Chlorogenic acid content, essential oil compositions, and in vitro antioxidant activities of Chromolaena odorata leaves

Abstract

Chromolaena odorata (L.) R. M. King and H. Rob. is a Thai medicinal plant used for the treatment of wounds, rashes, diabetes, and insect repellent. The leaves of C. odorata were collected from 10 different sources throughout Thailand. The chemical constituents of essential oils were hydro-distilled from the leaves and were analyzed by gas chromatography-mass spectrometry. Chlorogenic acid contents were determined by thin-layer chromatography (TLC) - densitometry with winCATS software and TLC image analysis with ImageJ software. The TLC plate was developed in the mobile phase that consisted of ethyl acetate:water:formic acid (17:3:2). Antioxidant activities were examined by 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging and β-carotene bleaching assays. C. odorata essential oil has shown the major components of pregeijerene, dauca-5, 8-diene, (E)-caryophyllene, β-pinene, and α-pinene. The chlorogenic acid content of C. odorata leaves was determined by TLC-densitometry and TLC image analysis. Results have shown that TLC-densitometry and TLC image analysis method were not statistically significantly different. DPPH radical scavenging and β-carotene bleaching assays of ethanolic extract of C. odorata leaves showed its antioxidant potential.

INTRODUCTION

Chromolaena odorata (L.) R. M. King and H. Rob. (syn. Eupatorium odoratum L.) was known in common name as Siam Weed, Christmas Bush, or Common Floss Flower which is a species in the family Asteraceae. This plant is widely distributed in Asia, Africa, and the Pacific. It may reach 1 m or more as a standing shrub. The three-nerved leaves are deltoid to ovate-lanceolate with a dentate margin. The leaves are aromatic when crushed. The inflorescences are corymbs of white, lavender, or pink.[1,2] In herbal medicine, leaf extracts with salt are used as a gargle for sore throats and colds. It is also used to scent aromatic baths.[3] Extracts of C. odorata have been shown to inhibit Neisseria gonorrhoeae that causes gonorrhea in vitro[4] and to accelerate blood clotting.[5] In Thailand, the plant is used for the treatment of wounds, rashes, diabetes, and insect repellent. From the literature, it was found that the leaf extracts of C. odorata are more beneficial than the other parts. The chemical constituents of which were isolated from this plant may be responsible for its pharmacological activities. C. odorata has been shown its pharmacological activities such as anti-inflammatory, antimicrobial, blood coagulating, insecticidal and antioxidant activities.[6] Polyphenolic extract exhibited a slight antioxidant effect.[7] Chlorogenic acid is a natural chemical compound which is known as an antioxidant. These polyphenols were found in a wide distribution of plants. Structurally, chlorogenic acid is a combination of a caffeic acid moiety bound to quinic acid [Figure 1].[8] Thereby, the aims of this study are to analyze the chemical constituents of essential oil by gas chromatography-mass spectrometry (GC-MS) and to investigate the content of chlorogenic acid. ImageJ free software was used to compare with thin-layer chromatography (TLC) densitometry while antioxidant activities testing by 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging assay and β-carotene bleaching assay for evidence-based efficacy of this crude drug.

Figure 1.

Structure of chlorogenic acid

MATERIALS AND METHODS

Crude extract preparation

The leaves of C. odorata were collected from 10 different sources throughout Thailand and authenticated by Assoc. Prof. Dr. Nijsiri Ruangrungsi, Chulalongkorn University. Voucher specimens were deposited at the College of Public Health Sciences, Chulalongkorn University, Thailand. Each authentic sample was dried in hot air oven at 45°C and ground to powders.

Preparation of standard solutions

The standard chlorogenic acid was purchased from Sigma-Aldrich, USA. The stock solution of chlorogenic acid was prepared in 95% ethanol and diluted to obtain the series of standard solution (0.05, 0.15, 0.25, 0.35, and 0.45 mg/ml). These solutions were stored in a refrigerator at 4°C.

