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Phenolic constituents, hepatoprotective and cytotoxic activities of Pluchea

dioscoridis

Article in International Journal of Applied Research in Natural Products · October 2014

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Sayed A El-Toumy Sayed Ahmed

National Research Center, Egypt Beni Suef University
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International Journal of Applied Research in Natural Products
Vol. 7 (4), pp. 1-10.
Directory of Open Access Journals
©2008-2014. IJARNP-HS Publication

Original Research

Phenolic constituents, hepatoprotective and cytotoxic

activities of Pluchea dioscoridis
El-Toumy SA1, Ahmed SA2*, Kamel EM2
1
Chemistry of Tannins Department, National Research Center, Dokki, Cairo, Egypt.
2
Chemistry Department, Faculty of Science, Beni Suef University, Beni Suef, Egypt.

Summary. The hepatoprotective effect of aqueous ethanol extract of Pluchea dioscoridis aerial parts was investigated against carbon
tetrachloride-induced acute hepatotoxicity in rats. The hepatoprotective activity of P. dioscoridis was evaluated by measuring levels of
serum marker enzymes activities: aspartat amino transferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and
gamaglutamyl (γ-GT). The histological studies were also carried out to support the above parameters. Administration of P. dioscoridis (100
mg/kg) markedly prevented CCl4 –indeduced elevation of serum AST, ALT, ALP and γ-GT activities. A phytochemical study of P.
dioscoridis resulted in the isolation of Kaempferol 3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (1), Quercetin 3-O-α-L-
rhamnopyranosyl-(1→6)-β-D-glucopyranoside (Rutin) (2), 5, 7, 4ʼ-trihydroxy 8-C-glucopyranoside (vitexin) (3), 5, 7, 4ʼ-trihydroxy 6-C-
glucopyranoside (isovitexin) (4), Quercetin-3-O-α-L- rhamnopyranoside (5), Kaempferol 3-O-β-D-glucopyranosyl (6), Quercetin 3-O-β-D-
glucopyranosyl (7), Kaempferol (8) and Quercetin (9). The structures of the isolated compounds were elucidated by chromatography, UV
and 1D/2D 1H/13C NMR spectroscopy. Hepatoprotective effect of P. dioscoridis is probably due to combined action of flavonoids. The
cytotoxicity of the aqueous ethanol extract was tested against five human tumor cell lines namely; HCT116, MCF7, HepG2, HeLa and
HEp2. The extract showed the highest activity against human epidermoid larynx carcinoma cells (HEp2 cell line) with IC50 of 10.2 µg/ml.
Industrial Relevance. Natural products of plant origin are important sources for the discovery of new drugs. The outcome of our study
depicts the potential of the aqueous ethanolic extract of P. dioscoridis as anticancer and hepatoprotective agent. The obtained data suggests
that P. dioscoridis extract showed a potent cell growth inhibition activity on all tested cancer cell lines, in a dose dependent manner and a
hepatoprotective activity.
Keywords. Pluchea dioscoridis; phenolics; flavonoids; cytotoxicity; hepatoprotective activity

INTRODUCTION

Natural products, mainly the plant-derived constituents, have long been considered as source of drugs, and a great part of
the pharmaceuticals available in modern medicine are directly or indirectly derived from natural sources. Natural products
are also of great interest for the programmers of drug discovery, due to their large diversity in nature (Farnsworth and
Bingel, 1997). Even today, natural products are important sources of drugs and almost 60% to 75% of the world's population
use plant-based medicines for initial pharmaceutical cure (WHO, 2002). Between 1981 and 2002, it is estimated that almost
50 % of new chemical entities authorized to initiate clinical studies were natural products or related natural products
(Balunas and Kinghorn, 2005). Moreover, natural products exhibiting various biological activities usually possess a complex
and diverse structure leading to strong challenges in organic synthesis (Rollinger, et al., 2004; Newman, et al., 2003).
Despite tremendous advances in modern medicine, hepatic disease remains a worldwide health problem, thus the search
for new patents is still ongoing. Numerous formulations of medicinal plants are used to treat liver disorders. Many of this of
these treatments act as radical scavengers, whereas others enzyme inhibitors or mitogens (Fadhel and Amran, 2002). Carbon
tetrachloride accumulates in hepatic paraenchyma cells and is metabolized to CCl3 M radicals by liver cytochrome P450-
dependent monooxygenases (Recknagel, 1983). Research shows that hepatoprotective effects have been associated with
plant extracts that are rich in phenolic compounds (De et al., 1996; Shannmugasundaram and Venkataraman, 2006; Jain et
al., 2008; Sabir and Rocha, 2008; Huang et al., 2010; Srivastava and Shivandappa, 2009; Zeashan et al., 2009). Many of the
pluchea species have been utilized as antioxidant (Jahangir et al., 2005; Sen et al., 2005), anticancer (Schmidt et al., 2009),
antiulcer (Pal and Chaudhuri, 1989), Antinociceptive (Perez et al., 1996), Anti-amoebic (Biswas et al., 2007), antibacterial
(Sittiwet, 2009; Pramanik and Chatterjee, 2008), antifungal (Mandeel and Taha, 2005) and a treatment for Alteration in the
Gastrointestinal Absorptive Characteristics (Burger et al., 2005). Previously phytochemical screening of plants from the
genus pluchea resulted in the isolation of triterpenes, sterols, flavonoids, glycosides and sesquiterpene lactones. Boehmerol
acetate, sorghumol acetate, monoterpenic ester, chromenone, steroidal latone, Pluchein and plucheinol (sequiterpenes)
(Ahmed and Kamel, 2013; Greca et al., 1990; Córdova et al., 2006; Suliman et al., 2006; Scholz et al., 1994; Alqasoumi,
20009; Arriaga-Giner et al., 1983; Chopra et al., 1996; Chawla et al., 1991; Dixit and Tewari, 1991; Ohtsuki et al., 2008).

