Hepatoprotective and Cytotoxic Activities of Anvillea Garcinii and Isolation of Four New Secondary Metabolites

Hepatoprotective and cytotoxic activities of Anvillea garcinii and isolation of four new secondary metabolites

Shagufta Perveen, Areej Mohammad Al-Taweel, Hasan Soliman Yusufoglu, Ghada Ahmed Fawzy, Ahmed Foudah & Maged Saad Abdel-Kader

Journal of Natural Medicines

ISSN 1340-3443 Volume 72 Number 1

J Nat Med (2018) 72:106-117 DOI 10.1007/s11418-017-1118-1

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J Nat Med (2018) 72:106–117 https://doi.org/10.1007/s11418-017-1118-1

ORIGINAL PAPER

Hepatoprotective and cytotoxic activities of Anvillea garcinii and isolation of four new secondary metabolites

1 1 2 Shagufta Perveen • Areej Mohammad Al-Taweel • Hasan Soliman Yusufoglu • 1,3 2 2,4 Ghada Ahmed Fawzy • Ahmed Foudah • Maged Saad Abdel-Kader

Received: 14 June 2017 / Accepted: 26 July 2017 / Published online: 9 August 2017 Ó The Japanese Society of Pharmacognosy and Springer Japan KK 2017

Abstract Anvillea garcinii is a medicinal plant tradition- fractions and compounds 3, 4, 7 and 8 displayed significant ally used for the treatment of dysentery, gastrointestinal cytotoxic activity against these cell lines. troubles, hepatitis, lung disease, colds, digestive problems and pulmonary affections and in liver diseases. Four new Keywords Anvillea garcinii Á Sesquiterpene lactones Á sesquiterpene lactones, garcinamines A–D, along with Hepatoprotective activity Á Cytotoxicity seven known compounds, were isolated from the leaves of A. garcinii. This is the first report of the isolation of amino acid analogues of parthenolide-type sesquiterpene lactones Introduction from the family Asteraceae. Total ethanol extract of leaves as well as the chloroform and n-butanol fractions were For centuries, medicinal plants have traditionally been used tested for their hepatoprotective effect using the carbon for treating liver diseases. Several natural leads with tetrachloride liver toxicity model. The chloroform fraction, diverse chemical structures were discovered as potential at a dose of 400 mg/kg, demonstrated a significant hep- hepatoprotective agents. Moreover, the development of atoprotective effect comparable to silymarin in all serum new natural anti-cancer drugs with specificity and higher and tissue parameters. The cytotoxicity of all extracts and potency against different cancer cells has become an compounds were evaluated against five human cancer cell important target in biomedical research. In continuation of lines: MCF-7, HCT-116, HepG2, Hela and A-549. The our work on medicinal Saudi plants, we selected Anvillea results indicated that the chloroform and n-butanol garcinii (Burm.f.) DC. (Anvillea garcinii subsp. radiata), which is a shrub with florescent yellow flowers. The genus Anvillea belongs to the family Asteraceae, and comprises Electronic supplementary material The online version of this four species distributed in the region from North Africa to article (doi:10.1007/s11418-017-1118-1) contains supplementary Iran, including a number of Middle Eastern countries, such material, which is available to authorized users. as Egypt, Palestine and Saudi Arabia [1]. Anvillea garcinii & Shagufta Perveen is widely used by local people for its medicinal properties [email protected]; [email protected] and is traditionally used for the treatment of dysentery,

1 gastrointestinal troubles, hepatitis, lung disease, colds, Department of Pharmacognosy, College of Pharmacy, King digestive problems and pulmonary affections, and in liver Saud University, P. O. Box 22452, Riyadh 11495, Kingdom of Saudi Arabia diseases [2]. Anvillea garcinii was proven to have signiﬁ- cant antidiabetic potential by decreasing blood glucose 2 Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University, P.O. Box 173, levels and maintaining serum lipid concentrations at nor- Al-Kharj 11942, Saudi Arabia mal [3]. Previous phytochemical studies on the various 3 Department of Pharmacognosy, Faculty of Pharmacy, Cairo parts of A. garcinii have led to the isolation of different University, Cairo 11562, Egypt biologically active compounds, including germacranolides 4 Department of Pharmacognosy, Faculty of Pharmacy, [4–8], ﬂavanoids and their glycosides [9, 10]. Germacra- Alexandria University, Alexandria 21215, Egypt nolides and their isomers isolated from A. garcinii were 123 Author's personal copy

J Nat Med (2018) 72:106–117 107 shown to possess anti-tumor, anti-HIV and cytotoxic sesquiterpene lactone attached with an aromatic moiety and activities in both in vitro and vivo assays [5, 6]. It has been closely resembled the 9a-hydroxyparthenolide, isolated reported recently that different extracts of A. garcinii previously from the chloroform-soluble fraction of the inhibit the oxidative burst of primary neutrophils in same plant A. garcinii [8]. 1 humans which, together with its high anti-inflammatory The H-NMR spectrum showed specific signals of an L- 0 potential, make it a promising candidate for further phenylalanine amino acid group at dH3.46 (1H, m, H-2 ), medicinal uses and applications [11]. Previous phyto- 2.92 (2H, d, J = 5.0 Hz, H-30), 7.27 (2H, m, H-50,90), 7.26 chemical work on the Saudi A. garcinii has revealed that (2H, m, H-60,80) and 7.21 (1H, m, H-70). The 13C-NMR this plant is a rich source of sesquiterpenes. In the present signals of an L-phenylalanine group appeared at dC174.0 study, we have isolated four new sesquiterpene lactones (C-10), 63.4 (C-20), 38.2 (C-30), 138.1 (C-40), 130.0 (C-50, along with seven known compounds from different frac- 90), 128.7 (C-60,80), 126.8 (C-70) and confirmed the pres- tions of the plant extract. In addition, the total ethanol ence of an L-phenylalanine amino acid group (Tables 1, 2) extract of the leaves as well as the chloroform and n-bu- [12]. The missing C-13 methyl signal in NMR and the tanol fractions were tested for their hepatoprotective effect appearance of an additional downfield methylene group at against liver toxicity induced by carbon tetrachloride. dH 2.91 (1H, J = 10.0 Hz, H-13a) and 2.74 (1H, m, H-13b) Additionally, the cytotoxic activities of A. garcinii extracts strongly suggested that C-13 is attached to the nitrogen of and its isolates were evaluated against five human cancer L-phenylalanine amino acid. The point of attachment of the cell lines (MCF-7 breast adenocarcinoma, HCT-116 colon L-phenylalanine group was further confirmed by the carcinoma, HePG-2 liver hepatocellular carcinoma, Hela HMBC spectrum, in which H-13a (dH 2.91) showed 2 cervical cells and A-549 lung carcinoma). J correlations with the methine carbon (dC 46.8, C-11) and 3J correlations with C-12 (177.6) and C-20 (63.4). Further confirmation of the position and presence of the L-pheny- Results and discussion lalanine moiety was made by the acid hydrolysis of compound 1. The a-orientation of the hydroxyl group at C-9 Structure elucidation of new compounds (1–4) was confirmed by the upfield chemical shift of the C-9 at