Preparation of ethanol extracts of Chromolaena odorata

The powders of C. odorata (5.0 g) were extracted with ethanol by Soxhlet apparatus. The extract was filtered and evaporated in vacuo to dryness. The yield was recorded. The extract was appropriately dissolved in 95% ethanol to obtain the concentration of 20 mg/ml.

Thin-layer chromatography-densitometry

Three microliters of 10 ethanol extracts and standard solutions were applied on the TLC plate (Silica gel 60 GF254) by Linomat 5 applicator (Camag, Switzerland). The plate was developed in a TLC chamber that consisted of ethyl acetate:water:formic acid (17:3:2) as mobile phase. The plate was scanned under wavelength at 330 nm using TLC scanner 3 (Camag, Switzerland) with winCATS software (Camag, Switzerland). Chlorogenic acid was quantitated by peak area. The test was done in triplicate.

Thin-layer chromatography image analysis by ImageJ software

TLC plate was photographed under ultraviolet (UV) light at 254 nm by a digital camera and saved as tiff files. Chromatographic peak and peak area was obtained using ImageJ free software (Department of Health and Human Services, National Institutes of Health (NIH) in the United State). The test was done in triplicate.

Method validation

TLC quantitation of chlorogenic acid in C. odorata leaves was validated. Calibration range, accuracy, repeatability, intermediate precision, limit of detection (LOD), limit of quantitation (LOQ), and robustness were performed according to the International Conference on Harmonisation guideline.[9]

Data analysis

The chlorogenic acid contents between TLC image analysis and TLC-densitometry were compared by paired t-test statistical analysis.

Extraction of essential oils

The fresh leaves of C. odorata were cut into small pieces and coarsely crushed. The essential oil was extracted by hydro-distillation with Clevenger apparatus.[10]

Gas chromatography/mass spectrometry analysis

The analysis was performed using a Finnigan trace GC ultra with DSQ Quadrupole detector. BPX5 fuse silica column (30 m × 0.25 mm, 0.25 μm film thicknesses) was used as stationary phase. The oven temperature started from 60°C to 240°C with a constant rate of 3°C/min. The carrier gas was helium with the flow rate of 1 ml/min. One microliter of the oil (1:100 in high-performance liquid chromatography grade methanol) was injected by Finnigan Autoinjector AI3000 with a split ratio of 100:1. MS was performed by EI positive mode at 70 electron volts. The chemical constituents were identified by matching mass spectra and retention time indices with Adams Essential Oils Mass Spectral library and NIST 05 Mass Spectral library. Peak area was shown in percentage.

1,1-diphenyl-2-picryl hydrazyl radical scavenging assay

One hundred microliters of various concentrations of the extract, standard chlorogenic acid, and positive controls (butylated hydroxytoluene [BHT] and quercetin) in ethanol were added to 100 μl of DPPH (120 μm in ethanol) in 96 well microplate.[11] The plate was incubated at room temperature for 30 min in the dark. The absorbance was measured at 517 nm. Each sample was done in triplicate. Percent scavenging activity was calculated:

% Inhibition = ([Absorbance control − Absorbance sample]/Absorbance control) × 100.

β-carotene bleaching assay

This assay was performed in 96 well plate. One milliliter of β-carotene solution (0.2 mg/ml in chloroform) was added with 20 μl of linoleic acid and 200 μl of Tween 20 in 96 well microplate.[12] Chloroform was removed at 40°C under vacuum. Ultra-pure water (50 ml) was added and shaken to form an emulsion. Aliquots (200 μl) of the emulsion were transferred into the 96 well plates containing 10 μl of the various concentrations of extract, standard chlorogenic acid, or positive controls (BHT and quercetin) and heated at 50°C. Absorbance at wavelength of 470 nm was recorded at 30 min intervals for 120 min. Each sample was performed in triplicate. The antioxidant activity was evaluated.