___________________
*Corresponding Author.
 sayed.hassan@science.bsu.edu.eg
 +201221799055
Available online http.//www.ijarnp.org
 El-Toumy et al.

MATERIALS AND METHODS

Plant Material. Aerial parts of P. dioscoridis were collected in April 2008 from the road Suez-Cairo desert. Identification
of the plants was confirmed by Prof. Dr. Salwa A. Kawashty. National Research Center, Cairo, Egypt and comparison with
herbarium specimens. Voucher Specimens were kept in herbarium, National Research Center, Cairo, Egypt
Extraction and Isolation. The comminuted air-dried aerial parts of P. dioscoridis (1.5kg) was defatted with chloroform (2
x 3 L) and exhaustively extracted with EtOH : H2O (7 : 3) under reflux over a boiling water bath for 10 hours. The extract
was then filtered and the solvent was removed in vacuo at ≈40°C. Finally, the extraction process was repeated to yield 150 g
of dark amorphous material. A sample (100 g) dissolved in 50 ml aqueous methanol (70%) was carefully applied to the top
of a column (150 x 3.5 cm) containing 500 gm of polyamide 6S. Gradient elution started with water followed by H2O/EtOH
mixtures of decreasing polarities at a flow rate 1 ml/minute was then carried out. The bands migrated along the column were
traced under UV light during elution to note their characteristics and to control the fractionation process as well. Six
fractions were then obtained, individually collected, dried under vacuo at ≈40 oC and subjected to detailed investigations by
TDPC. Phytochemical investigation of the first fraction revealed the presence of trace amounts of phenolic compounds and
the majority of this fraction is free sugars which were elucidated by comparative paper chromatography (CoPC) to be
glucose, and rhamnose. The residual material of the second fraction (200mg) obtained after the evaporation of the eluent
20% ethanol was applied on a polyamide column chromatography (80 x 3 cm), and eluted by the solvent system 30 %
ethanol-water which gave two successive sub-fractions, the two sub fractions were further purified by Sephadex LH-20
column chromatography (60 x 1.5 cm) and eluted by methanol HPLC to afford the purified samples of compound (1) (70mg)
and compound (2) (60mg).. The third fraction (180mg) obtained after the evaporation of the eluent 40% ethanol was applied
on a polyamide column chromatography (80 x 3 cm), and eluted by the solvent system 40 % ethanol-water which gave two
successive sub-fractions, the two sub fractions were further purified by Sephadex LH-20 column chromatography (60 x 1.5
cm) and eluted by methanol HPLC to afford the purified samples of compound (3) (45mg) and compound (4) (55mg). The
residual material of the fourth fraction (150 mg) obtained after the evaporation of the eluent (60 % EtOH) was applied on a
cellulose column and eluted by using H2O/EtOH mixtures for elution led to the desorption of two sub-fractions (i) and (ii)
which were individually collected and dried in vacuo. Repeated preparative PC of sub-fraction (i) on Whatman Paper No.
3MM and irrigation with BAW afforded chromatographically pure samples of both components (5) (30 mg) and (6) (34 mg),
While the second fraction (ii) afforded a pure sample of compound (7) (28mg). The under vacuum dried constituents of the
fifth fraction (100 mg) obtained after the evaporation of the eluent (80 % EtOH) was applied on a polyamide column using
80 % ethanol as an eluent, and by preparative paper chromatography using 15% AcOH solvent system which gave two
successive sub-fractions. The two sub fractions were further purified by Sephadex LH-20 column and eluted by methanol
HPLC to afford the purified samples of compound (8) (40mg) and compound (9) (35mg). Each purified compound was
subjected to detailed studies to elucidate its structure.