dC 70.4 ppm; in the case of 9b-hydroxyparthenolide, the Chromatographic separation of the chloroform and n-bu- C-9 b-orieanted hydroxymethine carbon usually appeared 13 tanol fractions of the ethanol extract of A. garcinii, using in the range of dC 78–79 ppm in the C-NMR spectrum silica, Sephadex LH-20, and RP-18 column chromatogra- [4]. In the nuclear Overhauser enhancement (NOE) phy, yielded four new (1–4) and seven known compounds experiment on 1, correlations were observed between the (5–11) (Fig. 1). Their structures were elucidated by spec- 6b-proton, 11b-proton and 9b-proton, which further con- tral data analysis, including IR, 1D, 2D NMR, and ESI– firmed the a-position of the hydroxyl group at C-9 carbon. MS. The relative configuration of C-4 and C-5 was determined Garcinamine A (1) obtained as yellow gummy solid by NOE experiments. NOE correlations were observed 25 [a] D = –27.0 (c = 0.10, CH3OH) and the molecular between the 5a-H and 7a-H, 7a-H and13a-H2,6b-H and formula was established as C24H30NO6 at m/z 428.2068 9b-H, and 6b-H and 15b-H3. Acid hydrolysis of 1 provided - (calcd 428.2073) [M–H] by negative ESI–MS. The IR the L-phenylalanine and 9a-hydroxyparthenolide [8] and spectrum showed characteristic signals for a hydroxyl this was confirmed through co-TLC, the 1H-NMR spectrum group (3442 cm-1), a c-lactone group (1762 cm-1) and a and the sign of their optical rotation with standards. double bond (1650 cm-1). The 1H-NMR spectrum showed Therefore, compound 1 was unambiguously elucidated as a downfield signal at dH 5.48 (d, J = 10.0 Hz) for an 13-L-phenylalanine-9a-hydroxyparthenolide, and named olefinic proton, three hydroxymethine protons at dH 2.51 garcinamine A. (m, H-5), 4.02 (m, H-6) and 3.98 (m, H-9) and character- Garcinamine B (2) was obtained as a yellow gummy 25 istic signals at dH 1.59 (s, H-14), 1.19 (s, H-15) were solid, [a] D =-29 (c = 0.15, CH3OH). The positive ion assigned for two methyl groups, which suggests the pres- mode ESI–MS gave a [M ? H]? peak at m/z 382 corre- 13 1 ence of a sesquiterpene lactone ring (Table 1). The C- sponding to the molecular formula C20H32NO6. The H- NMR and DEPT spectra of 1 showed two methyl groups at and 13C-NMR data were closely similar to compound 1 and dC 16.2 and 17.3, five methylene at dC 22.9, 36.1, 36.8, it showed features common to garcinamine A (1) except for 45.5 and 38.2, 12 methine at dC 120.7, 66.1, 82.2, 35.5, the characteristic signals of an L-valine amino acid group 70.4, 46.8, 63.4, 130.0 9 2, 128.7 9 2 and 126.8, and five instead of an L-phenylalanine amino acid moiety attached 1 13 quaternary carbon signals at dC 61.3, 138.3, 177.6, 174.0 at C-13 methylene carbon atom. The H- and C-NMR and 138.1 (Table 2). These NMR spectroscopic data sug- spectra showed signals of an L-valine amino acid group at 0 0 gested that compound 1 was a parthenolide-type dH 2.88 (1H, d, J = 5.0 Hz, H-2 ), 1.24 (1H, brs, H-3 ), 123 Author's personal copy

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OH OH 4' 4' 5' 3' 1 3' 9 8' 2 10 8 2' 2' HN HN 5 14 7 1' COOH 3 6 COOH 4 11 1' 13 13 O O O 12 O 15 1 2 O O

OH O 4' 3' 5' 2' N 1' COOH

13 O O O O 5 3 9-α OH 4 β O 9- OH O OH OH O

O O O O 6 7 9-α OH 8 9-β OH O O OR3

R2O O

9 β R=H,R1 =OCH3,R2 = -D-glucoside, R3 =CH3 10 R=H,R =OCH,R = β-D-glucoside, R =H R1 OR 1 3 2 3 11 β O α R= -D-glucose-6- - -L-rhamnoside, R1 =H,R2 =H,R3 =H OH O