% Antioxidant activity = (1−[A₀−A₁₂₀]/[C₀−C₁₂₀]) × 100

Where A₀, A₁₂₀ : The absorbance values measured at 0 time and end time of sample

C₀, C₁₂₀ : The absorbance values measured at 0 time and end time of control.

RESULTS

Quantitative analysis of chlorogenic acid

The yield of ethanolic extract of C. odorata leaves was 27.46 ± 2.21 g/100 g by dry weight. The quantitative analysis of chlorogenic acid in the leaf extracts was performed by TLC-densitometry and TLC image analysis using ethylacetate:water:formic acid (17:3:2) as mobile phase. TLC chromatogram under UV 254 is shown in Figure 2. TLC densitogram scanned in the range of 200–700 nm is shown in Figure 3. The chlorogenic acid content of C. odorata leaves determined by TLC-densitometry and TLC image analysis were 7.39 ± 0.40 and 7.31 ± 0.77 g/100 g of dry leaves, respectively. The chlorogenic acid contents by two methods were not significantly different (P > 0.05) using paired t-test.

Figure 2.

The thin-layer chromatography plate under ultraviolet 254 nm; standard chlorogenic acid (track 1–5), and Chromolaena odorata leaves extracts from 10 different locations

Figure 3.

The absorption spectra of chlorogenic acid in standard and sample bands

Method validation

The method validation consisted of the specificity, accuracy, precision, LOD, LOQ, and robustness. The specificity was confirmed by comparing UV spectrum of the peak in standard chlorogenic acid and all samples. The result showed the maximum absorbance at a wavelength of 330 nm [Figure 2]. The validity of TLC densitometry and TLC image analysis is presented in Table 1. The polynomial calibration curves ranged from 0.15 to 1.35 μg/spot [Figures 4 and 5]. The recovery was determined to evaluate the accuracy by spiking known three concentrations of chlorogenic acid in a sample. The recovery values of both methods were between 84.69% and 103.99%. The repeatability and the intermediate precision were determined in the same day and in three different days. The repeatability and the intermediate precision of both methods were <10% relative standard deviation (RSD). The LOD and LOQ of TLC-densitometry and TLC image analysis were calculated by the residual standard deviation of a regression line and were found to be 0.002 and 0.007 μg/spot, respectively. The robustness studied by changing the mobile phase ratio showed the values of 0.99% RSD for TLC-densitometry and 2.62% RSD for TLC image analysis.

Table 1.

Method validity of thin-layer chromatography-densitometry and thin-layer chromatography image analysis of chlorogenic acid content in Chromolaena odorata leaves

Figure 4.

The calibration curve of chlorogenic acid in Chromolaena odorata by thin-layer chromatography-densitometry

Figure 5.

The calibration curve of chlorogenic acid in Chromolaena odorata by thin-layer chromatography image analysis

Gas chromatography mass spectrometry analysis

The essential oils of C. odorata leaves were analyzed by GC/MS and at least 20 compounds were detected as shown in Table 2. Pregeijerene, dauca-5, 8-diene, α-pinene, (E)-caryophyllene, and β-pinene were found as major components of the essential oil. Their quantities were 40.60%, 16.75%, 9.67%, 6.11%, and 5.37%, respectively.

Table 2.

The chemical constituents of Chromolaena odorata essential oil

1,1-diphenyl-2-picryl hydrazyl radical scavenging assay

The results demonstrated that the ethanolic extract of C. odorata, chlorogenic acid, BHT, and quercetin showed the radical scavenging activity with IC₅₀ of 72.23, 10.59, 32.55, and 3.82 μg/ml, respectively [Figure 6].

Figure 6.

1,1-diphenyl-2-picryl hydrazyl scavenging activities of ethanolic extract of Chromolaena odorata leaves, chlorogenic acid, butylated hydroxytoluene, and quercetin

β-Carotene bleaching assay

C. odorata ethanolic extract at 1 mg/ml showed 35.61% antioxidant activity compared to 90.88 and 87.18% of BHT and quercetin at the same concentration, respectively. The antioxidant activities of these extract and positive controls demonstrated the dose-response relationship. On the contrary, chlorogenic acid expressed a reciprocal relationship with 20.8–0.3% antioxidant activities at the concentrations of 0.125–1 mg/ml [Figure 7].

