Phenolic compounds from Foeniculum vulgare (Subsp. Piperitum) (Apiaceae) herb and evaluation of hepatoprotective antioxidant activity
Abstract
Objective:
The study was designed to evaluate the antioxidant and hepatoprotective activities of the 80% methanolic extract as well as the ethyl acetate (EtOAc) and butanol (BuOH) fractions of the wild fennel (Foeniculum vulgare (Subsp; Piperitum)) and cultivated fennel (F. vulgare var. azoricum). In addition, quantification of the total phenolic content in the 80% methanol extract of fennel wild and cultivated herbs is measured.
Materials and Methods:
An amount of 400 g of air dried powdered herb of wild and cultivated fennel were sonicated with aqueous methanol (80%), successively extracted with Hexane, EtOAc, and n-BuOH. The EtOAc and n-BuOH were subjected to repeated column chromatography on silica gel and Sephadex LH-20. The antioxidant effect was determined in vitro using 1,1-diphenyl-2-picrylhydrazyl (DPPH). Hepatoprotective activity was carried out using a Wistar male rat (250–300 g). Total phenolic and flavonoid contents were determined as chlorogenic acid and rutin equivalents, respectively.
Results:
Two phenolic compounds, i.e., 3,4-dihydroxy-phenethylalchohol-6-O-caffeoyl-β-D-glucopyranoside and 3΄,8΄-binaringenin were isolated from the fennel wild herb, their structures were elucidated by spectral methods including 1D NMR, 2D NMR, and UV. The EtOAc and BuOH fractions of wild fennel were found to exhibit a radical scavenging activity higher than those of cultivated fennel. An in vitro method of rat hepatocytes monolayer culture was used for the investigation of hepatotoxic effects of the 80% methanol extract on the wild and cultivated fennel, which were >1000 and 1000 μg/mL, respectively. As well as, their hepatoprotective effect against the toxic effect of paracetamol (25 mM) was exerted at 12.5 μg/mL concentration.
Conclusions:
Fennel (F. Vulgare) is a widespread plant species commonly used as a spice and flavoring. The results obtained in this study indicated that the fennel (F. vulgare) herb is a potential source of natural antioxidant. Two phenolic compounds, i.e. 3,4-dihydroxy-phenethylalchohol-6-O-caffeoyl-β-D-glucopyranoside (A) and 3΄,8΄-binaringenin (B) were isolated from the fennel wild herb for the first time.
INTRODUCTION
Fennel (Foeniculum vulgare Miller, Apiaceae) is one of the most widespread plants of the Apiaceae family (formerly the Umbelliferae). It is a perennial herb and a well-known Mediterranean aromatic plant,[12] but nowadays it can be found in several temperate areas of the world, both as wild and cultivated species, commonly used as a spice and favoring and in folk medicine as a carminative, digestive, lactagoge, and diuretic.[3] Fennel has been used in the folk medicine for centuries to cure digestion and respiratory disorders, and its herbal drug preparations are used for dyspeptic complaints such as mild, spasmodic gastrointestinal complaints, bloating, and flatulence.[4] Fennel is also used for catarrh of the upper respiratory tract. Despite being widely studied for its essential oils by gas chromatography-mass spectrometry (GC-MS),[5] little information is available on the nonvolatile constituents of the fennel. Polyphenolic compounds are associated with the prevention of disease assumed to be induced by oxidative stress, such as cardiovascular diseases, cancer, and inflammation. The possible protective effects reported are generally associated with the antioxidant activity of the polyphenolics.[6] The purpose of this study was to evaluate the hepatoprotective and antioxidant activities of the 80% of methanol extracts of the fennel herb and to elucidate their antioxidative actions. In this report, we describe the isolation and structure elucidation of two phenolic compounds: 3,4-dihydroxy-phenethylalchohol-6-O-caffeoyl-β-D-glucopyranoside and 3′,8′-binaringenin which were isolated for the first time from F. vulgare subsp. Piperitum. The structural elucidation has been performed by spectral analysis, including 1D NMR, 2D NMR, and UV techniques.
MATERIALS AND METHODS
Plant material
Wild fennel (F. vulgare subsp. Piperitum) were collected during the flowering period (March 2009) from Marsa-Matroh area, Egypt. The cultivated fennel (F. vulgare var. azoricum)) was collected from the Sekem Company Plantation, Bilbase city, Egypt (March 2009). The plants were kindly authenticated by Prof. Dr. Ibrahim El-Garf, Department of Botany, Faculty of Science, Cairo University. The herb was dried in oven at 40 °C, finely powdered and kept for phytochemicals and biological studies.