Fig. 1 Structures of compounds 1–11 from the leaves of A. garcinii

0.89 (3H, d, J = 5.0 Hz, H-40), 0.86 (3H, d, J = 5.0 Hz, 68.3, C-20) of the L-valine moiety. Acid hydrolysis of 0 0 0 0 H-5 ) and dC 159.0 (C-1 ), 68.3 (C-2 ), 30.9 (C-3 ), 19.6 (C- compound 2 afforded 9a-hydroxyparthenolide [8] and L- 40), 18.8 (C-50) (Tables 1, 2). The methylene protons of valine amino acid, and this was conﬁrmed through TLC, 1 H-13a (d 2.63, m) and H-13b (dH 2.90) showed NMR and the sign of its optical rotation. Based on H- and 2 13 J correlations with the methine carbon (dC 47.6, C-11) and C-NMR and COSY data and correlations, the stereo- 3 J correlations with C-12 (177.6) and methine carbon (dC chemistry of compound 2 was assumed to be same as

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Table 1 1H-NMR data for Position 1234 compounds 1–4 (DMSO, J in d d d d Hz, d in ppm, 500 MHz) H H H H 1 5.48 (d, J = 10) 5.48 (d, J = 10.0) 5.48 (d, J = 10.0) 5.32 (dd 10.0, 5.0) 2a 2.08 (brd, J = 10) 2.03 M 2.03 (m) 2.07 m 2b 2.39 (brd, J = 10) 2.48 m 2.40 (t, J = 5.0) 2.40 m 3a 1.12 m 1.15 m 1.14 (m) 1.10 m 3b 1.88 m 1.85 m 1.80 (m) 2.0 m 4–––– 5 2.51 m 2.62 m 2.65 (d, J = 10.0) 2.70 (d, J = 10.0) 6 4.02 m 4.0 m 4.03 (m) 3.99 (m) 7 2.26 m 2.18 m 2.03 (m) 2.40 (m) 8 a 2.58 (d, J = 10.0) 2.66 m 1.89 (m) 1.80 (m) 8b 2.00 m 2.01 m 2.01 (m) 1.85 (m) 9 3.98 m 3.99 m 4.0 (m) 3.96 (m) 10–––– 11 2.51 m 2.50 m 2.70 (m) 2.65 (m) 12 – – – 2.10 (m) 13a 2.91 (d, J = 10.0) 2.63 m 3.12 (m) 3.13 (m) 13b 2.74 m 2.90 m 3.0 (m) 3.01 (m) 14 1.59 s 1.61 s 1.61 (m) 1.63 (s) 15 1.19 s 1.20 s 1.20 (m) 1.21 (s) 10 –––– 20 3.46 m 2.88 (d, J = 5.0) 3.48 (t, J = 10.0) 3.40 (t, J = 10.0) 30a 2.92 (d, J = 5.0) 1.24 brs 2.10 (m) 2.07 (m) 30b – – 1.86 (m) 1.85 (m) 40a – 0.89 (d, J = 5.0) 1.82 (m) 1.81 (m) 40b – – 1.71 (m) 1.73 (m) 50a 7.27 m 0.86 (d, J = 5.0) 3.15 (m) 3.11 (m) 50b – 2.70 (m) 2.63 (m) 60 7.26 m – – – 70 7.21 m – – – 80 7.26 m – – – 90 7.27 m – – – compound 1. Thus, the structure of 2 was elucidated as correlations with the methine carbon C-11 (45.3) and 3 13-L-valine-9a-hydroxyparthenolide, and named garci- J HMBC correlations with C-12 (177.2), C-7 (36.6), C-20 namine B. (67.5) and C-50 (53.6), which conﬁrmed the position of the Garcinamine C (3) was isolated as a yellow gum amino acid group at C-13 carbon. Acid hydrolysis of 25 [a] D =-20 (c = 0.17, CH3OH). The positive ion mode compound 3 afforded 9a-hydroxyparthenolide and L-pro- ESI-MS gave a molecular ion peak [M ? H]? at m/z 380 line amino acid, and this was conﬁrmed through TLC, 1 corresponding to molecular formula C20H30NO6. The H- NMR and the sign of its optical rotation. Therefore, com- 13 and C-NMR data were similar to those of 1and 2, and the pound 3 was elucidated as 13-L-proline-9a-hydroxy- only difference was observed in an amino acid moiety at parthenolide, and named garcinamine C. 1 C-13 carbon. In the H-NMR spectrum, the signals of the L- Garcinamine D (4) was isolated as a yellow gum 25 proline amino acid group appeared at dH 3.48 (t, [a] D =-22 (c = 0.15, CH3OH). The positive ion J = 10.0 Hz, H-20), 2.10 and 1.86 (each m, H-30a, b), 1.82, mode ESI-MS gave a quasi-molecular [M ? H]? peak at 0 0 1.71 (each m, H-4 a, b), 3.15 and 2.70 (each m, H-5 a, b). m/z 380 corresponding to molecular formula C20H30NO6. 13 The C-NMR spectrum gave signals for the L-proline Detailed comparison of the NMR spectra of 4 with those 0 0 0 moiety at dC 174.4 (C-1 ), 67.5 (C-2 ), 29.3 (C-3 ), 23.9 (C- of 3 indicated that the only differences in chemical shifts 40) and 53.6 (C-50) (Tables 1, 2). The methylene protons were C-9 oxymethine and C-14 methyl groups. According H-13a (3.12) and H-13b (3.0) showed 2J HMBC to the literature, if the 9-hydroxy group has a-orientation