Chemical Characterization of the Isolated Compound. Kaempferol 3-O-α-L-rhamnopyranosyl-(1→6)-β-D-
glucopyranoside (1). Yellow amorphous powder, UV Spectral Data, λ max (nm); MeOH: 260 nm, 300sh, 350 nm;
+NaOMe: 273 nm, 325sh, 409 nm; +NaOAc: 268 nm, 310, 380 nm; +NaOAc /H3BO3: 266 nm, 300 nm, 360 nm; +AlCl3:
278 nm, 310 nm, 360 nm, 398 nm; +AlCl3/ HCl: 278 nm, 309 nm, 362 nm, 398 nm. 1H NMR (DMSO-d6), Aglycone: δ
(ppm): 8.05 (2H, d, J = 8.8 Hz, H-2', H-6'), 6.9 (2H, d, J = 8.8 Hz, H-3', H-5'), 6.43(1H, d, J = 2.0 Hz, H-8), 6.2 (1H, d, J =
2.0 Hz, H-6). Sugar: δ (ppm): 5.32 (1H, d, J = 7.5 Hz, H-1''), 4.38 (1H, d, J = 2.0 Hz, H-1'''), 3-3.7 (9H, m, H-2''- H-6'', H-
2'''- H-5'''), 1.03 (3H, d, J = 6.15, CH3 of rhamnose). 13C NMR: Aglycone; δ (ppm): 157.6 (C-2), 133.5 (C-3), 177.7 (C-4),
161.5 (C-5), 99.0 (C-6) , 164.5 (C-7), 94.1 (C-8), 156.8 (C-9), 104.4 (C-10), 121.2 (C-1'), 131.2 (C-2'), 115.4 (C-3'), 160.2
(C-4'), 115.4 (C5'), 131.2 (C-6'). Sugar; Glucose at 3- position: δ (ppm): 101.7 (C-1''), 74.5 (C-2''), 76.1 (C-3''), 72.2 (C-4''),
76.7 (C-5''), 67.2 (C-6''). Rhamnose at 3- position as terminal sugar: δ (ppm): 101.1 (C-1'''), 70.3 (C-2'''), 70.7 (C-3'''), 70.9
(C-4'''), 68.5 (C-5'''), 18.0 CH3 of Rhamnose. Negative ESI-Mass Data; [M+Na] ¯ = m/z 617.14. From these data, compound
1 was identified as; Kaempferol 3-O α-L-rhamnopyranosyl)-(1→6))-β-D-glucopyranoside.
Quercetin 3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyanoside (Rutin) (2).Yellow powder, UV Spectral Data, λ max
(nm); MeOH: 258 nm, 358 nm; +NaOMe: 272 nm, 329sh, 410 nm; +NaOAc: 272 nm, 324 nm, 385 nm; +NaOAc /H3BO3:
261 nm, 378 nm; +AlCl3:274 nm, 338 nm, 433 nm; +AlCl3/ HCl: 269 nm, 297sh, 362, 398 nm. 1H NMR (DMSO-d6),
Aglycone: δ (ppm): 7.57 (1H, d, J = 2.1 Hz, H-2'), 7.54 (1H, dd, J = 2.1 Hz, H-6'), 6.85 (1H, d, J = 9Hz, H-5'), 6.39 (1H, d, J
= 2.1 Hz, H-8), 6.2 (1H, d, J = 2.1 Hz, H-6). Sugar: δ (ppm): 5.35 (1H, d, J = 7.5 Hz, H-1''), 4.4 (1H, d, J = 2.0 Hz, H-1'''),
3.16-3.65 (9H, m, H-2''- H-6'', H-2'''- H-5'''), 1.01 (3H, d, J = 6.3 Hz, CH3 of rhamnose). 13C NMR, Aglycone: δ (ppm): 158.6
(C-2), 135.6 (C-3), 179.4 (C-4), 163.0 (C-5), 100.1 (C-6) , 166.5 (C-7), 95.0 (C-8), 159.3 (C-9), 105.5 (C-10), 123.1 (C-1'),
117.7 (C-2'), 145.9 (C-3'), 149.9 (C-4'), 116.1 (C5'), 123.5 (C-6'). Sugar: Glucose at 3- position: δ (ppm): 104.8 (C-1''),
75.7 (C-2''), 78.2 (C-3''), 71.4 (C-4''), 77.2 (C-5''), 68.6 (C-6''). Rhamnose at 3- position as terminal sugar: δ (ppm): 102.4 (C-
1'''), 72.1(C-2'''), 72.2 (C-3'''), 73.9 (C-4'''), 69.7 (C-5'''), 17.9 CH3 of Rhamnose. Negative ESI-Mass Data; [M-H] ¯ = m/z
609.147. From the previous data, compound 2 was identified as, quercetin 3-O- α-L-rhamnopyranosyl-(1→6))-β-D-
glucopyranoside (rutin).
5, 7, 4-trihydroxy flavone 8-C-glucopyranoside (vitexin) (3). Yellow powder, UV Spectral Data, λ max (nm); MeOH: 334
nm, 272 nm; +NaOMe: 391 nm, 332 sh, 381 nm; +NaOAc: 391 nm, 305 sh, 281 nm; +NaOAc /H3BO3: 350 nm, 400 sh, 278
nm; +AlCl3:340 nm, 385 nm, 305 nm, 278 nm; +AlCl3/ HCl: 340 nm, 385 nm, 365 nm, 278 nm. 1H NMR (DMSO-d6),
Aglycone: δ (ppm): 8.02 (2H, d, J = 8.4 Hz, H-2', H-6'), 6.90 (2H, d, J = 8.4 Hz, H-3', H-5'), 6.75 (1H, S, H-3), 6.27 (1H, S,
H-6), 4.68 (1H, d, J = 9.1 Hz, H-1''). Sugar: δ (ppm): 5.61 (1H, d, J = 7.2 Hz, H-1''), 3.2-3.7 (5H, m, H-2''- H-6''). 13C NMR,
Aglycone: δ (ppm): 163.99 (C-2), 102.94 (C-3), 182.03 (C-4), 160.44 (C-5), 98.23 (C-6) , 162.69 (C-7), 104.76 (C-8),
156.05 (C-9), 104.07 (C-10), 121.66 (C-1'), 128.99 (C-2', C-6'), 115.88 (C-3', C-5'), 161.19 (C-4'). Sugar: 73.48 (C-1''),
70.93 (C-2''), 78.73 (C-3''), 70.63(C-4'''), 81.88 (C-5''), 61.38 (C-6''). Negative ESI-Mass Data; [M-H] ¯ = m/z 431. From
these data we can conclude that compound 3 is 5,7,4'- trihydroxyflavone 8-C-glucopyranoside (vitexin).

