Pharmacognostic Specification, Chlorogenic Acid Content, and In vitro Antioxidant Activities of Lonicera japonica Flowering Bud.

BACKGROUND: Lonicera japonica Thunb. or Japanese Honeysuckle has been widely used in traditional medicine for antipyretic.
OBJECTIVE: To establish the pharmacognostic specification of L. japonica flowering bud in Thailand and to determine its chlorogenic acid content and in vitro antioxidant activities.
MATERIALS AND METHODS: Dried L. japonica flowering bud from 15 various herbal drugstores throughout Thailand were investigated for pharmacognostic specification. Their chlorogenic acid contents were quantitatively analyzed by thin layer chromatography (TLC) densitometry with winCATS software. The mobile phase for TLC development consisted of ethyl acetate: formic acid: acetic acid: water (10:1.1:1.1:2.6). Antioxidant activities were investigated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferric ion reducing antioxidant power assay, nitric oxide scavenging assay, and β-carotene bleaching assays.
RESULTS: Qualified L. japonica flowering bud in Thailand was presented that the contents of loss on drying, total ash, acid-insoluble ash, and water should not be >10.11%, 6.59%, 1.14%, and 10.82% by weight, respectively. The ethanol and water soluble extractive values should not be < 16.46% and 28.88% by weight, respectively. Chlorogenic acid content in L. japonica flowering bud was found to be 2.24 ± 0.50 g/100 g of crude drug. L. japonica flowering bud showed DPPH and nitric oxide scavenging activities as well as reducing power property.
CONCLUSION: This pharmacognostic specification with special reference to the chlorogenic acid content can be used for quality control of L. japonica flowering bud in Thailand. The potential antioxidant of this crude drug was demonstrated in vitro.
SUMMARY: Pharmacognostic specification of Lonicera japonica flowering bud in Thailand has been establishedThe chlorogenic acid content has been quantified by thin layer chromatography-densitometryThe ethanolic extract of L. japonica flowering bud showed antioxidation potential, especially on reducing power property. Abbreviations Used: TLC: Thin layer chromatography, DPPH: 2,2-diphenyl-1-picrylhydrazyl, FRAP: Ferric ion Reducing Antioxidant Power, WHO: World Health Organization, ICH: International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use; LOD: Limit of detection; LOQ: Limit of quantitation; BHT: Butylated hydroxytoluene; FeSO4: Iron(II) sulfate; DMSO: Dimethyl sulfoxide; TPTZ: 2,4,6-tripyridyl-s-triazine.

INTRODUCTION

Lonicera japonica Thunb. (Caprifoliaceae), commonly known in English as “Japanese Honeysuckle” and called “Sai Nam Phueng” in Thai, is native to the eastern Asia and become naturalized in Argentina, Brazil, Mexico, Australia, New Zealand, and the United States.[12] The pharmacological studies of L. japonica flowering bud have shown a wide biological activity such as antibacterial,[34] α-glucosidase inhibitory,[5] antiviral,[6] anti-inflammatory,[78] antinociceptive,[9] antiangiogenic,[9] antioxidant,[1011] hepatoprotective,[12] antifibrotic,[13] and neuroprotective activities.[14] In Thailand, the plant is widely used for antipyretic effect.[2] The chemical constituents have been widely researched. The main compositions such as organic acids, flavones, saponins, iridoids, essential oil, and inorganic elements were isolated and identified. In Chinese Pharmacopoeia, the indicator compound of L. japonica is chlorogenic acid, which has been used as characteristics for the quality of this plant.[1]

Chlorogenic acid [Figure 1] is a kind of polyphenol derivative widely found in plants, fruits, and vegetables. Structurally, it is an ester form of caffeic acid and quinic acid.[15] Chlorogenic acid has been shown its biological and physiological activities such as antihyperalgesic,[16] radioprotective,[17] anti-inflammatory,[18] antiulcerogenic,[19] antihepatotoxic,[20] neuroprotective effects,[21] and antioxidant activity of chlorogenic acid-rich extract from Etlingera elatior leaves.[22]

Figure 1

The structure of chlorogenic acid

The standardization is important for quality, purity, and authentication of plant crude drug.[23] Therefore, the aims of this study were to determine the pharmacognostic specification of L. japonica flowering bud in Thailand, to investigate the content of chlorogenic acid using thin layer chromatography (TLC)–densitometry, and to examine its antioxidant activities by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferric ion reducing antioxidant power (FRAP) assay, nitric oxide scavenging assay, and β-carotene bleaching assay for evidence-based efficacy of this crude drug.