Extraction and isolation
First, 400 g of air dried powdered herb of wild and cultivated fennel were sonicated with aqueous methanol (80%) (3 × 1 L). The extracts were combined and evaporated under reduced pressure at 45 °C to yield a dark greenish black residue (about 72.5 g).
The residue was dissolved in 200 mL dist. H2O and was successively extracted with hexane, ethyl acetate (EtOAc) and n-butanol (n-BuOH) (3 × 500 mL for each). Fractions were evaporated under reduced pressure. The fractions were examined by thin-layer chromatography (TLC) using the solvent system: ethyl acetate:formic acid:acetic Acid:H2O (30:1.2:0.8:8). It was found that both EtOAc and n-BuOH contain the same compounds on TLC. Therefore, they were collected together (EB). About 10 g of the EB fraction was subjected to column chromatography using silica gel. The elution was carried out firstly with 100% hexane, followed by methylene dichloride/hexane in the ratio of 1:1, 100% CH2Cl2 and then increasing the polarity by 10% stepwise using MeOH until reaches 100% MeOH. The similar fractions were collected together, concentrated under reduced pressure at temperature 45 °C.
The fractions were monitored by TLC on silica gel plates (Merck) (Darmstadt, Germany) developed with the solvent system: EtOAc–HCOOH–AcOH–H2O (30:1.2:0.8:8). Spots were detected using Neu's spray reagent (1% diphenylboric acidethanolamine complex in methanol)[7] and visualized under UV light at 254 and 365 nm. The fractions eluted with CH2Cl2/MeOH (80:20) and MeOH were collected together and subjected to Sephadex LH-20 CC using H2O, H2O/EtOH, and EtOH as eluent. The fraction eluted with H2O/EtOH and EtOH were collected together and subjected again to Sephadex LH-20 CC using MeOH as eluent to afford compounds A and B.
Determination of total phenolic content
Eighty percent of the methanolic extract was prepared using 5 g of the dried powder herb of both wild and cultivated fennel. The yield of 80% methanolic extract was completed to 100 mL with methanol in a measuring flask. Total phenolic content (TPC) were measured as chlorogenic acid equivalents using (UV/VIS, 2041 spectrophotometer). The Folin–Ciocalteu method was used to determine the TPC according to the method described by Meda et al.[8] From each extract, 1 mL was mixed with 0.5 mL of 0.2 N Folin–Ciocalteu reagent for 5 min and 1 mL of saturated solution of Na2CO3 (40 g/100 mL) was then added. After incubation at room temperature for 30 min, the reaction mixtures were diluted to 10 mL with deionized water. Absorbance of the clear solution was measured at 725 nm. Concentration of the total phenolic content was determined from a standard calibration curve using chlorogenic acid as standard.
Determination of total flavonoid content
Total flavonoid content (TFC) was determined using a method adapted by Meda et al.[8] According to that method, 3 mL of 2% AlCl3 in methanol and 2 mL methanol were mixed separately with 3 mL of 80% alcoholic extract and absorption reading at 415 nm (UV/VIS, 2041 spectrophotometer) was taken after 30 min against a blank sample consisting of a 3 mL of AlCl3 with 3 mL of methanol.