2
 Phenolic constituents, hepatoprotective and cytotoxic activities of Pluchea dioscoridis

5, 7, 4-trihydroxy flavone 6-C-glucopyranoside (isovitexin) (4). Yellow powder, UV Spectral Data, λ max (nm); MeOH:
3342 nm, 272 nm; +NaOMe: 339 nm, 332 sh, 281 nm; +NaOAc: 391 nm, 305 sh, 281 nm; +NaOAc /H3BO3: 350 nm, 400
sh, 278 nm; +AlCl3:340 nm, 385 nm, 305 nm, 278 nm; +AlCl3/ HCl: 340 nm, 385 nm, 365 nm, 278 nm.1H NMR (DMSO-
d6), Aglycone: δ (ppm): 7.88 (2H, d, J = 8.4 Hz, H-2', H-6'), 6.901 (2H, d, J = 8.4 Hz, H-3', H-5'), 6.7 (1H, S, H-3), 6.44
(1H, S, H-6), 4.61 (1H, d, J = 9.1 Hz, H-1''). Sugar: δ (ppm): 5.61 (1H, d, J = 7.2 Hz, H-1''), 3.21-3.74 (5H, m, H-2''- H-6'').
13
C NMR, Aglycone: δ (ppm): 163.23 (C-2), 102.6 (C-3), 181.63 (C-4), 161.43 (C-5), 109.09 (C-6) , 161.44 (C-7), 94.02
(C-8), 156.47 (C-9), 102.60 (C-10), 121.06 (C-1'), 128.37 (C-2', C-6'), 116.10 (C-3', C-5'), 160.71 (C-4'). Sugar: 73.32 (C-
1''), 70.64 (C-2''), 79.11 (C-3''), 70.27(C-4'''), 81.44 (C-5''), 61.47 (C-6''). Negative ESI-Mass Data [M-H] ¯ = m/z 431.
Compound 4 was identified as 5,7,4- trihydroxy flavone 6-C-glucopyranoside (isovitexin).
Quercetin-3-O-α-L-rhamnopyranoside (5). Yellow powder, UV Spectral Data, λ max (nm); MeOH: 253 nm, 263sh, 344
nm; + NaOMe: 272 nm, 322sh, 372 nm; + NaOAc: 260 nm, 300sh, 367 nm; + H3BO3: 272 nm, 382 nm; +AlCl3: 272 nm,
304sh, 333sh, 430 nm; +HCl: 272 nm, 303sh, 353 nm, 401 nm. 1H NMR (DMSO-d6), Aglycone: δ (ppm): 7.33 (1H, d, J =
2.0 Hz, H-2' ), 7.28 (1H, dd, J = 2.0 Hz, and J=8.5Hz, H-6' ), 6.9 (1H, d, J = 8.5Hz, H-5'), 6.42 (1H, d, J = 2.0 Hz, H-8), 6.23
(1H, d, J = 2.0 Hz, H-6). Sugar: δ (ppm): 5.29 (1H, d, J = 1.41 Hz, H-1''), 3.16-3.94 (4H, m, H-2''- H-5''),1.03 (3H ,d, J =
6.15 Hz, CH3 of rhamnose) 13C NMR, Aglycone: δ (ppm): 157.34 (C-2), 134.27 (C-3), 177.79 (C-4), 161.35 (C-5), 98.78
(C-6), 164.34 (C-7), 93.70 (C-8), 156.86 (C-9), 104.16 (C-10), 121.17 (C-1'), 115.52 (C-2'), 145.25 (C-3'), 148.50 (C-4'),
115.72 (C-5'), 120.80 (C-6'). Sugar: δ (ppm): 101.87 (C-1''), 70.43 (C-2''), 70.63 (C-3''), 71.25 (C-4''), 70.11 (C-5''), 17.54
(C-6''). Negative ESI-Mass Data [M-H] ¯ = m/z 447. These data when compared with the published data, compound 5 was
identified as quercetin-3-O-α-L-rhamnopyranoside.
Kaempferol 3-O-β-D-glucopyranosyl, (Astralagin) (6). Greenish yellow amorphous powder, UV Spectral Data, λ max
(nm); MeOH: 266 nm, 346 nm; +NaOMe: 274 nm, 327sh, 401 nm; +NaOAc: 274 nm, 305 nm, 393 nm; +NaOAc/H3BO3:
267 nm, 352 nm; +AlCl3: 274 nm, 304 nm, 349 nm, 396 nm; +AlCl3/ HCl: 274 nm, 345 nm, 394 nm. 1H NMR (DMSO-d6),
Aglycone: δ (ppm): 8.1 (2H, d, J = 8.8 Hz, H-2', H-6'), 6.98 (2H, d, J = 8.8 Hz, H-3', H-5'), 6.54(1H, d, J = 2.0 Hz, H-8), 6.31
(1H, d, J = 2.0 Hz, H-6). Sugar: δ (ppm): 5.57 (1H, d, J = 7.2 Hz, H-1''), 3.23-3.65 (5H, m, H-2''- H-6''). 13C NMR,
Aglycone: δ (ppm): 156.76 (C-2), 133.57 (C-3), 177.82 (C-4), 161.59 (C-5), 99.10 (C-6), 164.63 (C-7), 94.04 (C-8), 156.76
(C-9), 104.34 (C-10), 121.28 (C-1'), 131.24 (C-2') 115.47, (C-3'), 160.31 (C-4'), 115.47 (C-5'), 131.24 (C-6'). Sugar: δ
(ppm): 101.29 (C-1''), 70.28 (C-2''), 74.59 (C-3''), 76.80 (C-4''), 77.83 (C-5''), 61.23 (C-6''). From the previous data
compound 6 is identified as kampferol-3-O-β-D-glucopyranoside.
Quercetin 3-O -β-D- glucopyranosyl (7). Yellow amorphous powder, UV Spectral Data, λ max (nm); MeOH: 253 nm,
263sh, 294sh, 351 nm; +NaOMe: 271 nm, 328sh, 410 nm; +NaOAc: 273 nm, 321 nm, 375 nm; +NaOAc /H3BO3:262 nm,
300sh, 377 nm; +AlCl3:275 nm, 305sh, 332 nm, 435 nm; +AlCl3/ HCl: 275 nm, 305sh, 361sh, 403 nm. 1H NMR (DMSO-
d6), Aglycone: δ (ppm): 7.67 (1H, dd, J = 2.12 Hz, and J=8.62 Hz, H-6' ), 7.53 (1H, d, J = 2.12 Hz, H-2' ), 6.82 (1H, d, J =