MATERIALS AND METHODS

Plant material and chemicals

Dried L. japonica flowering bud was purchased from 15 various herbal drugstores throughout Thailand during July-September 2014 and authenticated by one of the authors, Ruangrungsi N. Voucher specimens were deposited at College of Public Health Sciences, Chulalongkorn University. Crude drugs were grounded into coarse powder before use. The standard chlorogenic acid (CAS no. 327-97-9) was purchased from Sigma-Aldrich, USA. The chemicals used were of analytical grade.

Morphological identification

The macroscopic study of crude drugs was observed based on the shape, size, color, texture, odor, and taste. The flowering bud powder and cross-section of the corolla were observed under microscope (Carl Zeiss, Germany) to identify tissue and cell structures for microscopic characteristics. Pictures were illustrated by hand drawing in proportional scale related to the original size.

Physicochemical characteristics

Determination of loss on drying, total ash, acid-insoluble ash, ethanol and water extractive values, water content, and volatile oil content were evaluated to establish the pharmacognostic specification of L. japonica flowering bud.[2324] All samples were analyzed in triplicate. Grand mean and pooled standard deviation were calculated.

Quantitative analysis of chlorogenic acid by thin layer chromatography–densitometry

The powders of flowering bud were extracted with 95% ethanol by Soxhlet apparatus till exhaustion. The extract was filtered, evaporated to dryness, and calculated the percent yield of dried crude drugs. Ten milligrams of the extract was dissolved with 1 ml of 95% ethanol for TLC analysis.

Three microliters of various standard concentrations and 15 ethanolic extracts were spotted on the silica gel 60 GF254 TLC plate (20 cm × 10 cm) and developed in a TLC chamber containing ethyl acetate:formic acid:acetic acid:water (10:1.1:1.1:2.6) as mobile phase. The developed TLC plate was scanned by TLC scanner 4 (Camag, Switzerland) at a wavelength of 325 nm and expressed as chromatographic peak by winCATS software. The test was done in triplicate.

Method validation

According to the International Conference on Harmonisation guideline,[25] calibration range, specificity, accuracy, repeatability, intermediate precision, limit of detection (LOD), limit of quantitation (LOQ), and robustness of chlorogenic acid quantitative analysis in flowering bud were validated.

Antioxidant activities

2, 2-diphenyl-1-picrylhydrazyl radical scavenging assay

One hundred microliters of various ethanolic extract concentrations, standard chlorogenic acid, and positive controls (quercetin and butylated hydroxytoluene [BHT]) dissolved in methanol were added to 100 μl of 120 μM DPPH methanolic solution in 96-well microplate.[26] The plate was incubated for 30 min in the dark at room temperature. The absorbance at 517 nm was measured. Each sample was done in triplicate. Percent scavenging activity was calculated:

Ferric ion reducing antioxidant power assay

Twenty-five microliters of ethanolic extract and standard chlorogenic acid (1 mg/ml) were mixed with 175 μl of FRAP reagent in 96-well microplate and incubated for 30 min in the dark at room temperature. The absorbance at 593 nm was measured using microplate reader[27] Quercetin and BHT was used as a positive control. The FRAP value of samples was calculated using the linear relationship from the calibration curve of FeSO4 methanolic solutions in the range of 0.5–1.5 mM. Each sample was done in triplicate. The samples and positive controls were expressed as mM of ferrous iron (Fe(II)/mg crude extract).

Nitric oxide scavenging assay

The mixture containing 50 μl of ethanolic extract, standard chlorogenic acid, or positive controls (quercetin) at concentration 800 μg/ml dissolved in dimethyl sulfoxide and 50 μl of sodium nitroprusside (10 mM) in phosphate-buffered saline was incubated at 25°C for 150 min. Then, the mixture reacted with 50 μl of Griess reagent (0.33% sulphanilamide in 20% glacial acetic acid) and incubated for 10 min. After incubation, 50 μl of 0.1% naphthyl ethylenediamine dihydrochloride was added and allowed to stand for 30 min.[2829] The absorbance at 540 nm was measured. Each sample was done in triplicate. Percent scavenging activity was calculated as aforementioned.