Free radical scavenging activity (DPPH)
The sample was measured in terms of hydrogen-donating or radical-scavenging ability using the stable radical DPPH•. Briefly, 1.5 mL of a DPPH methanolic solution (20 mg/L) was added to 0.75 mL of extract, at different concentrations ranging from 10 to 200 μL/mL (methanol for the control). The absorbance was measured at 517 nm after 20 min of reaction. The percent of DPPH of the sample was calculated according to the following equation: % decolorization [1 – (Abs. sample/Abs. control) × 100. The decoloration was plotted against the sample extract concentration, and a logarithmic regression curve was established to calculate the IC50, which is the amount of sample necessary to decrease by 50% the absorbance of DPPH.[9]
Rat hepatocytes monolayer culture
Isolation and preparation of rat hepatocytes monolayer culture
A primary culture of rat hepatocytes was prepared according to the Seglen method,[10] which was modified by Kiso et al.,[11] using a Wistar male rat (250–300 g). The rat was obtained from the animal house of the NRC (National Research Center, Cairo). Animal procedures were performed in accordance with the Ethics Committee of the National Research Centre and followed the recommendations of the National Institutes of Health Guide for Care and Use of Laboratory Animals.[12]
IC50 determination on rat hepatocytes monolayer culture
After 22–24 h, the rat hepatocyte monolayer was washed twice with phosphate buffer saline (PBS). In order to determine IC50, different concentrations were prepared for each sample (100–1000 μg mL-1 ). After 2 h of cells incubation with the extract, cell viability was determined using the MTT assay. The assay was performed according to the method of Mosmann,[13] modified by Carmichael et al.[14] Absorbance of formasan crystals produced by viable cells was read at 540 and 630 nm dual wavelength using the Automatic Kinetic Microplate Reader (Labsystems Multiskan RC reader). Each experiment was repeated three times, and the mean absorption of each concentration was calculated. A graph plotted with x-axis showing the different concentrations of the extract used and the y-axis showing the absorbance percentage of viable cells. The IC50 was graphically determined from the concentration that yielded an absorption coinciding with the 50% of cells that received no extract.
Evaluation of hepatoprotective activity
The primary rat hepatocyte monolayer was prepared as in Section (Rat hepatocytes monolayer culture Different concentrations were prepared from the 80% methanol (12.5–100 μg/mL) using the serial dilutions technique by dissolving in DMSO (1% maximum concentration). For each concentration, three replicates were carried out; in addition to positive control, that was 50 μg/mL Silymarin. The plate was incubated for 2 h at 37°C and 5% CO2, then washed twice with PBS. A 200 μL of 25 mM paracetamol was added to each well. After 1 h of cells incubation with the paracetamol, cell viability was determined using the MTT assay. The concentration of the extract that was able to protect the cells from the hepatotoxic effect of paracetamol by 100% was considered hepatoprotective.
RESULTS
Spectrometric identification of compounds A and B
Compounds A and B were identified as the 3,4-dihydroxy-phenethylalchohol-6-O-caffeoyl-β-D-glucopyranoside and 3′,8′-binaringenin depend on the spectral data and compared with the published data.[1516]
Quantitative estimation of TPC and TFC
TPC
TPC of both 80% methanol extracts expressed as mg of chlorogenic acid equivalents/1 g herb could be calculated from the following equation
Y = 6.4X + 0.065
where Y is the absorbance and X is the corresponding concentration mg/mL. The wild fennel contains 2.4% and the cultivated one contains 3.1%.
TFC
TFC was determined using a calibration curve with rutin as standard. TFC of 80% alcoholic extract was expressed as mg of rutin equivalents/1 g of herb could be calculated from the following equation:
Y = 6.24X – 0.01
Flavonoid content of wild is 1.2% and cultivated fennel is 1.6%.
Hepatotoxicity
The assay was applied with a broad range of concentrations of the studied extracts (from 125 to 1000 μg/mL) on the monolayer of rat hepatocytes. It revealed that the 80% methanolic extract of the wild and cultivated fennel had IC50 effects at a concentration of >1000 and 1000 μg/mL, respectively [Figure 1].
Evaluation of hepatoprotective activity
The hepatoprotective effects of both 80% methanolic extracts of wild and cultivated fennel herb against the toxic effect of 25 mM paracetamol on the monolayer hepatocyte cells was 12.5 μg/mL.
From the results of the hepatoprotective and hepatotoxic effect of the methanolic extract of wild and cultivated fennel, we can conclude that they showed a safety margin, as the hepatotoxicity dose is 80 folds that of the hepatoprotection dose [Figure 2].
Free radical scavenging activity (DPPH)
Radical scavenging activity (expressed as absorbance percentage) of F. vulgare subsp. piperitum (wild) and F. vulgare subsp. azoricum (cultivated) extracts [Table 1 and Figure 3] as well the two isolated compounds [Table 2].
Radical scavenging activity (expressed as absorbance percentage) of Foeniculum vulgare subsp. piperitum (wild) extracts and Foeniculum vulgare subsp. azoricum (cultivated)
Radical scavenging activity (expressed as absorbance percentage) of Foeniculum vulgare subsp. piperitum (wild) extracts and Foeniculum vulgare subsp. azoricum (cultivated) in comparison with Trolox.