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Table 2 13C-NMR data for compounds 1–4 (DMSO, d in ppm, Hepatoprotective activity 125 MHz) Position 1234In the current investigation, hepatotoxicity was induced dC dC dC dC using carbon tetrachloride, which converted in the endoplasmic reticulum into trichloromethyl free radical (CCl ) 1 120.7 120.6 121.1 123.7 3 and Cl COO which, with the aid of oxygen, conjugated 2 22.9 23.4 23.4 23.6 3 with essential cellular macromolecules, cellular lipids and 3 36.1 36.1 36.1 36.4 proteins to induce lipid peroxidation [15]. Lipid peroxi- 4 61.3 61.3 61.6 61.7 dation forms reactive aldehydes that can form adducts with 5 66.1 66.4 66.1 65.7 proteins. These reactions increase the endoplasmic reticu- 6 82.2 81.9 82.1 81.8 lum’s and other membranes’ permeability to Ca2?, result- 7 35.5 36.6 36.6 42.5 ing in severe disturbances of calcium homeostasis and 8 36.8 36.8 35.7 38.0 consequently necrotic cell death [16]. Treatment of animals 9 70.4 70.5 70.5 78.3 with the hepatotoxic agent carbon tetrachloride resulted in 10 138.3 137.8 137.6 138.1 significant increase of transaminases (AST and ALT) and 11 46.8 47.6 45.3 46.4 alkaline phosphatase (ALP) levels that reflect the func- 12 177.6 177.6 177.2 177.0 tional status of the liver and consequently indicate hepa- 13 45.5 46.0 51.4 51.9 tocyte damage [17]. Severe jaundice was reflected by the 14 16.2 16.5 16.4 11.2 elevated levels of serum bilirubin (Table 3). 15 17.3 17.5 17.3 17.3 The use of silymarin at a dose of 10 mg/kg (20.7 lmol/ 0 1 174.0 159.0 174.4 174.3 kg) prior to the administration of CCl4 resulted in a sig- 20 63.4 68.3 67.5 67.5 nificant decrease (p \ 0.001) in the elevated AST, ALT, 30 38.2 30.9 29.3 29.2 gamma-glutamyl trans-peptidase (GGT), ALP and biliru- 40 138.1 19.6 23.9 24.2 bin levels in rats (Table 3). Silymarin acts as an antioxidant 50 130.0 18.8 53.6 53.9 by scavenging pro-oxidant free radicals and by increasing 60 128.7 – – – the intracellular concentration of glutathione. It also 70 126.8 – – – enhances the cellular membrane’s permeability to protect 80 128.7 – – – against xenobiotic injury. Moreover, it promotes the syn- 90 130.0 – – – thesis of ribosomal RNA by stimulating DNA polymerase-I as well as having a steroid-like action in regulating DNA transcription and stimulating protein synthesis leading to then the C-9 carbon shows the signal at dC 70-72 ppm, regeneration of liver cells [18, 19]. while in the case of the b-oriented 9-hydroxy group, the Rats supplemented with the total ethanol extract of A. 13 C-9 carbon signal appeared at dC 78–79 ppm in the C- garcinii at 400 mg/kg counteracted the toxic effect of CCl4 NMR spectrum. The downfield 13C-NMR signal of C-9 and resulted in a significantly (p \ 0.01, 0.001) moderate carbon atom of 4 at dC 78.3 confirmed the b-orientation decrease in the elevated levels of AST, ALT, GGT, ALP of the hydroxyl group at position 9 [4]. The methyl group and bilirubin. The total extract at 400 mg/kg also signifi- at C-10 carbon atom was shifted from dC 16.4 to dC 11.2, cantly reduced the concentration of malonaldehyde which further confirmed the b-orientation of the hydroxyl (MDA). The level of total protein was significantly group at C-9 position. Acid hydrolysis of compound 4 (p \ 0.001) increased (Table 4). The fractions obtained afforded 9b-hydroxyparthenolide and L-proline amino after liquid–liquid fractionation were also tested for their acid, and this was confirmed through TLC, NMR and the hepatoprotective effect at 2 doses, 200 and 400 mg/kg. The sign of its optical rotation. Therefore, compound 4 was n-butanol fraction did not show any protective effect at the elucidated as 13-L-proline-9b-hydroxyparthenolide, and two doses tested. However, the chloroform-soluble fraction named garcinamine D. at the higher dose showed highly significant (p \ 0.001) The known compounds isolated from the chloroform improvement in all the serum parameters, comparable to and n-butanol fractions were identified as parthenolide-9- the effect of the standard drug silymarin (Table 3). The one (5), 9a-hydroxy-1b,10a-epoxyparthenolide (6), 9a- effect on restoring non-protein sulfhydryl groups (NP-SH) hydroxyparthenolide (7), 9b-hydroxyparthenolide (8), spi- (p \ 0.01) was equal to that of silymarin. Restoration of nacetin-7-glucoside (9)[5], patuletin-7-glucoside (10)[13] the normal levels of MDA and total protein was highly and rutin (11)[14] by comparing the NMR spectra with significant (p \ 0.01, 0.001) and close to that produced by published literatures. silymarin (Table 4).