β-carotene bleaching assay

One milliliter of β-carotene solution (2 mg/ml in chloroform) was mixed with 40 μl of linoleic acid (10 mg/ml) and 400 μl of Tween 20. The chloroform was removed at 40°C under vacuum. The mixture was diluted with 50 ml of ultrapure water and shaken to form an emulsion. Two hundred microliters of the emulsion was transferred into the 96-well microplate which contained 10 μl of the various concentrations of ethanolic extract, standard chlorogenic acid, or positive controls (quercetin and BHT) and heated at 50°C.[3031] The absorbance at 470 nm was recorded at 30 min intervals for 120 min. Each sample was done in triplicate. The antioxidant activity was calculated:

Where A0, A120: The absorbance values measured at zero time and end time of incubation for sample, C0, C120: The absorbance values measured at zero time and end time of incubation for control.

RESULTS AND DISCUSSION

Pharmacognostic specification

Macroscopic and microscopic characteristics are the first step to establish the identity of materials.[23] A dried flowering bud was yellowish-green color, clavate shape, and 2–3 cm in length with velvet surface [Figure 2a]. Histological and anatomical characters including corolla, glandular and nonglandular trichomes, pollen grain, spiral, parenchyma, and calcium oxalate were illustrated in Figure 2b and c.

Figure 2

(a) Dried Lonicera japonica flowering bud crude drug, (b) anatomical characteristics of Lonicera japonica flowering bud (corolla part); (1) Glandular trichome, (2) Unicellular trichome, (3) Epidermis, and (4) Vascular bundle, (c) histological characteristics of the powder of Lonicera japonica flowering bud; (5) Corolla fragment, (6) Glandular and nonglandular trichome, (7) Pollen grain, (8) Spiral vessel, (9) Parenchyma and vascular bundle, (10) Prism of calcium oxalate, and (11) Petal parenchyma

The physicochemical characteristics are an essential for qualification of crude drug. The pharmacognostic specification of L. japonica flowering bud was demonstrated in Table 1. The ethanol and water soluble extractive values should not be <16.46% and 28.88% by weight, respectively, while the loss on drying, total ash, acid-insoluble ash, and water content should not be >10.11%, 6.59%, 1.14%, and 10.82% by dry weight, respectively. The total ash, acid-insoluble ash, and water content were less than previously reported (10.6%, 1.2%, and 17.5%, respectively).[32] In Chinese Pharmacopoeia, the total ash, acid-insoluble ash, and water content were not >10.0%, 3.0%, and 12.0%, respectively.[33] The volatile oil was undetected in this study because the samples were dried crude drug, and the previous study showed the lowest volatile oil content at flowering bud stage.[34]

Table 1

Physicochemical characteristics of Lonicera japonica flowering bud

The yield of ethanolic extract of L. japonica flowering bud was 39.44 ± 5.83 g/100 g crude drug. Chlorogenic acid content in the ethanolic extract was found to be 0.06 ± 0.01 g/g and in the crude drug was found to be 2.24 ± 0.50 g/100 g which was less than previously reported as 2.62%–3.66%.[35] However, according to Chinese Pharmacopoeia, the content of chlorogenic acid in L. japonica flowering bud should not be <1.5%.[33]

The calibration range of chlorogenic acid was polynomial ranged from 0.6 to 3.0 μg/spot [Figure 3]. The specificity was shown by comparing ultraviolet (UV) spectrum of the peak apex among all samples and standard chlorogenic acid for peak identity, and the comparison of UV spectrum recorded at up-slope, apex, and down-slope of the peak for peak purity. The spectra showed the maximum absorbance at the wavelength of 325 nm [Figure 4]. The validity of chlorogenic acid quantification in L. japonica flower buds was displayed in Table 2. The recovery was determined to evaluate the accuracy of the method by spiking known three concentrations of standard chlorogenic acid in a sample extract. The recovery value was 94.10% ± 4.09%. The repeatability and the intermediate precision were determined on the same day and in three different days. The repeatability and the intermediate precision were 1.17%RSD and 3.73%RSD, respectively. The LOD and LOQ of TLC–densitometry were calculated by the residual standard deviation of a regression line and was found to be 0.07 and 0.21 μg/spot, respectively. The robustness performed by varying the mobile phase ratio showed the values of 8.59%RSD. The results were close to the previous study reported that the accuracy, repeatability, intermediate precision, LOD, and LOQ were 99.66%–101.59% recovery, 1.01–1.32%RSD, 3.21%RSD, 0.12 μg/spot, and 0.20 μg/spot.[35] Thus, this method was suitable and reliable to evaluate the quantitative analysis.