8.62 Hz, H-5'), 6.4 (1H, d, J = 1.8 Hz, H-8), 6.2 (1H, d, J = 1.8 Hz, H-6). Sugar: δ (ppm): 5.37 (1H, d, J = 7.63 Hz, H-1''),
3.28-3.65 (5H, m, H-2''- H-6''). 13C NMR, Aglycone: δ (ppm): 156.80 (C-2), 133.60 (C-3), 177.50 (C-4), 161.60 (C-5), 98.90
(C-6), 164.60 (C-7), 93.80 (C-8), 156.60 (C-9), 104.00 (C-10), 121.60 (C-1'), 115.80 (C-2'), 145.80 (C-3'), 148.80 (C-4'),
116.20 (C-5'), 122.00 (C-6'). Sugar: δ (ppm): 101.20 (C-1''), 71.60 (C-2''), 74.40 (C-3''), 70.02 (C-4''), 77.70 (C-5''), 61.50
(C-6''). Negative ESI-MS Data [M-H] ¯ = m/z 462.9. Data of 7 was compared with the published data, and it was found to
be quercetin 3-O-β-D-glucopyranoside.
Kaempferol (8). Greenish yellow amorphous powder, UV Spectral Data, λ max (nm); MeOH: 266 nm, 292sh, 319sh, 366
nm; +NaOMe: 276 nm, 320sh, 411 nm; +NaOAc: 274 nm, 306 nm, 378 nm; +NaOAc/H3BO3: 265 nm, 294 nm, 319 nm, 369
nm; +AlCl3: 269 nm, 305 nm, 350 nm, 423 nm; +AlCl3/ HCl: 267 nm, 305 nm, 350 nm, 424 nm. 1H NMR (DMSO-d6), δ
(ppm): 8.03 (2H, d, J = 8.5 Hz, H-2', H-6'), 6.93 (2H, d, J = 8.5 Hz, H-3', H-5'), 6.41 (1H, d, J = 2.0 Hz, H-8), 6.17 (1H, d, J
= 2.0 Hz, H-6). 13C NMR: δ (ppm): 146.80 (C-2), 135.84 (C-3), 176.20 (C-4), 161.60, (C- 5), 98.60 (C-6), 164.59 (C-7),
93.85 (C-8), 156.40 (C-9), 103.70, (C-10), 121.90 (C-1’), 129.90 (C-2’, C-6’), 115.80 (C-3’, C-5’), 159.5 (C-4’).EI-Mass
Data: [M]+ = m/z 286. From these data compound 8 is identified as kampferol.
Quercetin (A9). Yellow amorphous powder, UV Spectral Data, λ max (nm); MeOH: 256 nm, 295 nm, 370 nm; +NaOMe:
269 nm, 331sh, 428 nm; +NaOAc: 276 nm, 319sh, 380 nm; +NaOAc /H3BO3:260 nm, 301sh, 383 nm; +AlCl3:271 nm,
302sh, 349 nm, 442 nm; +AlCl3/ HCl: 267 nm, 302sh, 358, nm 428 nm. 1H NMR (DMSO-d6), δ (ppm): 7.69 (1H, d, J = 2.1
Hz, H-2' ), 7.57 (1H, dd , J = 2.1 Hz, and 8.4 Hz, H-6' ), 6.9 (1H, d, J = 8.4 Hz, H-5'), 6.42 (1H, d, J = 1.8 Hz, H-8), 6.2 (1H,
d, J = 1.8 Hz, H-6). 13C NMR: 147.50 (C-2), 136.44 (C-3), 176.55 (C-4), 161.43 (C-5), 98.88 (C-6), 164.59 (C-7), 94.05
(C-8), 156.83 (C-9), 103.71 (C-10), 122.66 (C-1'), 116.31 (C-2'), 145.76 (C-3'), 148.40 (C-4'), 115.76 (C-5'), 120.68 (C-6').
Negative ESI-Mass Data: [M-H] ¯ = m/z 301.2. In accordance with the published data compound 9 was found to be
quercetin.
Hepatoprotective Studies. Animals. Thirty-six male Sprague- Dawley rats weighing (120- 150 g) were purchased from
the Animal House,National Research Centre, Dokki, Cairo, Egypt. They were housed at standard environmental condition
andwere allowed free access to tap water and pellet diet. The study has got the approval from the Local Ethical Committee,
in the National Research Centre. Acute toxicity study was performed on rats during 24-72 hrs. Rats were divided into six
groups, each of six
Group A: Rats served as normal control and were orally administered saline for 30 days.
Group B: Rats were administered aqueous ethanol extract of P. dioscoridis (100 mg/kg b.w., oral) alone for 30 days.
Group C: Rats were administered CCl4 (1.5 mg/kg b.w., oral, 50 % w/w paraffin oil) twice weekly for 30 days(Saraf and
Dixit, 19991; Mohideen, et al., 2003).
Group D: Rats were administered aqueous ethanolic extract of P. dioscoridis (100 mg/kg b.w., oral) for 15 days followed
by administration of a CCl4 (1.5 mg/kg b.w., oral) twice weekly until day 30 (Saraf and Dixit, 19991; Mohideen, et al.,
2003).
Group E: Rats were administered CCl4 (1.5 mg/kg b.w., oral) twice weekly for 15 days followed by administered aqueous
ethanolic extract of P. dioscoridis (100 mg/kg b.w., oral) until day 30(Saraf and Dixit, 19991; Mohideen, et al., 2003).

