Table of Contents  
ORIGINAL ARTICLE
Year : 2012  |  Volume : 11  |  Issue : 1  |  Page : 59-65

Bioactive lignans and other phenolics from the roots, leaves and seeds of Arctium lappa L. grown in Egypt


1 Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo, Egypt
2 Department of Pharmacognosy, National Organization for Drug Control and Research, Cairo, Egypt
3 Department of Pharmacology, National Research Center, Giza, Egypt

Date of Submission02-Jan-2012
Date of Acceptance15-Mar-2012
Date of Web Publication18-Jul-2014

Correspondence Address:
Elsayed A. Aboutabl
Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Kasr-el-Aini Str., 11562 Cairo
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.7123/01.EPJ.0000415466.17860.6a

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  Abstract 

Aim

Investigation of the phenolic constituents of roots, leaves and seeds of A. lappa L. cultivated in Egypt.

Methods

Qualitative and quantitative analysis of lignans and other phenolics by LC-MS/MS and HPLC/PDA-LC-MS/MS, as well as evaluation of potential bioactivities.

Results and conclusions

LC-MS/MS of the ethyl acetate fractions from Arctium lappa seeds, leaves and roots showed the presence of 13 phenolics, including lignans, distributed in the different organs. The following phenolics were identified: caffeic acid 1, genistein 2, biochanin A 3, luteolin 4, chlorogenic acid 5, materisenol 6, arctigenin 7, quercetin 8, cynarin 9, arctiin 10, lappaol A 11, rutin 12 and lappaol F 13. Quantification of these compounds by HPLC-PDA-MS/MS showed that arctiin was the major compound in seeds. The antioxidant, antihepatotoxic, anti-inflammatory and cytotoxic activities of ethanolic extracts of the different organs were evaluated.

Keywords: antihepatotoxicity, anti-inflammatory, antioxidant, Arctium lappa L., coumarin, cytotoxicity, lignans, phenolics


How to cite this article:
Aboutabl EA, El Mahdy ME, Sokkar NM, Sleem AA, Shams MM. Bioactive lignans and other phenolics from the roots, leaves and seeds of Arctium lappa L. grown in Egypt. Egypt Pharmaceut J 2012;11:59-65

How to cite this URL:
Aboutabl EA, El Mahdy ME, Sokkar NM, Sleem AA, Shams MM. Bioactive lignans and other phenolics from the roots, leaves and seeds of Arctium lappa L. grown in Egypt. Egypt Pharmaceut J [serial online] 2012 [cited 2020 Nov 29];11:59-65. Available from: http://www.epj.eg.net/text.asp?2012/11/1/59/136972


  Introduction Top


Arctium lappa L. or burdock (Asteraceae) is native to Europe and north Asia. It is a safe and edible food product in Asia 1,2. In traditional medicine, decoctions of powdered seeds, leaves and roots of burdock have been used in the treatment of cold, catarrh, gout and rheumatism, as a diuretic, diaphoretic and laxative, and for skin problems 3. Burdock is one of the herbs widely used by cancer patients in some Canadian populations to improve the quality of life and reduce cancer progression 4. Evaluation of the activities of extracts of different organs of burdock and the compounds isolated thereof were carried out including antipyretic, antimicrobial, diuretic, diaphoretic, antihyperglycaemic 5, in-vitro antioxidant 3,6–9, anti-inflammatory, antihepatotoxicity 10–13, antiulcer 14, antimutagenicity 15,16 and antitumour activities 17–19. Phytochemical investigations indicated the presence of fixed oil (15–30%), phenolic acids, flavonoids and lignans 2, 7, resin, mucilage, essential oil 20, polyacetylenes 20 and caffeoylquinic acid derivatives 8.

The present study aimed to investigate the constituents of roots, leaves and seeds of A. lappa L. cultivated in Egypt to explore any variations because of environmental changes. Lignans and other phenolics were investigated qualitatively and quantitatively by LC-MS/MS and HPLC/PDA-LC-MS/MS. The potential bioactivities of extracts from seeds, leaves and roots of the plant were evaluated, namely, in-vivo antioxidant, hepatoprotective, anti-inflammatory and cytotoxic activities.