Radical scavenging activity (expressed as absorbance percentage) of the two isolated compounds from Foeniculum vulgare subsp. piperitum (wild)
DISCUSSION
Two compounds A and B were isolated for the first time from the wild fennel and identified as 3,4-dihydroxy-phenethylalchohol-6-O-caffeoyl-β-D-glucopyranoside and 3′,8′-binaringenin, respectively. Their structures were elucidated by spectral methods including 1D NMR, 2D NMR and UV. The total phenolic and flavonoid contents of the wild fennel and cultivated fennel were determined according to the Meda method.[8] It revealed that the cultivated fennel had more phenolic and flavonoid contents (3.1% and 1.6%, respectively) than that found in the wild fennel (2.4% and 1.2%; respectively). The 80% methanolic extract of the wild and cultivated fennel showed a hepatoprotective effect at a concentration of 12.5 μg/mL and a hepatotoxic effect at a concentration of 1000 μg/mL. From this we can conclude that they both show a safety margin, as the hepatotoxicity dose is 80 folds that of the hepatoprotective dose.
The antioxidant activity was measured as free radical scavenging activity (DPPH). The ethyl acetate and butanol fractions of the wild fennel were found to exhibit a radical scavenging activity higher than those of the cultivated fennel. At the same time, compounds A showed a very high radical scavenging activity comparing with that of trolox while compound B gave moderate activity.
There is little information available on the nonvolatile constituents of fennel and the polyphenolic compounds are associated with the prevention of disease thought to be induced by oxidative stress, such as cardiovascular diseases, cancer, and inflammation. In this study, we aimed to highlight on the antioxidant and hepatic-activity of phenolic compounds of the wild and cultivated fennel.
Footnotes
Source of Support: Work is partially supported by Science and Technology Development Fund STDF, Egyptian Academy of Scientific Research and Technology “ID# 245”.
Conflict of Interest: None declared.
ACKNOWLEDGMENTS
This work is partially supported by Science and Technology Development Fund STDF, Egyptian Academy of Scientific Research and Technology “ID# 245”.
References
- 1. Screening the essential oil composition of wild Sicilian fennelBiochem Syst Ecol20103821323[Google Scholar]
- 2. Comparison of the essential oils from ten Foeniculum vulgare Miller samples of fruits of different originFlavour Fragr J19991437982[Google Scholar]
- 3. Separation and characterization of phenolic compounds in fennel (Foeniculum vulgare) using liquid chromatography–negative electrospray ionization tandem mass spectrometryJ Agric Food Chem200452367987[PubMed][Google Scholar]
- 4. European Pharmacopoeia19972nd ed
- 5. GC–MS analysis of essential oil of some commercial Fennel teasFood Chem20027630710[Google Scholar]
- 6. Phenolic composition and antioxidant activities in flesh and achenes of strawberries (Fragaria ananassa)J Agric Food Chem200553403240[PubMed][Google Scholar]
- 7. The Flavonoids: Advances in Research1982LondonChapman and Hall19134
- 8. Determination of total phenolic, flavonoid and proline content in Burkina Fasan honey, as well as their radical scavenging activityFood Chem2005915717[Google Scholar]
- 9. Observations on the estimation of scavenging activity of phenolic compounds using radical 1,1-Diphenyl-2-picrylhydrazyl (DPPH•) testJ Am Oil Chem Soc20027912[Google Scholar]
- 10. Preparation of isolated rat liver cellsMethods Cell Biol1976132983[PubMed][Google Scholar]
- 11. Assay method for antihepatotoxic activity using galactosamine-induced cytotoxicity in primary-cultured hepatocytesJ Nat Prod1983468417[PubMed][Google Scholar]
- 12. NIH Guide for the Care and Use of Laboratory AnimalsNIH Publication Number. 85-23, Revised 1985, U.S. Department of Health, Education and WelfareResearch Triangle Park, North Carolina[Google Scholar]
- 13. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxic assayJ Immunol Methods19836526371[Google Scholar]
- 14. Evaluation of tetrazolium-based semiautomated colourimetric assay: Assessment of chemosensetivity testingCancer Res19874793642[PubMed][Google Scholar]
- 15. Phenylethanoid glycoside from Plantago asiaticaPhytochemistry1995387413[PubMed][Google Scholar]
- 16. Biflavonoids from Fruits of Poison ivy Toxicodendron RadicansPhytochemistry19781721401[Google Scholar]