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Table 3 Effect of A. garcinii leaf extracts on serum marker enzymes of control and experimental rats Treatments Dose SGOT (AST) U/L SGPT (ALT) U/L ALP U/L GGT (U/L) Bilirubin (mg/dl) mg/kg Mean ± SE % Mean ± SE % Mean ± SE % Mean ± SE % Mean ± SE % Change Change Change Change Change Author's Normal 110.33 ± 2.21 31.20 ± 1.55 340.0 ± 9.69 4.08 ± 0.11 0.56 ± 0.01 a CCl4 311.83 ± 7.52*** 314.83 ± 569.1 ± 16.0 ± 2.9 ± 5.6***a 10.68***a 0.5***a 0.05***a b b b Silymarin ? CCl4 10 156.00 ± 9.64*** 49.97 99.77 ± 3.6*** 68.31 401.1 ± 5.6*** 27.93 7.7 ± 51.97 1.0 ± 65.02 0.25***b 0.04***b Total ethanol extract ? 200 295.33 ± 1.99b 5.29 294.16 ± 5.3*b 6.56 551.5 ± 5.91b 3.1 13.9 ± 0.4*b 13.30 2.74 ± 0.04b 5.57 personal CCl4 Total ethanol extract ? 400 233.16 ± 7.14***b 25.22 271.33 ± 13.81 517.8 ± 4.98**b 9.0 11.5 ± 27.75 2.5 ± 12.98 b b b CCl4 2.8*** 0.3*** 0.03*** Chloroform fraction ? 200 239.83±5.90***b 23.08 150.66 ± 52.14 449.3 ± 7.3***b 21.0 10.1 ± 36.59 1.8 ± 36.24 b b b CCl4 5.7*** 0.2*** 0.06*** copy Chloroform fraction ? 400 177.83 ± 6.19***b 42.97 126.66 ± 59.76 423.0 ± 6.2***b 25.6 8.2 ± 0.3***b 48.33 1.3 ± 54.91 b b CCl4 3.0*** 0.02*** b b b b b n-Butanol fraction? CCl4 200 303.33 ± 4.16 - 299.83 ± 4.4* 5.08 595.5 ±.41* 4.6 15.78 ± 0.50 1.55 2.91 ± 0.05 – b b b b b n-Butanol fraction ? CCl4 400 306.00 ± 4.39 - 287.50 ± 4.8** 8.68 576.6 ± 8.8 1.3 14.71 ± 0.26 8.21 2.7 ± 0.02* 5.57 All values represent mean ± SEM. * p\0.05, ** p\0.01, *** p\0.001; ANOVA, followed by Dunnett’s multiple comparison test a As compared with control group b As compared with CCl4 group 123 111 Author's personal copy

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Table 4 Effect of A. garcinii Treatments (n = 6) Dose mg/kg MDA (nmol/g) NP–SH (nmol/g) Total protein leaf extracts on MDA, NP-SH (g/L) and total protein in liver tissue Normal 1.06 ± 0.02 4.12 ± 0.16 123.75 ± 3.36 a a a CCl4 7.07 ± 0.20*** 2.72 ± 0.17*** 52.69 ± 1.74*** b b b Silymarin ? CCl4 10 1.68 ± 0.07*** 3.55 ± 0.08** 111.37 ± 1.82*** b b b Total ethanol extract ? CCl4 200 5.79 ± 0.47* 2.57 ± 0.08 59.88 ± 2.76 b b b Total ethanol extract ? CCl4 400 4.70 ± 0.21*** 2.85 ± 0.20 72.25 ± 2.17*** b b b Chloroform fraction ? CCl4 200 3.30 ± 0.22*** 3.67 ± 0.30* 82.23 ± 2.94*** b b b Chloroform fraction ? CCl4 400 2.12 ± 0.16*** 3.55 ± 0.09** 100.99 ± 2.08*** b b b n-butanol fraction ? CCl4 200 7.09 ± 0.14 2.56 ± 0.15 54.29 ± 1.35 b b b n-butanol fraction ? CCl4 400 5.30 ± 0.25*** 2.58 ± 0.07 63.87 ± 1.18*** All values represent mean ± SEM. * p\0.05, ** p\0.01, *** p\0.001; ANOVA, followed by Dunnett’s multiple comparison test a As compared with control group b As compared with CCl4 group

Fig. 2 Hepatoprotective effect of A. garcinii leaf extract and fractions

The histopathological results in the liver of rats intoxi- with multiple areas of necrosis, vacuolization of hepato- cated with carbon tetrachloride in the presence or absence cytes and fatty changes (Fig. 2e). The best protection was of A. garcinii extracts are depicted in Fig. 2. The achieved in the group supplemented with 400 mg/kg body histopathology of the liver of rats treated with A. garcinii weight of the chloroform fraction, where only slight total ethanol extract at 400 mg/kg showed little protection, necrosis and vacuolization of hepatocytes was observed

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(Fig. 2g). Thus, the hepatoprotective effect of the chloro- with the literature: parthenolides have attracted particular form fraction may be related to its phytoconstituents, attention owing to their promising potential as anticancer exempliﬁed by the isolated sesquiterpene lactones. This is agents [23]. The cytotoxic effect of 6 on cervical and liver in agreement with previous reports on the marked biolog- carcinomas was even stronger than that of the vinblastine ical activities of sesquiterpene lactones, including their positive standard. Although IC50 values for 6 and 7 were hepatoprotective effects [20–22]. The decrease in activity comparable, 6 was shown to be a better cytotoxic com- of the n-butanol fraction may be related to its constituents, pound, which demonstrates the effect of the stereochem- with the amino acids coupled to the sequiterpene lactones. istry of 9-hydroxyparthenolide at the C-9 position on cytotoxic activity. Additionally, it was proved that the Cytotoxic activity replacement of the 9-hydroxy group in the aglycone 7. Thus, we could deduce that the glycosylation decreased the The cytotoxic activities of total ethanol extract, fractions cytotoxic activity of the sesquiterpene lactone. and compounds isolated from A. garcinii were studied using Concerning the two epimers 3 and 4, the results demon- the chorionic villus sampling method and vinblastine sulfate strated the higher cytotoxic activity of 4 (IC50 ranging from as a reference drug. Five human cancer cell lines were used 2.23 to 13.40 lg/ml), as well as the high selectivity of this and the response parameter (IC50) was calculated for each compound on cervical and liver carcinomas. It is worth cell line. From the cytotoxicity results shown in Table 3, the noting that the coupling of the sesquiterpene lactone with chloroform and the n-butanol fractions showed moderate to amino acids affected the cytotoxic activity variably. On signiﬁcant cytotoxic activity. The chloroform fraction was comparing compounds 1 (coupled to L-phenylalanine), 2 strongly selective on the hepatocellular carcinoma, with (coupled to L-valine) and 3 (coupled to L-proline), it could be

IC50 of 5.80 lg/ml compared to vinblastine (IC50 3.48 lg/ seen that compound 3 with L-proline amino acid is the ml). This further confirmed the strong hepatoprotective strongest cytotoxic agent. Based on these in vitro results, we activity of the chloroform fraction. Moreover, the chloro- are planning to investigate the in vivo efficacy of active form and n-butanol fractions demonstrated significant constituents of A. garcinii concerning cytotoxic activity. activity against colon, liver, cervical and lung carcinomas. with IC50 values ranging from 10.6 to 19.8 lg/ml). The total ethanol extract showed relatively weak cytotoxic activity Materials and methods

(IC50 ranging from 37.6 to 94.4 lg/ml), which might be due to antagonistic action between the compounds (Table 5). General As for the compounds isolated, 6 and 7 demonstrated signiﬁcant cytotoxic effect against the ﬁve cell lines (IC50 All the instruments used in the study are provided in the ranging from 2.98 to 12.40 lg/ml). This is in agreement supplementary material.