  Subjects and methods Top


Plant material

Seeds of A. lappa L. were kindly provided to Prof. Dr E. Aboutabl by the Botanical Garden, Bohn, Germany. The plant was raised in the Experimental Station, Faculty of Pharmacy, Cairo University. Seeds, leaves and roots were annually obtained from the cultivated plant collected during the fruiting stage in July, air-dried and kept in closed containers.

Reference standards

Standard samples of arctigenin and arctiin were kindly supplied to Prof. Dr E.A. Aboutabl by the late Prof. Dr Martin Luckner, Institute of Pharmaceutical Biology, Martin-Luther University, Halle/S, Germany. Chlorogenic acid, caffeic acid, coumarin, cynarin, quercetin, rutin and luteolin were obtained from the National Organization of Drug Control and Research Reference Standards Department, and genistein, biochanin A, carrageenan and alloxan were obtained from Sigma (USA). Indomethacin was obtained from Epico, (Egypt), α-tocopheryl acetate from Pharco Pharmaceutical Co. (Egypt) and carbon tetrachloride BDH (England).

Biochemical kits and reagents

Glutathione kits for the assessment of antioxidant activity and transaminase kits for the assessment of serum aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) were obtained from Biodiagnostic Co. (Cairo, Egypt).

Animals

Albino mice weighing 25–30 g and adult albino rats of the Sprague–Dawley strain (130–150 g) were kept under hygienic conditions and on standard laboratory diet and water, supplied ad libitum.

Methods

Thin-layer chromatography (TLC) was performed on precoated silica gel F254 plates (Fluka, Germany). The solvent systems used were: (S1) chloroform–methanol (9 : 1); (S2) ethyl acetate–water–formic acid (85 : 15 : 10) for lignans, phenolic acids and coumarin; and (S3) hexane–ethyl acetate–formic acid (20 : 19 : 1) for flavonoids. Visualization of flavonoids and phenolic acids was carried out under UV (254 and 366 nm), that of lignans was performed by spraying with 50% H2SO4 and that of coumarins by spraying with 10% alcoholic KOH solution.

Column chromatography was performed using silica gel H 60 (Sigma); for VLC, Sephadex LH-20 (Pharmacia, Sweden) and silica gel 60 (Fluka, Germany) were used.

NMR spectra were run on JOEL GLM, and JOEL TMS route instrument, 500 MHz (Japan).

EI-MS, mass spectrophotometer (FINNIGAN MAT SSQ.70000, USA) and (Shimadzu QP 1000EX, Japan) ionization mode 70 eV equipment were used.

LC-MS/MS was performed on a Thermo ion trap mass spectrophotometer LCQ Advantage. The analysis was performed using the following settings: drying gas (air) was heated to 400°C; capillary voltage was set at 4 kV; air was the nebulizer gas; the curtain gas was N2; the collision gas was He; ionization was performed in negative mode [M–H] and collision energy was 35%. The full-scan mass infusion was performed using a syringe pump (Hamilton syringe, 500 µl) directly connected to electrospray ionization at a flow rate of 10 µl/min. The total ion mapping technique was used in LC-MS/MS [Figure 1], [Table 1].
Table 1: LC-MS/MS of phenolics in the extracts of Arctium lappa L. grown in Egypt

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Figure 1: Total Ion chromatograms of LC-MS/MS of (a) seed extract, (b) leaf extract and (c) root extract.

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HPLC analysis was performed using a PDA detector and an Intersil ODS-2 C18 column, 2.1×50 mm, particle size 3 µm (Alltech, Deerfield, Illinois, USA), using the solvent system A: water 0.2% formic acid and B: acetonitrile/methanol (60:40 v/v). The gradient program was as follows: 70% A (2 min), 50% A (4 min), 30% A (3 min) and 70% A (9–12 min); a flow rate of 0.2 ml/min; and an injection volume of 20 µl. X calibur version 1.4 (Thermo Fisher Scientific Inc., USA) was the software linked to the instrument for the calculation of the corresponding concentrations.