3
 El-Toumy et al.

Group F: Rats were administered aqueous ethanolic extract of P. dioscoridis (100 mg/kg b.w., oral) concomitant CCl4
(1.5 mg/kg b.w., oral twice weekly) for 30 days(Saraf and Dixit, 19991; Mohideen, et al., 2003).
Biochemical Assessment. At the end of the experiment, blood samples were obtained from the retro-orbital vein plexuses,
under ether anaesthesia. ALT and AST activities in serum were measured according to Reitman and Frankel (1957),
determination of ALP activity was done according to the method of Belfield and Goldberg (1971) and γ- glutamyl
transferase by Sazasz (1976).
Histopathological Studies. Liver samples were excised, washed with normal saline and processed separately for
histopathological observation. Initially the materials were fixed in 10% buffered neutral formalin and paraffin sections were
taken at 5 μm thickness processed in alcohol-xylene series and was stained with alum hematoxylin and eosin. The sections
were examined microscopically for histopathology changes.
Statistical analysis. One way ANOVA (PC – STAT, 1985) was used for data analysis. Results were expressed as mean ±
standard error (SE). Values of P>0.05 were considered statistically non-significantly different, while values of P<0.05,
P<0.01 and P<0.001 were significantly, highly significantly and very highly significantly different respectively.
Cytotoxicity Activity. Cancer cell lines. All cell lines were purchased from the American Type Culture Collection
(ATCC). Five cell lines were used in this study; HepG2 cells (Human cell line of a well differentiated hepatocellular
carcinoma isolated from a liver), HeLa (Cervical carcinoma cells), HCT 116 (Colon carcinoma cells), MCF-7 (Breast
carcinoma cells), HEp2 (Human epidermoid larynx carcinoma cells) and Vero cell line (Normal kidney cells) to evaluate the
cytotoxic effect of ethanolic extract of P. dioscoridis. Cells were grown or maintained upon arrival at 37°C in a humidified
incubator with 5% CO2 and 95 % atmosphere as recommended by ATCC. The cells were propagated in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine, HEPES buffer and
50 µg/ml gentamycin.
Chemicals used: Dimethyl sulfoxide (DMSO), crystal violet and trypan blue dye were purchased from Sigma (St. Louis,
Mo., USA). DMEM, RPMI-1640, FBS, HEPES buffer solution, L-glutamine, gentamycin and 0.25% Trypsin-EDTA were
purchased from (Bio Whittaker® Lonza, Belgium).
Crystal violet stain (1%). It composed of 0.5% (w/v) crystal violet and 50% methanol then made up to volume with
ddH2O and filtered through a Whatmann No.1 filter paper.
Cytotoxicity evaluation using viability assay. For cytotoxicity assay, the cells were seeded in 96-well plate at a cell
concentration of 1x 104 cells per well in 100 µl of growth medium. Fresh medium containing different concentrations of the
extract was added after 24 hours of seeding. Serial two-fold dilutions of the tested extract were added to confluent cell
monolayer dispensed in 96-well, flat-bottomed microtiter plates (Falcon, NJ, USA) using multi channel pipette. The
microtiter plates were incubated at 37°C in a humidified incubator with 5% CO2 for a period of 48 h. Three wells were
used for each concentration of the tested extract. Control cells were incubated without tested sample and with or without
DMSO. The little percentage of DMSO present in the wells (maximal 0.1%) was found not to affect the experiment. After
incubation of the cells for 24 h at 37°C, various concentrations of the extract (50, 25, 12.5, 6.25, 3.125 and 1.56 µg)
were added and the incubation was continued for 48 h and viable cells yield was determined by colorimetric method.
After the end of the incubation period, media were aspirated and the crystal violet solution (1%) was added to each
well for at least 30 minutes. The stain was removed and the plates were rinsed using tap water until all excess stain
is removed. Glacial acetic acid (30%) was then added to all wells and mixed thoroughly, then the absorbance of the
plated were measured after gently shaken on Microplate reader (TECAN, Inc,), using a test wavelength of 490nm.
All results were corrected for background absorbance detected in wells without adding stain. Treated samples were
compared with the cell control in the absence of the tested extract. All experiments were carried out in triplicate. The
cell cytotoxic effect of each tested concentration was calculated (Mosmann, 1983).

RESULTS AND DISCUSSION

Phytochemical studies. Fractionation of the ethanolic extract of P. dioscoridis resulted in isolation and identification of
nine flavonoids (1-9) (Fig. 1). The structure of the isolated compounds was elucidated by chromatography, conventional
chemical and spectroscopic methods of analysis (UV, 1/2D NMR). By comparison of the spectral data of the isolated
compounds with those reported, these isolated compounds were identified as: Kaempferol 3-O-α-L-rhamnopyranosyl-
(1→6)-β-D-glucopyranoside (1) (Markham and Geiger, 1994), Quercetin 3-O-α-L-rhamnopyranosyl-(1→6)-β-D-
glucopyranoside (Rutin) (2) (Markham and Geiger, 1994), 5, 7, 4ʼ-trihydroxy 8-C-glucopyranoside (vitexin) (3) (Velandia et
al., 2002), 5, 7, 4ʼ-trihydroxy 6-C-glucopyranoside (isovitexin) (4) (Velandia et al., 2002), Quercetin-3-O-α-L-
rhamnopyranoside (5) (Ye and Huang, 2006), Kaempferol 3-O-β-D-glucopyranosyl (6) (Markham and Geiger, 1994),
Quercetin 3-O-β-D-glucopyranosyl (7) (Vvedenskaya et al., 2004), Kaempferol (8) (Mabry et al., 1970; Markham, 1982)
and Quercetin (9) (Zheng et al., 2008).
Cytotoxicity assay. The ethanolic extract was tested for cytotoxicity to five human cancer cell lines, namely, colon
(HCT116), breast (MCF7), liver (HEPG2), Cervical (HeLa) and larynx (HEp2) cell lines. The activities were expressed by
the IC50 value and their results are shown in (Fig. 2) and table 1. According to the American National Cancer Institute
guidelines (Suffness and Pezzuto, 1990) extracts with IC50 values <30 μg/ml were considered active. It was found that the
ethanolic extract was active against colon (HCT116), breast (MCF7), liver (HEPG2), Cervical (HeLa) and larynx (HEp2)
human cancer cell lines with an IC50; 21.2, 13.9, 22.6, 15.2 and 10.2μg/ml; respectively.
Cytotoxicity screening models provide important preliminary data to help selecting plant extract with potential
antineoplastic properties for future work (Cardellina et al., 1999). In view of the present data, P. dioscoridis extract showed
a potent cell growth inhibition activity on all tested cancer cell lines, in a dose dependent manner, with more potent
antiproliferative activity against the human epidermoid larynx carcinoma cells (Hep2 cell line) with IC50 of 10.2 µg/ml, as
compared to the control cells. Flavonoids, terpenoids and phenolics were identified in the current tested plant. This
observation is of particular importance since flavonoids are ingredients of many vegetables and fruits and the association of

