Scholarly article on topic 'Bioactivities, phenolic compounds and in-vitro propagation of Lippia citriodora Kunth cultivated in Egypt'

Bioactivities, phenolic compounds and in-vitro propagation of Lippia citriodora Kunth cultivated in Egypt Academic research paper on "Chemical sciences"

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Abstract of research paper on Chemical sciences, author of scientific article — Seham S. El-Hawary, Miriam F. Yousif, Amira A. Abdel Motaal, Lamia M. Abd-Hameed

Abstract The aqueous and alcoholic extracts of the fresh aerial parts of Lippia citriodora Kunth, family Verbenaceae, cultivated in Egypt, exhibited variable antiinflammatory, antipyretic, analgesic and antioxidant properties. Three phenolic compounds; two phenolic acids, dihydrocaffeic acid (1) and 4-hydroxycinnamic acid (3) and a flavonoid glycoside, luteolin-7-O-glucoside (2), were isolated and identified from the ethyl acetate fraction of the plant. The structures of the isolated compounds were elucidated on the basis of spectral analysis. The effects of explant type, method of sterilization and growth regulators on the in-vitro callus formation of L. citriodora were studied. Shoot tips and leaf explants (cut in the midrib region) sterilized by soaking in 0.2% mercuric chloride for 5min, then washed twice with sterilized distilled water gave callus, on Murashige and Skoog (MS) medium impregnated with 4mg/L 6-benzyl amino purine (BAP). Spectrophotometric estimation of the total flavonoids showed that the fresh in-vitro formed callus contained 68.4% of the total flavonoids of the fresh aerial parts of the conventional plant.

Academic research paper on topic "Bioactivities, phenolic compounds and in-vitro propagation of Lippia citriodora Kunth cultivated in Egypt"

Bulletin of Faculty of Pharmacy, Cairo University (2012) 50, 1-6

Cairo University Bulletin of Faculty of Pharmacy, Cairo University

www.elsevier.com/locate/bfopcu www.sciencedirect.com

ORIGINAL ARTICLE

Bioactivities, phenolic compounds and in-vitro propagation of Lippia citriodora Kunth cultivated in Egypt

Seham S. El-Hawary a, Miriam F. Yousif a, Amira A. Abdel Motaal a'*, Lamia M. Abd-Hameed b

a Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Kasr-El-Ainy St., Cairo 11562, Egypt b Defence Industry Medical Center, Ismail Kamel Street, East Helwan, Egypt

Received 25 October 2011; accepted 29 December 2011 Available online 30 January 2012

KEYWORDS

Lippia citriodora; Antiinflammatory; Dihydrocaffeic acid; In-vitro propagation

Abstract The aqueous and alcoholic extracts of the fresh aerial parts of Lippia citriodora Kunth, family Verbenaceae, cultivated in Egypt, exhibited variable antiinflammatory, antipyretic, analgesic and antioxidant properties. Three phenolic compounds; two phenolic acids, dihydrocaffeic acid (1) and 4-hydroxycinnamic acid (3) and a flavonoid glycoside, luteolin-7-O-glucoside (2), were isolated and identified from the ethyl acetate fraction of the plant. The structures of the isolated compounds were elucidated on the basis of spectral analysis. The effects of explant type, method of sterilization and growth regulators on the in-vitro callus formation of L. citriodora were studied. Shoot tips and leaf explants (cut in the midrib region) sterilized by soaking in 0.2% mercuric chloride for 5 min, then washed twice with sterilized distilled water gave callus, on Murashige and Skoog (MS) medium impregnated with 4 mg/L 6-benzyl amino purine (BAP). Spectrophotometric estimation of the total flavonoids showed that the fresh in-vitro formed callus contained 68.4% of the total flavonoids of the fresh aerial parts of the conventional plant.

© 2012 Faculty of Pharmacy, Cairo University. Production and hosting by Elsevier B.V. All rights

reserved.

* Corresponding author.

E-mail address: a_motaal@hotmail.com (A.A. Abdel Motaal).

1110-0931 © 2012 Faculty of Pharmacy, Cairo University. Production and hosting by Elsevier B.V. All rights reserved.

