarinic acid, and caffeic acid that is taken as the lowest concentration of analyte in a sample that could be detected was 1.64, 2.22, and 0.65 µg/mL, respectively. The LOQ of chlorogenic acid, rosmarinic acid, and caffeic acid that is taken as the lowest concentration of analyte in a sample that could be accurately quantitated was 4.97, 6.72, and 1.97 µg/mL, respectively.

The robustness of sample and standard compounds was determined during the analysis of the RP-HPLC method when the flow rate of the mobile phase varied from 1.195-1.205 mL/min, the column temperature varied from 29-31 ºC, and the wavelength varied from 322-328 nm. The results demonstrated no differences (%RSD <4) in the area of the curve and retention time as shown in Tables IV and V. However, the method validation in this study used L. japonica flowering bud ethanolic extract as a sample matrix which might not represent all of the plant samples. It was recommended that further quantification of chlorogenic acid, rosmarinic acid, and caffeic acid in each plant material extract as stated in this study should be verified for each sample matrix.

TABLE IV
Robustness of chlorogenic acid, rosmarinic acid and caffeic acid quantitation in L. japonica flowering bud

Compounds	% RSD of sample
	Flow rate			Temperature			Wavelength
	Rt	Area		Rt	Area		Rt	Area
Chlorogenic acid	0.31	0.50		0.79	0.78		0.06	1.14
Rosmarinic acid	0.19	0.66		2.63	0.89		0.02	1.33
Caffeic acid	0.27	0.95		1.11	3.14		0.07	2.08

• * Rt = Retention time

TABLE V
Robustness of standard chlorogenic acid, rosmarinic acid and caffeic acid quantitation

Compounds	% RSD of standard compounds
	Flow rate			Temperature			Wavelength
	Rt	Area		Rt	Area		Rt	Area
Chlorogenic acid	0.24	0.33		0.78	0.14		0.14	1.13
Rosmarinic acid	0.11	1.38		2.57	1.10		0.03	2.41
Caffeic acid	0.23	0.29		1.20	0.25		0.09	0.87

• * Rt = Retention time

The RP-HPLC analysis in this study demonstrated the contents of 3 phenolic compounds in selected plants that could be useful as a chemical marker for quality control of plant material. The interesting plants with special reference to these markers could be further investigated for their biological activities involving hydroxycinnamic acid derivatives.

ACKNOWLEDGEMENTS

The authors are supported the scholarship from “The 100^th Anniversary Chulalongkorn University Fund for Doctoral Scholarship”. The authors are also thankful to College of Public Health Sciences, Chulalongkorn University and all the staff members for necessary assistance and instrumental support.

REFERENCES

• Ayelign A, Sabally K. Determination of chlorogenic acids (CGA) in coffee beans using HPLC. Am J Res Commun. 2013;1(2):78-91.
• Bravo L. Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutr Rev. 1998;56(11):317-333.
• Chaowuttikul C, Palanuvej C, Ruangrungsi N. Pharmacognostic specification, chlorogenic acid content, and in vitro antioxidant activities of Lonicera japonica flowering bud. Pharmacognosy Res. 2017;9(2):128-132.
• Food and Drug Administration. FDA. Department of Health and Human Services. Center for Drug Evaluation and Research (CDER) . Reviewer guidance: validation of chromatographic methods. FDA, 1994. [cited 2018 July 16]. 30 p. Available from: https://www.fda.gov/downloads/Drugs/Guidances/UCM134409.pdf
  » https://www.fda.gov/downloads/Drugs/Guidances/UCM134409.pdf
• Haghi G, Hatami A. Simultaneous quantification of flavonoids and phenolic acids in plant materials by a newly developed isocratic high-performance liquid chromatography approach. J Agric Food Chem. 2010;58(20):10812-10816.
• Hansen S, Pedersen-Bjergaard S, Rasmussen K. Quantification and quality of analytical data. In: Introduction to pharmaceutical chemical analysis. West Sussex: John Wiley & Sons; 2011. p.309-326.
• Köseoglu Yilmaz P, Kolak U. SPE-HPLC determination of chlorogenic and phenolic acids in coffee. J Chromatogr Sci. 2017;55(7):712-718.
• Lafay S, Gil-Izquierdo A. Bioavailability of phenolic acids. Phytochem Rev.2008;7(2):301-311.
• Ma T, Huang C, Meng X, Zhang Q, Zhang L, Lv X, et al. Fingerprint analysis of Hawk-tea by high-performance liquid chromatography. Food Chem. 2011;129(2):551-556.
• Magnani C, Isaac VLB, Correa MA, Salgado HRN. Caffeic acid: a review of its potential use in medications and cosmetics. Anal Methods. 2014;6(10):3203-3210.
• Manach C, Scalbert A, Morand C, Rémésy C, Jiménez L. Polyphenols: food sources and bioavailability. Am J Clin Nutr. 2004;79(5):727-747.
• Petersen M, Simmonds MS. Rosmarinic acid. Phytochemistry. 2003;62(2):121-125.
• Shan Q, Cao G, Cai H, Cai B. Simultaneous determination of four bioactive compounds in Glechoma longituba extracts by high performance liquid chromatography. Pharmacogn Mag. 2013;9(35):216-219.
• Shekarchi M, Hajimehdipoor H, Saeidnia S, Gohari AR, Hamedani MP. Comparative study of rosmarinic acid content in some plants of Labiatae family. Pharmacogn Mag. 2012;8(29):37-41.
• Teixeira J, Gaspar A, Garrido EM, Garrido J, Borges F. Hydroxycinnamic acid antioxidants: an electrochemical overview. Biomed Res Int.2013;2013:251754.
• The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. ICH. ICH Harmonised Tripartite Guideline, Validation of Analytical Procedures: Text and Methodology Q2(R1). Geneva: The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use; 2005. 17 p.
• Upadhyay R, Mohan Rao LJ. An outlook on chlorogenic acids-occurrence, chemistry, technology, and biological activities. Crit Rev Food Sci Nutr. 2013;53(9):968-984.
• U.S. Department of Health and Human Services. Guidance for Industry: Bioanalytical Method Validation. Maryland: U.S. Food and Drug Administration; 2001. 25 p.
• Zeng JG, Tan ML, Peng X, Luo Q. Standardization and quality control of herbal extracts and products. In: Liu WJH, editor. Traditional herbal medicine research methods: identification, analysis, bioassay, and pharmaceutical and clinical studies. New Jersey: John Wiley & Sons; 2011. p.377-427.

Publication Dates

• Publication in this collection
  16 Mar 2020
• Date of issue
  2020

History

• Received
  11 July 2017
• Accepted
  10 Oct 2018