4
 Phenolic constituents, hepatoprotective and cytotoxic activities of Pluchea dioscoridis

vegetable and fruit consumption with reduced cancer risk has been reported (Ferguson et al., 2004; Kanadaswami et al.,
2005). Flavonoids have been shown to exhibit a series of biological effects among which stand out the inhibition of lipid
peroxidation and platelet aggregation, due to their antioxidant properties and their ability of removing free radicals and
chelating divalent cations (Hanasaki et al., 1994). Data of the phytochemical screening showed the presence of the
flavonoids quercetin and kaempferol. Quercetin and its derivatives, have received special attention as dietary constituents
during the last few years. The epidemiological studies pointed out to their possible role in preventing cardiovascular diseases
and cancer (Kamalakkanan and Prince, 2006). Also, quercetin has been reported to exert numerous pharmacological
activities, such as free radical scavenging (Horvathova et al., 2003), TNF- α inhibition (Park et al., 2000), anti-carcinogenic
effects (Van der logt et al., 2003) and arrest or G1 arrest in different cell types (Beniston and Campo, 2003). Moreover,
quercetin-mediated apoptosis may result from the induction of stress proteins, disruption of microtubules and mitochondrial,
release of cytochrome c, and activation of caspases (Ong et al., 2004). Moreover, Russo (2007) reported that the antioxidant,
anti-inflammatory, antiproliferative, and apoptotic effects of quercetin have been largely analyzed in cell culture models, and
it is known to block NF-κB activation.

Figure 1. Structures of the isolated compounds

5
 El-Toumy et al.

Figure 2. Cytotoxicity assay of the aqueous ethanolic extract of P. dioscoridis against Vero, MCF-7, HCT 116, HeLa, HepG2 and HEp2
cells.

Table 1. Cytotoxic activity of aqueous ethanolic extract of p. dioscoridis

IC50 value (μg/ml)

Cell lines HepG2 HEp2 HeLa HCT MCF-7

aq. ethanolic extract 22.6±1.41 10.2±1.21 15.2±0.94 21.8±2.01 13.9±1.11

Hepatoprotective activity. P. dioscoridis total alcoholic extract had no toxic signs up to 3 gm/kg b.wt oral administration.
Pilot experiment was performed to evaluated effective dose to be used in hepatoprotective study. It was shown that 100
mg/kg the lowest effective. Results in table 2 showed that ALT, AST, ALP and γ-GT in CCl4-injected group significantly
increased compared with control. Significant decrease in plasma enzyme activity of AST, ALT, ALP and γ-GT was
observed in the groups administrated P. dioscoridis extract. Histopathological study of liver in control group showed a
normal hepatic architecture with distinct hepatic cells, sinusoidal spaces, prominent nucleus and a central vein (Fig. 3). The
histological architecture of liver sections in rat treated with 100 mg/kg plant extract showed normal lobular pattern with a
mild lymphocyte infiltration (Fig 3B). However, CCl4–intoxicated group exhibited sever histopathological changes, such as
centrilobular hepatic necrosis, fatty change. Degeneration and broad infiltration of the lymphocytes and Kupffer cells around
the central vein and portal tract, with the loss of cellular boundaries were also observed (Fig. 3C). Pretreatment-rats with P.

6
 Phenolic constituents, hepatoprotective and cytotoxic activities of Pluchea dioscoridis

dioscoridis showed restoration and apparently normal organ with very few hemorrhage in central vein with binucleated cells
were found (Fig. 3D). Thus P. dioscoridis pretreatment greatly inhibited liver morphological changes and necrosis due to
CCl4 hepatotoxicity. Post-treatment of plant extract after CCl4 intoxication also attenuated the hepatic damage induced by
CCl4 with dialed of blood sinusoids with the presence of inflammatory cells (Fig. 3E). Fatty change and lymphocyte
infiltration were improved in the histological sections of group treated with plant extract concomitant with CCl4. Fewer
necrotic zones were also observed (Fig.3). The maximum protection was seen in the group pretreatment with P. dioscoridis
extract.

Table 2. Effect of the ethanolic extract of the aerial parts of P. dioscoridis (100 mg/ Kg) on Serum enzymes of liver (ALT, AST and γ-GT)
and (ALP) in normal and carbon tetrachloride- treated rats (n = 6, means± SE of the means).