Peer review under responsibility of Faculty of Pharmacy, Cairo University.

doi:10.1016/j.bfopcu.2011.12.001

1. Introduction

Lemon verbena, Lippia citriodora Kunth, [syn. Lippia triphylla (L'Her.) Kuntze; Aloysia triphylla (L'Her.) Britton] was cultivated in Egypt for its lemon-like aroma to be used in herbal tea preparations, as it is known for its antispasmodic, antipyretic, sedative and digestive properties.1-4

Phenolic compounds (mainly flavonoids, phenolic acids and phenylpropanoids) were reported to be responsible for most of the pharmacological activities of lemon verbena,2,3,5 such as analgesic, antiinflammatory and antioxidant effects.6,7 In spite of attributing many of the activities of lemon verbena to its main component, the phenyl propanoid verbascoside, we cannot assume that all the bioactivities of the plant extract are

due to just one compound since several researches have shown interactions between phenolic compounds, mainly synergistic and antagonistic effects.5 Flavonoids and other phenolic compounds were isolated from L. citriodora obtained from different localities.8'9 However, hardly any datum was available concerning L. citriodora cultivated in Egypt except for the study of the essential oil and its bioactivities.10

Although L. citriodora can be cultivated in any soil yet it cannot withstand salty ones. The seed being minute, the plant propagation is mainly by cuttings in late spring.11 Foliation is in spring and summer and the plant loses its leaves and shrivels at the end of winter looking really dead.12 Research articles concerning the micropropagation of Lippia were confined to species other than L. citriodora.13'14 Trials of in-vitro propagation deemed, therefore, necessary as a first step for an Egyptian cultivar with a high content of active ingredients.

The present study aimed to evaluate the medicinal potentiality of the alcoholic and aqueous extracts of L. citriodora Kunth, cultivated in Egypt, as well as, to investigate the phenolic composition of the most active extract. This report stressed also on establishment of suitable conditions for the in-vitro callus formation of the cited plant.

2. Materials and methods

2.1. Plant material

Samples of aerial parts of L. citriodora were obtained from plants cultivated in a private farm in Mounufia, Egypt. Identification of the plant was confirmed by Professor Dr. Abd El-Salam Al-Nowaihi, Professor of Plant Taxonomy, Faculty of Science, Ain Shams University. Voucher specimens are deposited at the herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Cairo University.

2.2. Biological screening 2.2.1. Experimental animals

Male albino mice (25-30 g) were used for the acute toxicity testing, and adult male albino rats (130-150 g) for studying the analgesic, antipyretic, anti-inflammatory and antioxidant activities. Animals were obtained from the animal-breeding unit of National Research Center, El-Dokki, Giza, Egypt. They were fed on a standard laboratory diet under hygienic conditions and water supplied ad libitum.

2.2.2. Tested extracts

The aqueous and alcoholic extracts were prepared by boiling the aerial parts (65 and 110 g) in distilled water and macerating them in successive portions of 70% ethanol till exhaustion, respectively. The extracts were separately evaporated till dry-ness and the residues (8 and 14.5 g, respectively) were redis-solved in distilled water (containing few drops of Tween 80 in the case of the alcoholic extract) to give solutions of 25% (w/v) concentration.

2.2.3. Acute toxicity

LD50 of both aqueous and alcoholic extracts of L. citriodora was estimated according to the procedure developed by Kar-ber, 1931.15

2.2.4. Pharmacological experiments

Animals were divided into four groups, each of six. The first group was considered as a control, the second was given orally the appropriate standard drug while the other two groups each received 100 mg/kg b.wt. of the test extracts. The anti-inflammatory activity was determined according to the method described by Winter et al.16 Carrageenan, 0.1 ml of 1% solution, (Sigma-Aldrich, MO, USA) was used to induce inflammation and 20 mg/kg b.wt. indomethacin (Epico Pharmaceutical Co., Egypt) was used as a standard anti-inflammatory drug. The analgesic activity was evaluated according to Charlier et al.17 using dipyrone-metamizole, 50 mg/kg b.wt., (Novalgin, Hoechst Orient, Cairo, Egypt) as a standard analgesic. The antipyretic effect was tested following the method of Bush and Alexander.18 Brewers' dry yeast, 1 ml/100 g b.wt. of 40% suspension, (Rehab Food Company, Cairo, Egypt) was given by intramuscular injection to induce hyperthermia and 20 mg/kg b.wt. paracetamol (Paramol, Misr Pharmaceutical Co., Mataria, Egypt) was the standard antipyretic.

For evaluation of the antioxidant activity, animals were divided into five groups, each of 10. Four of the groups were treated as for the previously mentioned bioactivity tests, where as the fifth group was given Alloxan I. P., 150 mg/ kg b.wt. (Sigma-Aldrich, MO, USA) to induce diabetes melli-tus. The antioxidant activity was estimated by determining the blood glutathione level of the diabetic rats19 as compared to dl a- tocopheryl acetate as a positive control (Vitamin E, Pharco Pharmaceutical Co., Egypt). All data were expressed as mean ± SE and the statistical significance was evaluated by Student's t test.20

Table 1 Acute antiinflammatory activity of the alcoholic and aqueous extracts of L. citriodora and indomethacin in male albino rats

(n = 6).