HPLC-PDA-LC-MS/MS quantification of major phenolics

Defatted air-dried powdered seeds, leaves and roots (1 g, each) were separately extracted with 70% methanol by sonication at room temperature. Stock solutions were prepared by dissolving each of the concentrated extracts in 5 ml methanol. The sample solutions were injected into the mass detector against isolated compounds considered as external standards of caffeic acid, cynarin, chlorogenic acid, arctigenin and arctiin at different dilutions (0.031–2 mg/ml, each) and arctigenin (0.018–2 mg/ml). The stock solutions were stored at −20°C. The stock and standard solutions were filtered through 0.45 μm filters, before injection, and diluted as necessary with methanol. Each concentration of the standards was analysed in triplicate. Quantification of the compounds was performed by measuring the peak area against six concentrations of the standards, plotting the standard curves and determining the compounds as the mean values of three replicate injections. Quantitative determination of caffeic acid, cynarin, arctigenin, chlorogenic acid and arctiin was carried out [Figure 1] and [Figure 2]; [Table 2].
Table 2: Quantitative HPLC determination of phenolics (mg/g powder)a in the seeds, leaves and roots of Arctium lappa L. grown in Egypt

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Figure 2: HPLC-PDA chromatogram of (a) seed extract, (b) leaf extract, (c) root extract.

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Colorimetric determination of total phenolics

Samples of air-dried and defatted powdered seeds, leaves and roots (5 g, each) were extracted with 70% methanol by sonication at room temperature. A stock solution (1 mg/ml) was prepared from the concentrated residue by dissolution in distilled water. Phenolics were determined using the Folin–Ciocalteau colorimetric method 21 and were expressed as mg of gallic acid equivalent/g of the air-dried plant material. The standard solution was prepared by dissolving 50 mg of gallic acid in 100 ml water. A calibration curve was constructed over the range of 15–100 µg/ml by diluting the stock solution in water. The prepared solutions from seeds, leaves and roots (0.1 ml, each) were separately mixed with 0.5 ml Folin–Ciocalteau reagent and diluted to 25 ml using an aqueous sodium carbonate solution (290 g/l). The absorbance was measured after 30 min at 730 nm against a blank prepared at the same time using 0.5 ml water as a compensating liquid. From the linear regression analysis (y=0.0169x+0.0358), r 2=0.9871, the phenol content of each extract was expressed as mg gallic acid/g. Results [Table 2] were the means of three determinations.

Sample preparation for the screening of phenolics by LC-MS/MS

Air-dried and defatted powdered seeds, leaves and roots (5 g, each) were extracted with 70% methanol by sonication at room temperature. The concentrated aqueous residue was fractionated between chloroform and ethyl acetate. The ethyl acetate fraction of each of the three organs was filtered over anhydrous sodium sulphate, concentrated to dryness and dissolved in 100 ml methanol (HPLC grade) for LC/MS analysis, as it contained lignans, flavonoids and phenolic acids as determined by TLC.

Isolation of phenolics

Air-dried and defatted powdered seeds (600 g) and roots (300 g) were repeatedly suspended in 70% methanol until exhaustion. The concentrated extracts (250 and 135 g; yield=41.7 and 45%, respectively) were separately suspended in water and successively fractionated with chloroform and ethyl acetate. The concentrated fractions yielded 27.6 and 4 g for seeds and 0.7 and 2 g for roots, respectively. The chloroform fraction of seed (10 g) was subjected to VLC (30×5 cm, silica gel G). Gradient elution was carried out using n-hexane; n-hexane/diethyl ether till 100% diethyl ether; diethyl ether/dichloromethane till 100% dichloromethane. Fractions (100 ml, each) were collected; similar fractions were pooled as monitored by TLC. The diethyl ether subfraction (140 mg) of seeds was subjected to preparative layer chromatography, followed by filtration through Sephadex and crystallization from methanol to yield compound 7 (20 mg). The concentrated dichloromethane subfraction (640 mg) was extracted with ethyl acetate to yield a yellow residue (500 mg); the latter was placed in a polyamide column eluted with 30% aqueous methanol and crystallization from a mixture of chloroform/ethyl acetate to yield compound 10 (100 mg). The ethyl acetate fractions of seeds (4 g) and roots (2 g) were placed in polyamide and Sephadex LH20 columns to yield compound 6 (80 mg) and compound 1 (6 mg) from seeds and compound 14 (10 mg) and compound 9 (20 mg) from roots.