Figure 3

The calibration curve of chlorogenic acid by thin layer chromatography–densitometry

Figure 4

(a) The ultraviolet absorbance spectra of chlorogenic acid in samples and standard chlorogenic acid bands, (b) peak purity determination using up-slope, apex, and down-slope of the peak

Table 2

The validity of chlorogenic acid in Lonicera japonica flower buds

The antioxidant activities of L. japonica flowering bud ethanolic extract and standard chlorogenic acid were summarized in Table 3.

Table 3

The antioxidant activities of Lonicera japonica flower buds extract, standard chlorogenic acid, and positive control

The character of DPPH is a stable free radical for evaluate the ability of substances that can donate a hydrogen atom or free radical scavengers, caused by the delocalization of DPPH, and to estimate the antioxidant activity.[36] The result of DPPH radical scavenging activity demonstrated that L. japonica flowering bud extract showed IC50 as 54.78 μg/ml. The scavenging activity of L. japonica flowering bud was less potent than chlorogenic acid, quercetin, and BHT with IC50 of 7.83, 4.84, and 24.82 μg/ml, respectively. The previous study also presented higher potential DPPH radical scavenging activity of dried L. japonica flowering bud as 19.45 μg/ml.[37]

The FRAP assay is antioxidant method to measure reducing the power of ferric iron and 2,4,6-tripyridyl-s-triazine to a colored product in plasma and botanicals.[38] In this study, L. japonica flowering bud extract showed the FRAP value as 1.24 ± 0.02 mM FeSO4/mg crude extract which similarly to chlorogenic acid as 1.17 ± 0.02 mM FeSO4/mg substance. Quercetin and BHT showed FRAP value of 1.48 ± 0.06 and 1.51 ± 0.01 mM FeSO4/mg substance, respectively.

Nitric oxide scavenging assay evaluates the ability of the substance to scavenge nitric oxide. The nitric oxide (NO •) reacts with oxygen, under aerobic conditions, to produce stable products (nitrate and nitrite), Griess reagent was applied to determine the quantity of nitric oxide scavenging assay.[39] The result showed percent nitric oxide inhibition of L. japonica flowering bud as 49.86% compared to chlorogenic acid and quercetin which showed percent inhibition of 75.97% and 72.27%, respectively.

β-carotene bleaching assay measures the ability of an antioxidant to inhibit lipid peroxidation which produced by linoleic acid. The discoloration of yellow color of β-carotene is due to peroxide free radicals. The activity of β-carotene bleaching can be decrease with the presence of antioxidants.[39] In this study, L. japonica flowering bud at a concentration of 1 mg/ml showed antioxidant activity of 2.71% compared to 1 mg/ml of quercetin and BHT which showed the antioxidant activity of 78.12% and 91.81%, respectively. The antioxidant activities of these extracts, chlorogenic acid, and positive controls demonstrated the dose-response relationship [Figure 5].

Figure 5

The antioxidant activity of Lonicera japonica flowering bud, standard chlorogenic acid, quercetin, and butylated hydroxytoluene by α-carotene bleaching assay

CONCLUSION

This study established the scientific information for authentication and quality control of L. japonica flowering bud in Thailand including the pharmacognostic specification as well as the chlorogenic acid content by TLC–densitometry. In addition, the ethanolic extract of L. japonica flowering bud showed antioxidation potential, especially on reducing power property.

Financial support and sponsorship

This study was financially supported by The 100th Anniversary Chulalongkorn University Fund for Doctoral Scholarship.

Conflicts of interest

There are no conflicts of interest.