Group Dose (mg/kg b.wt) % edema

Mean ± SE % of changea

Control 1 ml saline 61.7 ± 1.9 -

Alcoholic extract 50 26.2 ± 0.5* 57.536

Aqueous extract 50 29.3 ± 0.8* 52.512

Indomethacin 20 21.4 ± 0.6* 65.316

a Calculated as regard to the control group; b.wt, body weight.

P < 0.01 vs. control group.

Table 2 Analgesic activity of alcoholic and aqueous extracts of L. citriodora and novalgin in male albino rat (n = 6).

Group Dose (mg/kg b.wt) Volts needed after single oral dose

Volts needed before treatment(zero time) 1 h 2 h

Mean ± SE % of changea Mean ± SE % of changea

Control 1 ml saline 74.3 ± 1.4 75.1 ± 1.6 1.077 74.8 ± 2.1 0.673

Alcoholic extract 50 76.2 ± 1.5 126.2 ± 4.3* 65.617 143.2 ± 5.7* 87. 927

Aqueous extract 50 79.3 ± 1.4 112.6 ± 3.7* 41.992 131.8 ± 5.2* 66.204

Novalgin 50 78.2 ± 1.2 151.6 ± 5.8* 93.862 172.4 ± 6.3* 120.460

a Calculated as regard to the control group; b.wt, body weight. P < 0.01 vs. control group.

Table 3 Antipyretic activity of the alcoholic and aqueous extracts of L. citriodora and paracetamol in male albino rats (n = 6).

Group Dose (mg/kg b.wt) Body temperature change

Induced rise in temperature 1h 2h

Mean ± SE % of changea Mean ± SE % of changea

Control 1 ml saline 38.7 ± 0.2 39.1 ± 0.2 1.034 39.2 ± 0.3 1.292

Alcoholic extract 50 39.4 ± 0.3 38.1 ± 0.3* 3.299 37.2 ± 0.1* 5.584

Aqueous extract 50 39.8 ± 0.4 38.3 ± 0.4* 3.769 37.1 ± 0.2* 6.784

Paracetamol 20 39.2 ± 0.3 37.8 ± 0.1* 3.571 36.8 ± 0.1* 6.122

a Calculated as regard to the control group; b.wt, body weight.

P < 0.01 vs. control group.

Table 4 Antioxidant activity of the alcoholic and aqueous extracts of L. citriodora and vitamin E (n = 10).

Group Blood glutathione (mg%) % of changea

Control 36.2 ± 1.3 -

Diabetic 22.3 ± 0.9* 38.398

Diabetic + alcoholic extract(50 mg/kg b.wt) 35.2 ± 1.4 2.762

Diabetic + aqueous extract(50 mg/kg b.wt) 34.7 ± 0.8 4.144

Diabetic + vitamin E(75 mg/kg) 35.8 ± 1.1 1.105

a Calculated as regard to the control group.

P < 0.01 vs. control group.

Table 5 Spectral data of compounds 2 and 3 isolated from L. citriodora.

Compounds UV

'H NMR (300 MHz, DMSO-d6)

3C NMR (75 MHz, DMSO-d6)

kmax (MeOH): 250, 291.4sh, 335; (MeOH + NaOMe) 263, 299.6sh, 380; (MeOH + AlCl3) 264.2, 300sh, 374, 466; (MeOH + AlCl3/HCl) 262, 278sh, 329, 414; (MeOH + NaOAc) 255, 289sh, 342, 388sh; (MeOH + NaOAc/H3BO4) 257, 295.4sh, 359

kmax (MeOH): 283 and 323 nm

d 6.65 (1H, S, H-3); 6.48(1H, d, J = 2.7 Hz, H-6); 6.76 (1H, d, J = 2.7 Hz, H-8); 7.05 (lH, S, H-5'); 7.44 (1H, S, H-2'); 7.49 (1H, S, H-6'); the sugar moiety: d 5.12 (1H, d, J = 8.1 HZ, H-1''); 3.4-3.5 (overlapped peaks, H-3'' and H-4''); 3.6 (1H, m, H-5''); 3.65 (1H, m, H6''a); 3.84 (1H, m, H6''b)

d 8.5(1H, S, H-1); 6.7

(2H, d, J = 2.6 Hz, H2 and

H-6); 7.74 (2H, d, J = 2.7 Hz, H-3

and H-5); 7.5 (1 H d, J =14 Hz, H-7);