Evaluation of bioactivities

Air-dried powdered seeds, leaves and roots (100 g, each) were separately macerated in 70% ethanol until exhaustion. The ethanol extracts of seed (EES), leaf (EEL) and root (EER) were separately concentrated for bioactivity evaluation.

Toxicity

The median lethal dose (LD50) of each of the plant extracts was determined 22. Antioxidant activity: glutathione in blood was determined 23. Thirty rats were intraperitoneally administered Alloxan (150 mg/kg body weight) to induce hyperglycaemia 24. Hyperglycaemia was assessed after 72 h by measuring the blood glucose level 25. The results are presented in [Table 3].
Table 3: Antioxidant activity of ethyl acetate extract of the seeds (EES), leaves (EEL) and roots (EER) of Arctium lappa L. grown in Egypt in comparison with vitamin E in diabetic male albino rats (n=6)

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Hepatoprotective activity

Liver damage in rats was induced 26 by an intraperitoneal injection (5 ml/kg body weight) of 25% CCl4 in liquid paraffin. Serum ALT, AST 27 and ALP 28 were determined. Animals were randomly divided into five groups: the first group received a daily dose of 1 ml saline for 1 month before and after liver damage was induced (control); in groups 2–5, liver-damaged rats were pretreated daily with ethyl acetate extract of seeds, leaves and roots (EES, EEL and EER, respectively) (100 mg/kg body weight), as well as silymarin (25 mg/kg body weight), respectively, for 2 weeks. Administration of the extract was continued after liver damage for another 2 weeks, followed by overnight fasting; whole blood was obtained from the retro-orbital venous plexus through the eyes canthus of anaesthetized rats. Blood samples were collected at zero time, 72 h and 2 weeks after the CCl4 injection and at 1-month intervals. Serum was isolated by centrifugation [Table 4].
Table 4: Effect of ethyl acetate extract of the seeds (EES), leaves (EEL) and roots (EER) of Arctium lappa L. and silymarin on serum enzyme level (AST, ALT and ALP) in liver-damaged rats (n=6)

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Acute anti-inflammatory activity 29

Thirty male albino rats were divided into five groups (six animals each). Group 1 received 1 ml of saline and served as the control group; groups 2–5 received ethyl acetate extract of seeds, leaves and roots (EES, EEL and EER, respectively) (100 mg/kg body weight) and indomethacin (20 mg/kg body weight), respectively. One hour later, all the animals received a subplanter injection of a 1% carrageenan solution in saline in the right hind paw and 0.1 ml saline in the left hind paw. Four hours after drug administration, the rats were sacrificed; both hind paws were separately excised and weighed [Table 5].
Table 5: Acute anti-inflammatory activity of ethyl acetate extract of the seeds (EES), leaves (EEL) and roots (EER) of Arctium lappa L. grown in Egypt and indomethacin in male albino rats (n=6)

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Cytotoxicity

The potential cytotoxicity of the ethyl acetate extract of seeds, leaves and roots (EES, EEL and EER, respectively) of A. lappa L. was tested 30 on liver, breast and colon cancer cell lines. IC50 for each extract was calculated [Table 6]. The relations between the surviving fractions and extracts concentrations were tabulated [Table 7] and plotted to construct the survival curves of each tumour cell line [Figure 3].
Table 6: IC50 of ethyl acetate extract of the seeds (EES), leaves (EEL) and roots (EER) of Arctium lappa L.a on cell lines

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Table 7: Cytotoxic activity of ethyl acetate extract of the seeds (EES), leaves (EEL) and roots (EER) of Arctium lappa L.