Table 5 In vitro cytotoxic activity (IC50 values) of A. garcinii extract, fractions and compounds on ﬁve human cancer cell lines Sample conc. (lg/ml) MCF-7 HCT-116 HepG-2 HeLa A-549

Ethanol extract 93.90 ± 9.50a 37.60 ± 5.72a 39.80 ± 12.4a 61.80 ± 3.55a 94.40 ± 9.19a a a b b c CHCl3 fraction 24.50 ± 5.56 10.60 ± 0.89 5.80 ± 0.60 12.0 ± 0.79 14.70 ± 4.16 n-Butanol fraction 27.70 ± 4.96a 11.30 ± 0.81a 12.20 ± 1.04a 48.80 ± 4.35a 19.80 ± 6.28c 1 78.30 ± 4.30a 14.25 ± 0.93a 35.22 ± 1.23a 21.22 ± 1.43a 18.33 ± 0.98c 2 183.0 ± 18.40a 12.50 ± 0.86a 28.0 ± 1.07a 24.90 ± 0.07a 19.80 ± 2.97c 3 24.20 ± 1.43a 11.70 ± 1.54a 18.70 ± 2.74a 6.24 ± 0.49 6.20 ± 0.24b 4 13.40 ± 1.06a 3.40 ± 0.14c 3.49 ± 0.28 2.23 ± 0.66a 3.77 ± 0.44 5 402.0 ± 14.45a 211.0 ± 2.56a 388.0 ± 9.10a 403.0 ± 10.8a 178.0 ± 20.4a 6 85.32 ± 1.56a 58.98 ± 1.25a 12.33 ± 2.47a 35.66 ± 1.54a 125.30 ± 3.12a 7 6.55 ± 0.35 3.97 ± 1.52 3.01 ± 0.55 2.98 ± 0.42a 3.70 ± 0.24 8 6.70 ± 1.00 5.54 ± 0.24a 7.60 ± 0.71a 12.40 ± 1.52b 4.97 ± 1.16 Vinblastine sulfate 5.44 ± 0.57 2.58 ± 0.43 3.48 ± 0.22 6.54 ± 0.39 3.58 ± 0.48

Bold values show strong IC50 of particular compound

*IC50: concentration of extract required to reduce cell survival by 50% # Mean of IC50 values ± standard deviation; mean of three assays a p \ 0.001, b p \ 0.01, c p \ 0.05 compared to reference drug

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Plant material H2O–MeOH (7:3) was a binary mixture which was separated through HPLC with a ﬂow rate 1.0 ml/min using

The leaves of A. garcinii were collected in March 2014 H2O:MeOH (3:7) as eluent, to afford 10 (14 mg, from the new industrial area 17 km south-west of Al-Kharj Rt16.8 min). Compound 11 (20 mg) was isolated by direct city and identiﬁed by taxonomist Dr. M. Atiqur Rahman, precipitation of fractions B-3 and B-4. College of Pharmacy, Medicinal, Aromatic and Poisonous Plants Research Center, King Saud University. A voucher Garcinamine A (1) specimen (PSAU-CPH-6-2014) has been deposited in the 25 herbarium of the College of Pharmacy, Prince Sattam Bin Yellow gummy solid; [a] D = –27.2 (c 0.10, CH3OH); Abdulaziz University. (-) HRESIMS m/z 428.2068 [M-H]- (calcd. for 1 13 C24H30NO6, 428.2073); H- and C-NMR data, see Extraction and isolation Tables 1 and 2.