6.2 (1H, d, J = 14 Hz, H-8); 9.1 (1H, S, H-9)

166.35 (C-2), 102.75 (C-3), 182 (C-4), 161.33 (C-5), 101.65 (C-6), 163.5 (C-7), 94.67 (C-8), 157 (C-9), 109.8 (C-10), 120.14 (C-1'), 113.96 (C-20), 145.33 (C-3'), 149 (C-40), 116.34(C-5'), 116.8 (C-6'), 100.67(C-1''), 72.8 (C-2''), 75 (C-3''), 70.75 (C-4''), 79.86 (C-5''), 61.14 (C-6'')

d d d d d d

n n n n n n

a a a a a a

PhPHQH^H PHPHQHPH

AAAA m « pq «

a) b) c) d) a) b) c) d) e) f) g) h) a) b) c) d)

PhPHPHPHPH^PHPH

A A A A A BA A A BrnfflfflB^Bn

12 dd nn

< pq < <

4 10 4 4

pu o r

pu o r

2.3. Isolation of phenolics

The air-dried aerial parts (1 kg) of L. citriodora were extracted with successive portions of 70% ethanol till exhaustion. The eth-anolic extract was collected and concentrated under reduced pressure to give a viscous residue (130 g). This residue was suspended in the least amount of distilled water and partitioned successively with petroleum ether, chloroform and ethyl acetate. The ethyl acetate fraction (6.66 g) was chromatographed on a vacuum liquid chromatography column (VLC) (92 g, 5 x 14 cm, silica gel G, E-Merck). Elution was carried out by gradiently increasing the polarity using chloroform, ethyl acetate and methanol. The collected fractions (39 fractions, 100 ml each) were monitored by TLC (silica gel GF254 precoated plates, Fluka, Germany) under UV light (at 254 and 366 nm) before and after exposure to ammonia vapor. Spraying with AlCl3 or p-anisaldehyde-sulfuric acid spray reagent, followed by heating at 110 0C, was also carried out. Solvent system used was chloroform: methanol: formic acid, 8:2:0.5. Fractions were pooled to give four major collective fractions (1-4). Fraction 2 (2 g) was rechromatographed on a Sephadex LH-20 column (1 x 20 cm, Pharmacia Fine Chemicals AB Uppsala, Sweden) using methanol as an eluent to give compound 1 (5 mg) and compound 2 (25 mg). Fraction 3 (1.2 g) was purified on successive Sephadex LH-20 columns (1 x 16 cm) using methanol: water (50:50) to give compound 3 (10 mg). Mass spectra of the compounds were measured using Shimadzu QP-2010 Plus, 70 eV. 1H NMR (300 MHz) and 13C NMR (75 MHz) were measured on a Varian Mercury-VX-300 NMR instrument. The NMR spectra were recorded in DMSO-d6, and chemical shifts were given in d (ppm) relative to TMS as an internal standard. All solvents used were of analytical grade.

2.4. In-vitro propagation

The fresh aerial parts of L. citriodora were washed with soapy water, rinsed several times with running tap water, and divided into three groups for sterilization: group (1), was treated with acetone (2 min) (Adwic, Cairo, Egypt) followed by Chlorex® 30% (1.5% sodium hypochlorite) for 5 min, HgCl2 0.2% (5 min) (Universal Fine Chemical Pvt. Ltd.), then rinsed twice with sterilized distilled water; group (2), was treated with acetone (2 min) followed by HgCl2 0.2% (5 min), then rinsed twice with sterilized distilled water; group (3), was treated with HgCl2 0.2% (5 min), then rinsed twice with sterilized distilled water.

The sterilized aerial parts were aseptically excised to give four types of explants: shoot tips, nodes and internodes (0.5-1 cm each), and leaves cut into 1 cm square pieces (some cut in the midrib region and others in the lamina region).

Murashige and Skoog (MS) medium, in full strength, was used as a basal culture medium.21 The media were enriched with different concentrations of 6-benzyl amino purine (BAP) (Oxford) as an auxin, and different concentrations of 1-naphthaleneacetic acid (NAA) (Sigma) and/or indole-3-bu-tyric acid (IBA) (Sigma) as cytokinins.

2.5. Spectrophotometry estimation of total flavonoids

The percentage of total flavonoids was calculated as luteolin adopting a colorimetric method modified after Abou Zid and

Elsherbeiny.22 A solution of 0.1 M aluminium chloride was prepared according to US Pharmacopoeia, 2004.23 A standard calibration curve for luteolin was established. One gram of each of the fresh aerial parts and callus of L. citriodora was defatted with petroleum ether, then extracted with 95% etha-nol to be used for the assay.