Figure 7.

The antioxidant activity of Chromolaena odorata, butylated hydroxyloluene, quercetin, and chlorogenic acid by β-carotene bleaching assay

DISCUSSION

TLC-densitometry is a quantitative TLC method with high reliability due to selective optical characteristics of tested compound. Wavelength specific light absorption or light emission and its intensity are related to the amount of the compound. TLC-densitometric analysis of chlorogenic acid in C. odorata leaves was developed and found to be valid. Furthermore, the quantitation of TLC chromatogram can be performed using image analysis which is inexpensive and convenient technique. The image analysis is processed by acquiring a digital image of TLC chromatogram using simple digital camera computing the pixel intensity, converting to corresponding peak, and calculating peak area using ImageJ free software.[13] The quantitative analysis of chlorogenic acid in C. odorata leaves by TLC image analysis was also developed and its validity was demonstrated. The contents of chlorogenic acid in C. odorata leaves by two methods were not statistically significant different. Therefore, TLC image analysis could be used as an alternative method instead of TLC-densitometry.

For GC/MS analysis, the chemical constituents of C. odorata essential oil in this study were in accordance with the previous study in Thailand which reported the constituents of the essential oil from aerial parts as pregeijerene, α-pinene, germacrene D, β-caryophyllene, and β-pinene.[14] Likewise, the previous study in Nigeria showed that the main constituents in the stem essential oil were α-pinene, β-pinene, caryophyllene, bicyclo (7.2.0) undec-4-ene, and germacrene D.[15] However, the chemical compositions of samples from different parts and localities expressed the individual amount of components.[16]

DPPH is a stable free radical that is used to evaluate the ability of compounds to act as free radical scavengers or hydrogen donors that cause decoloration of DPPH and to estimate the antioxidant activity.[17] In this study, the ethanolic extract of C. odorata could scavenge DPPH free radical with IC₅₀ of 72.23 μg/ml. The scavenging activity of C. odorata was less potent than quercetin, chlorogenic acid, and BHT with IC₅₀ of 3.82, 10.59, and 32.55 μg/ml, respectively. The previous study also reported DPPH radical scavenging activity of C. odorata and showed nearly IC₅₀ value.[18]

In β-carotene bleaching assay, linoleic acid produces hydrogen peroxide. The bleaching of yellow color of β-carotene is due to peroxide free radicals. The rate of β-carotene bleaching can be delayed with the presence of antioxidants. In this study, the ethanolic extract of C. odorata at a concentration of 1 mg/ml showed antioxidant activity of only 35.61% compared to BHT and quercetin, which showed the antioxidant activity of 90.88% and 87.18%, respectively. The bleaching inhibitory activities of C. odorata, BHT, and quercetin were concentration-dependent, whereas chlorogenic acid expressed reciprocal relationship. In the previous study, phenolic acids presented in foods were reported to act as pro-oxidants such as caffeic, chlorogenic, and ferulic acids.[19,20] Pro-oxidants are chemicals that induce oxidative stress, either by generating reactive oxygen species or inhibiting antioxidant systems.[21]

CONCLUSION

The present study indicated that pregeijerene, dauca-5,8-diene, (E)-caryophyllene, β-pinene, and α-pinene were found as major components of the essential oil from C. odorata leaves in Thailand. The chlorogenic acid content of C. odorata leaves was established and TLC image analysis could be used for chlorogenic acid quantitation. Furthermore, the ethanolic extract of C. odorata leaves showed less potent of both free radical scavenging activity and inhibitory activity of β-carotene bleaching compared to the standard BHT.

Financial support and sponsorship

Tuition fees Scholarship for Master to Doctoral of Graduate School, Chulalongkorn University.

Conflicts of interest

There are no conflicts of interest.