Group ALT (µ/L) AST (µ/L) ALP (µ/L) γ-GT

Group A 47.3±0.81 126.5±1.99 215.2±0.91 6.06 ± 0.41
Group B 49.1±0.9** 128.7±0.2** 216.7±0.12* 5.05 ± 0.31
Group C 195.95±1.11*** 193±2.15** 300.3±2.19* 9.54 ± 0.7*
Group D 70.2±0.19** 135±0.22** 245±0.20** 6.8+ 0.51
Group E 150.6±0.7*** 190±0.1*** 290±0.11** 7.5 ± 0.44
Group F 140.1±1.21*** 189±0.99*** 250.1±0.98** 7.2 ± 0.42
Values statistically significant *p < 0.05, **p < 0.01, ***p < 0.001

Figure 3. A. Normal photomicrograph of the liver section of a rat showing the architecture of the hepatic lobule, central vein (CV),
hepatocytes (H), nuclus and blood sinusoids (S) (H&E. 400). B. Photomicrograph of the liver section of a rat received extract of P.
dioscoridis (100 mg/kg), showing normal architecture of the hepatic lobules (H&E .400). C. Photomicrograph of the liver section of carbon
tetrachloridetreated rats showing the distortion of architecture in hepatic cells. Congestion of portal tract (long arrow) and sever cellular
infiltration around portal tract (Head arrow). D. Photomicrograph of the liver section of a rat treated with ethanolic extract of P. dioscoridis
(100 mg/kg) for 15 days followed by administration of CCl4 (1.5 mg/kg) until day 30, showing congestion of central vein with dialated
sinusoids and binucleated cells. E. Photomicrograph of the liver section of a rat treated with CCl4 (1.5 mg/kg) for 15 days followed by
administered ethanolic extract of P. dioscoridis (100 mg/kg) until day 30, showing dialated of central vein with dialated sinusoids and
binucleated cells. F. Photomicrograph of the liver section of a rat treated with ethanolic extract of P. dioscoridis (100 mg/kg) and CCl4 (1.5
mg/kg) at the same time for 30 days, showing congestion of central vein with dialated sinusoids and binucleated cells.
Much attention has been focused on the protective biochemical function of naturally occurring antioxidants in biological
system and on the mechanisms of their action (Freil and Higdon, 2003; Manach et al., 2005). Liver injury induced by CCl4

7
 El-Toumy et al.

are the best characterized system of xenobiotic-induced hepatotoxicity and commonly used models for the screening of
antihepatotoxic and/or hepatoprotective activities of drugs (Lee, et al, 2004). Carbon tetrachloride, a widely used
experimental hepatotoxicant, it is biotransformed by cytochrome P-450 systems to produce the trichloromethyl free radical
(CCl3•) that causes lipid peroxidation and, thereby, produce liver damage (Recknagal. 1967). When administered, carbon
tetrachloride accumulates in hepatic parenchymal cells, which is metabolized to free radical CCl3•. The free radicals react
with molecular oxygen to produce peroxy radicals (H2O 2, O2 and •OH due to incomplete reduction of molecular oxygen),
thereby causing oxidative destruction of polyunsaturated fatty acids (Gebhardt, 2002). These activated radicals bind
covalently to the macromolecules and induce peroxidative degradation of membrane lipids of endoplasmic reticulum, rich in
polyunsaturated fatty acids. Lipid peroxidative degradation of biomembrane is one of the principle causes of hepatotoxicity
(Dhawan et al., 1991). Elevated levels of serum enzymes, ALT, AST and γ-GT are indicative of cellular leakage and loss of
functional integrity of cell membrane in liver (Drotman and Lawhorn, 1978). Treatment with aqueous ethanol extract of P.
dioscoridis decreased the serum levels of ALT, AST and γ-GT towards their respective normal value; that is an indication of
stabilization of plasma membrane as well as repair of hepatic tissue from the damage caused by CCl4. On the other hand
ALP is an indicator of pathological alteration in biliary flow (Ploa and Hewitt, 1989). CCl4 induced elevation of serum ALP
is in line with high levels of serum bilirubin. Effective control of ALP levels in aqueous ethanol extract of P. dioscoridis
treatment groups points towards an early improvement in the secretary mechanism of hepatocytes. These biochemical
findings were further substantiated by histopathological studies. Histopathology of liver of the normal control rats showed
prominent central vein and normal arrangement of hepatic cell (Fig 3A). CCl4 treatment rats showed massive fatty changes,
gross necrosis and broad infiltration section of rats treated with aqueous ethanol extract of P. dioscoridis showed significant
regeneration against CCl4 induced liver damage (Fig 3D and 3F). This is in agreement with data report by Rienke et al.,
(1988), Obi et al., (1998) and Yan-Jun et al., (2004). When hepatecytes are damaged, a variety of enzymes, normally located
in the cytosol, are released into the blood and their estimation is a useful quantitative marker of the extent and type of hepatic
cell damage (Mitra, et al., 1998). Adminsteration of aqueous ethanol extract of P. dioscoridis before CCl4 injection
improved liver enzymes. Phytochemical studies revealed that the presence of flavanoids in extract of P. dioscoridis. Several
flavonoids have been shown to have potential as hepatoprotective agents (Middleton and Kandaswarmi, 1994; Wegner and
Fintelmann, 1999; Palanivel et al., 2008). It is concluded that treatment with aqueous ethanol extract of P. dioscoridis.
decreases the CCl4-induced elevation in biochemical parameters (liver AST, ALT and ALP). These findings suggest that the
aqueous ethanol extract of P. dioscoridis was effective in bringing about functional improvement of hepatocytes. The
healing effect of this extract was also confirmed by histological observations. Our results demonstrated that the possible
hepatoprotective mechanisms of aqueous ethanol extract of P. dioscoridis on CCl4-induced liver damage in rats might be due
to the following effects: (1) inhibiting the cytochrome P450-dependent oxygenase activity; and (2) stabilizing the hepatocyte
membrane. The active compounds of P. dioscoridis which are responsible for the observed hepatoprotective effects, have
been isolated Kaempferol 3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside, Quercetin 3-O-α-L-rhamnopyranosyl-
(1→6)-β-D-glucopyranoside (Rutin) , 5, 7, 4ʼ-trihydroxy 8-C-glucopyranoside (vitexin), 5, 7, 4ʼ-trihydroxy 6-C-
glucopyranoside (isovitexin), Quercetin-3-O-α-L- rhamnopyranoside, Kaempferol 3-O-β-D-glucopyranosyl, Quercetin 3-O-
β-D-glucopyranosyl, Kaempferol and Quercetin.

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