The air-dried leaves (1.0 kg) were powdered and extracted Garcinamine B (2) three times with a mixture of ethanol–water in 8:2 ratio at 25 30 °C, and then filtered. The filtrate was evaporated in Yellow gummy solid; [a] D = –22.5 (c 0.15, CH3OH); vacuo to give a dark brown residue (115 g), which was (?) HRESIMS m/z 382.2224 [M ? H]? (calcd. for 1 13 suspended in water and fractionated with chloroform (35 g) C20H32NO6, 382.2230); H- and C-NMR data see and n-butanol (65 g). A part of the chloroform fraction Tables 1 and 2. (25 g) was loaded on silica gel column and eluted with n- hexane, n-hexane–ethyl acetate and with pure ethyl acetate Garcinamine C (3) to yield three sub-fractions, C-1, C-2, and C-3. The sub- 25 fraction C-1 (1.0 g) which was eluted with n-hexane–ethyl Yellow gummy solid; [a] D = –29.3 (c 0.15, CH3OH); acetate (9.0:1.0) was further subjected to silica gel columns (?) HRESIMS m/z 380.2065 [M ? H]? (calcd. for 1 13 eluted with n-hexane–ethyl acetate (8.0:2.0) as eluent to C20H30NO6, 380.2073); H- and C-NMR data, see afford 5 (10 mg). The sub-fraction C-2 (0.5 g) which was Tables 1 and 2. eluted with n-hexane–ethyl acetate (8.0:2.0) was a mixture of two components. It was further subjected to preparative Garcinamine D (4) TLC over silica gel plates using n-hexane–ethyl acetate 25 (7.0:3.0) as eluent to afford 6 (28 mg) and 7 (30 mg). The Yellow gummy solid; [a] D = –25.6 (c 0.10, CH3OH); sub-fraction C-3 was subjected to silica gel columns eluted (?) HRESIMS m/z 380.2048 [M ? H]? (calcd. for 1 13 with n-hexane–ethyl acetate (7.0:3.0) as eluent to afford 8 C20H30NO6, 380.2073); H- and C-NMR data, see (8 mg). Tables 1 and 2. A part of the n-butanol fraction (45 g) was subjected to Sephadex column and eluted with a mixture of water and Acid hydrolysis of compounds 1–4 methanol (10% methanol ? 100% methanol) as mobile phases, and pooled in four major sub-fractions B1–B4. Methanol solutions of compounds 1–4 (each 5 mg) con- Fraction B-1 (0.5 g) was applied to medium pressure RP- taining 1N HCl were refluxed for 1 h. After completion of 18 column and eluted with a mixture of water and methanol the reaction, the mixture was concentrated and diluted with (9:1) to yield 1 (10 mg). Fraction B-2 (1.0 g) was subjected water and extracted with ethyl acetate. The ethyl acetate to Sephadex LH-20 column eluted with water and metha- extract was subjected to silica gel column chromatography nol (8:2) to obtain two sub-fractions B-2a and B-2b. Sub- and on elution with CHCl3:MeOH (9:1) it gave 9a-hy- fraction B-2a (100 mg) was further purified by HPLC (flow droxyparthenolide from compounds 1, 2 and 3, while 9b- rate 3 ml/min; wavelength 375 nm; CH3OH–0.01%TFA: hydroxyparthenolide was obtained from compound 4. The H2O, 1:1) to afford 2 (12 mg, Rt 13.4 min). Sub-fraction water extract was concentrated and subjected to RP-C18 B-2b (150 mg) was purified by HPLC (flow rate 1.0 ml/ column chromatography (water:methanol 7:3) and the min; wavelength 375 nm; CH3OH–0.01%TFA: H2O, 1:1) fractions gave positive ninhydrin tests to confirm the to afford 3 (12 mg, Rt 12.7 min) and 4 (15 mg, Rt 13.1). presence of amino acid. The amino acid was L-phenylala- Fraction B-3 (500 mg) was rechromatographed on Sepha- nine from compound 1, L-valine from compound 2 and L- dex LH-20 (flow rate 1.0 ml/min) and eluted with H2O– proline from compounds 3 and 4, and these amino acids MeOH (7.5:2.5) to afford compound 9 (20.0 mg, Rt were identified from NMR and their signs of optical rota- 18.5 min). The sub-fraction B-4 (1.5 g) obtained from tions by comparison with standards [12].

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Animals Determination of NP-SH, MDA and total protein

Male Wistar albino rats (150–200 g) of about the same The liver samples were separately cooled in a beaker age (8–10 weeks) were obtained from the Experimental immersed in an ice bath. The tissues were homogenized in Animal Care Center, College of Pharmacy, King Saud 0.02 M ethylene-diamine tetra-acetic acid (EDTA) in a Pot- University. The animals were housed under optimum ter–Elvehjem type C homogenizer (Sigma-Aldrich). Homo- conditions of humidity, temperature and light/dark con- genates equivalent to 100 mg tissues were used for the ditions, and were allowed access to Purina chow (Lab- measurements. Non-protein sulfhydryl groups (NP-SH) were Diet, St. Louis, MO, USA) and drinking water ad libitum. quantified as reported previously, while concentrations of The procedures used in this study were approved by the MDA were determined according to the procedure of Utley Ethical Committee of the College of Pharmacy, Prince et al. [25]. For TP determination, parts ofthe homogenate were Sattam Bin Abdulaziz University, based on the existing treated with 0.7 ml of Lowry’s solution, mixed and incubated international regulations for the handling and care of for 20 min in the dark at room temperature. Diluted Folin laboratory animals. reagent (0.1 ml) were then added and samples were incubated at room temperature in the dark for 30 min. The absorbance of the resulting solutions was then measured at 750 nm. Chemicals Histopathology Silymarin, EDTA, Mayer hematoxylin, eosin-phloxine, Folin reagent, trichloroacetic acid, 5,50-dithio-bis-(2-ni- The fixed liver samples were put in cassettes and loaded trobenzoic acid), 2-thiobarbituric acid and paraffin were into an automated vacuum tissue processor (ASP 300 S, obtained from Sigma-Aldrich, St. Louis, MO, USA. The Leica Biosystems, Nussloch, Germany). The samples were solvents used in the investigation were of analytical blocked in paraffin wax and thin sections (3 lm) were cut grade. using a microtome (TBS SHUR/cut 4500, Triangle Biomedical Sciences, Durham, NC, USA). The sections Hepatoprotective activity obtained were stained with Mayer’s hematoxylin solution and counterstained in eosin–phloxine solution [26]. Slides Male Wistar rats were divided into nine groups of five were examined using Slide Scanner (SCN 400 F Leica animals each. Group I was the control group, taking normal Microsystems, Wetzlar, Germany) for microscope imaging saline. Groups II–IX were administered a single dose of using objective magnifications of 109 and 409. CCl4 (1.25 ml/kg body weight). Group II received only CCl4 treatment, while Group III was administered sily- Statistical analysis marin at a dose of 10 mg/kg per os (20.7 lmol/kg). Groups IV–IX were treated with two doses: 200 and 400 mg/kg of Results are expressed as mean ± standard error (SE) of the total ethanol extract, CHCl3 and n-butanol fractions, mean. Statistical analysis was performed using one-way respectively. In each group, treatment started 5 days before analysis of variance (ANOVA). When the F value was CCl4 administration and continued till day six. After 24 h found to be statistically significant (p \ 0.05), further of CCl4 administration, the animals were killed using ether comparisons between groups were made using Dunnett’s anesthesia. Blood samples were obtained by heart puncture multiple comparison test. All statistical analyses were and the serum was separated for determining the different performed using SPSS software v.17.0 (released Aug. 23, biochemical parameters. 2008), Chicago, IL, USA.