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Figure 3: Cytotoxic activity of ethyl acetate extract of seeds (a), leaves (b) and roots (c) of

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  Results Top


Seeds were found to be the richest in total polyphenols (20.35%), followed by the leaves and roots (9.28 and 5.33%, respectively). Screening of phenolics in ethyl acetate extracts of seeds, leaves and roots of A. lappa was run on LC-MS/MS. Total ion chromatograms [Figure 1]a–c] using the negative ion mode showed the following phenolics [Table 1]: caffeic acid 1, genistein 2, biochanin A 3, luteolin 4, chlorogenic acid 5, materisenol 6, arctigenin 7, quercetin 8, cynarin 9, arctiin 10, lappaol A 11, rutin 12 and lappaol F 13. As no commercial standards for materisenol 6, lappaol A 11 and lappaol F 13 were available, they were identified by their fragmentation pattern and comparison with literature data 7. The other compounds were identified by a comparison of their TLC, UV and MS data with the reference samples and literature data. Six compounds were isolated from the seeds and roots of A. lappa and identified as arctigenin 7, arctiin 10, chlorogenic acid 5, caffeic acid 1, coumarin 14 and cynarin 9 by comparing their UV, MS and 1HNMR data with the reference samples and literature 7, 31, 32.

Although A. lappa L. is known to be rich in lignans and phenolic acids, no studies in the literature has reported the presence of flavonoids in the seeds. This study dealing with the plant recently introduced in Egypt has shown that seeds contain biochanin A, genistein and quercetrin. However, arctiin was found to be absent in the root of the plant grown in Egypt, which is significantly different from that reported in a previous work 7. Quantitative analysis of the isolated compounds caffeic, cynarin, arctigenin, chlorogenic acid and arctiin by HPLC-PDA-LC-MS/MS showed that arctiin [Table 1] and [Table 2]; [Figure 1] and [Figure 2] was the major compound and concentrated mainly in seeds, followed by chlorogenic acid in different concentrations in the three organs.

Toxicity

The results indicated the safety of seeds (LD50 up to 9.3 mg extract/kg body weight), the leaves and roots (up to 10 mg extract/kg body weight).

Antioxidant activity

The results [Table 3] showed that the blood glutathione level was considerably reduced in the untreated diabetic rats. The blood glutathione level was restored by the treatment of animals with vitamin E (with percentage change=2.2). EES was the most potent extract, exerting almost the same effect as that of vitamin E, with percentage change=5.26, followed by EEL and EER. Previous in-vitro antioxidant studies have shown a higher potency of roots 8 and leaves 6 of A. lappa; this variation can possibly be attributed to differences in the concentrations of flavonoids and lignans in seeds.

Antihepatotoxic activity

Liver damage by 25% CCl4 (5 ml/kg) showed an increase in the levels of AST, ALT and ALP in blood. A significant decrease in serum enzymatic levels was observed [Table 4] after the administration of different extracts of A. lappa (100 mg/kg, daily) for 1 month, in the order seeds>leaves>roots. Previous investigations have reported the antihepatotoxicity of roots 10 and total herb 11.

Anti-inflammatory activity

All the extracts [Table 5] showed significant in-vivo anti-inflammatory effects as compared with the control group. The potency increased in the order: seeds>leaves>roots, showing percentage change of 54.2, 36.3 and 31.01%, respectively. In-vitro anti-inflammatory activity has been attributed to arctigenin 12. Previous studies 10,13 have studied the anti-inflammatory activity of root extracts.

Cytotoxic activity

Ethanolic extracts of different organs of A. lappa [Figure 3] and [Table 6] and [Table 7] showed highly potent cytotoxic activities in liver carcinoma. Ethanolic extract of seeds showed more potency in colon carcinoma, while that of leaves being the most potent in breast carcinoma. The antiproliferative activity of the prepared extracts was mainly because of arctigenin and arctiin 4, 17, 33. An anticarcinogenic effect was reported for seed 19 (using the cell line LNCaP) and root extracts 9 (using cell lines K562, MCF7 and 786-0).


  Conclusion Top


A. lappa L. was successfully cultivated in Egypt. In this report, its phytochemical and bioactive profile is presented as a comparative study of its different organs in order to investigate its safe consumption. Evaluation of antioxidant, hepatoprotective, anti-inflammatory and cytotoxicity activities showed that the most significant activities were found for extracts of seeds, which can possibly be attributed to its high content of phenolics, including flavonoids, lignans and phenolic acids.[33]

 
  References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

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