3. Results and discussion

The LD50 of the aqueous and alcoholic extracts were 6.4 g/ kg b.wt. and 5.9 g/kg b.wt., respectively, thus suggesting that both could be considered as safe. Simon et al.24 stated that L. citriodora Kunth is listed in USA as generally recognized as safe (GRAS) for human consumption in the form of alcoholic beverages and herbal teas.

3.1. Biological activities and isolation of active constituents

Both extracts possessed variable antiinflammatory, analgesic, antipyretic and antioxidant activities. The alcoholic extract showed higher antiinflammatory, analgesic and antioxidant activities (Tables 1, 2 and 4, respectively), while the aqueous extract possessed a higher antipyretic activity (Table 3). The percent of change in edema for the alcoholic extract was 57.5% reaching nearly 88% that of Indomethacin which caused a change of 65.3% (Table 1). The antiinflammatory activity of Lippia alba was previously reported and attributed to the presence of theviridoside, an iridoid glucoside.25 Iridoids are common constituents in family Verbenaceae26 and their occurence in lemon verbena was mentioned by Bilia et al.27 As reported by Nakamura et al.,6 the analgesic activity of L. citriodora may be due to the presence of verbascoside (acteoside) a phenyl-ethanoid glycoside which was previously separated from the Peruvian plant. Concerning the antipyretic activity, the aqueous extract exerted an effect slightly exceeding that of paracetamol (Table 3). As a matter of fact, L. citriodora is traditionally known to be a good herb for fever.28 The high antioxidant activity observed for the alcoholic extract compared to vitamin E (Table 4) could be attributed to its content of phenolic compounds.29'30

These findings stimulated the authors to investigate the phenolic composition of the alcoholic extract and especially its ethyl acetate fraction. The three major phenolic compounds, as detected by TLC, were isolated by repeated column chromatography.

Compound 1 (5 mg) was obtained as yellow crystals, soluble in methanol, ethanol and insoluble in petroleum ether and n-hexane (Rf value 0.93). UV spectra revealed one absorption band at 286 nm similar to authentic dihydrocaffeic acid.31 Mass spectra showed 182 (M + )32 and base peak at 55 m/z. Compound 1 was identified as dihydrocaffeic acid. To the best of our knowledge, this is the first report on the isolation of dihydrocaffeic acid from L. citriodora.

Compound 2 (25 mg) was obtained as yellow amorphous powder. By comparing its spectral data (Table 5) to those reported for luteolin-7-O-glucoside,33-35 it was concluded that compound 2 is luteolin-7-O-glucoside, previously isolated from L. citriodora.9^7

Compound 3 (10 mg) was obtained as yellowish brown crystals. Mass spectrum showed the molecular ion (M + ) at164 m/z and the base peak at 92 m/z (quinonium ion). Comparing its

spectral data (Table 5) to those of 4-hydroxycinnamic acid,36'37 it was concluded that compound 3 is 4-hydroxycinnamic acid. This compound was previously isolated from Lippia scaberr-ima.38 In fact such phenol carboxylic acids are precursors to more complex secondary metabolites as flavonoids.39 Collectively, compounds 1 and 3 were isolated for the first time from L. citriodora.

3.2. In-vitro propagation and spectrophotometry estimation of total flavonoids

The effects of explant type, method of sterilization and growth regulators on the in-vitro callus formation of L. citriodora were studied (Table 6). Although the first sterilization method, group (1), gave the least percentage of contamination (Fig. 1), it hindered callus formation probably being too vigorous to the explants' tissues. So group (3) sterilization method was the best as it allowed callus formation, in spite of leading to the highest contamination percentage. Both shoot tips and leaf explants (cut at the midrib) gave callus on MS media impregnated with 4 mg/L BAP. Neither using higher concentrations of BAP nor addition of NAA and/or IBA did promote callus formation in this study (Table 6). Ahmed et al.13 reported the induction of shoots from nodal explants of Lippia nodiflora on MS medium supplemented with 2.5 mg/L BAP and 0.5 mg/L kinetin. Also Gupta et al.14 produced multiple shoots from stem nodal segments of L. alba on MS medium impregnated with 2 ig/ml 6-benzyl adenine. The percent of total flavonoids in the fresh callus was 0.78% calculated as luteolin compared to 1.14% in the fresh aerial parts of the cultivated plant. However, further studies are still required to increase the flavonoidal content of the callus.

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