Determination of biochemical enzymatic parameters Cytotoxicity assay AST, ALT, GGT, ALP and bilirubin Cell culture The biochemical parameters, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl Human cell lines MCF-7 cells (breast cancer cell line), trans-peptidase (GGT), alkaline phosphatase (ALP) and HepG-2 (hepatocellular carcinoma), A-549 (human lung total bilirubin were determined according to the previously carcinoma), HELA cells (human cervical carcinoma) and reported methods of Edwards and Bouchier [24]. The HCT-116 (colon carcinoma) were obtained from The enzyme activities were measured using diagnostic strips Holding Company for Biological Products and Vaccines (Reflotron, Roche, Basel, Switzerland) and were read on a (VACSERA) Tissue Culture Unit. The cells were cultured ReflotronÒ Plus instrument (Roche). in Dulbecco’s modified Eagle’s medium (DMEM) with 123 Author's personal copy

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10% heat-inactivated fetal bovine serum, HEPES buffer, traditionally by local people for the treatment of many 1% L-glutamine and 50 lg/ml gentamycin. The cells were diseases including liver problems. Our results justified the kept at 37 °C under humidified condition with 5% CO2 and use of the plant for improving liver aliments. This is the were subcultured twice a week. first report to confirm the use of A. garcinii as a hepatoprotective traditional medicine. The chloroform and the n- Evaluation of cytotoxicity using viability assay butanol fractions showed moderate to significant cytotoxic activity. The chloroform fraction was strongly selective on The cells were seeded in 96-well plates at a concentration hepatocellular carcinoma compared to vinblastine refer- of 1 9 104 cells per well in 100 ll of growth medium. ence drug. Moreover, the chloroform, n-butanol fractions Different concentrations of the test sample were added and compounds 3, 4, 7 and 8 demonstrated significant after 24 h of seeding in fresh medium. Serial two-fold activity against colon, hepatocellular, cervical and lung dilutions of the tested sample were added to confluent cell carcinomas. monolayers dispensed into 96-well microtiter plates (Fal- con, NJ, USA). The microtiter plates were incubated at Acknowledgements This research project was supported by a grant 37 °C with 5% CO for 48 h. Three wells were used for from the ‘‘Research Center of the Female Scientific and Medical 2 Colleges’’, Deanship of Scientific Research, King Saud University. each concentration of the test sample. Control cells were incubated without test sample and with or without DMSO. Compliance with ethical standards By the end of the incubation period, media were aspirated and crystal violet solution (1%) was added to each well for Conflict of interest The authors declare they have no conflicts of interest. 30 min. The plates were rinsed using distilled water. Gla- cial acetic acid (30%) was then added to all wells and mixed thoroughly. Colorimetric evaluation of fixed cells was carried out by absorbance measurements in an auto- References matic microplate reader (TECAN, Inc.) at 490 nm. Treated samples were compared with the cell control in the absence 1. Chaudhary SA (2000) Flora of the Kingdom of Saudi Arabia, of the tested samples. All experiments were performed in Volume II, Ministry of Agri. and Water, Riyadh, Saudi Arabia 2. A guide to medicinal plants in North Africa (2005) IUCN Centre triplicate. The optical density was measured using the for Mediterranean Cooperation, Malaga, Spain, pp 35 microplate reader to determine the number of viable cells. 3. Kharjul M, Gali V, Kharjul A (2014) Antidiabetic potential of The percentage of viability was calculated as [1 - (ODt/ ethanolic extracts of Citrus maxima fruit peel and Anvillea gar- ODc)] 9 100%, where ODt is the mean optical density of cinii. Int J Pharm Inno 4:8–18 4. Essam AS, Ahmed MG, Gaber SM (1996) Antitumor germa- wells treated with the tested sample and ODc is the mean cranolides from Anvillea garcinii. J Nat Prod 59:403–405 optical density of untreated cells, and was illustrated as a 5. Essam AS, Andrew TM (2000) Cis-parthenolid-9-one from dose–response curve. The 50% inhibitory concentration Anvillea garcinii. J Nat Prod 63:1587–1589 (IC ) was estimated from graphic plots of the dose–re- 6. Tyson RL, Chang CJ, McLaughlin JL, Cassady JM (1981) 9a- 50 Hydroxyparthenolide, a novel antitumor sesquiterpene lactone sponse curve using Graphpad Prism software (San Diego, from Anvillea garcinii (Burm.) DC. Experientia 37:441–442 CA. USA). The reference antitumor drug used was vin- 7. Rustaiyan A, Dabiri M, Jakupovic J (1986) Germacranolides blastine sulfate [27]. from Anvillea garcinii. Phytochemistry 25:1229–1230 8. Hassany B, Hanbali F, Akssira M, Mellouki F, Haidour A, Bar- rero AF (2004) Germacranolides from Anvillea radiata. Fitoter- apia 75:573–576 Statistical analyses 9. Emilie D, Meryem AB, Sandrine Z, Mohamed A, Lhoucine R, Claire E (2015) Centrifugal partition chromatography elution Data were expressed as mean ± SD. For multivariate gradient for isolation of sesquiterpene lactones and flavonoids from Anvillea radiata. J Chromat B 985:29–37 comparisons, one-way ANOVA was conducted, followed 10. Ulubelen A, Mabry TJ, Aynehchi Y (1989) Flavonoids of by Tukey–Kramer testing using the GraphPad InStat (ISI Anvillea garcinii. J Nat Prod 42:624–626 Software) computer program. Differences were considered 11. Hanane B, Margarita NH, Viviana M, Dalila B, Jamel EB, Jean- significant at p \ 0.05. 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