Int J Pharm Pharm Sci, Vol 6, Issue 8, 187-196Original Article

CHEMO-PROTECTIVE EFFECT ON HEPATO-RENAL TOXCICITY AND CYTOTOXIC ACTIVITY OF LIPOIDAL MATTER OF ATRIPLEX LINDLEYI MOQ

AZZA A. MATLOUB1*, MANAL A. HAMED2, SAHAR S. M. EL SOUDA3

1*Pharmacognosy Dept., 2Therapeutic Chemistry Dept., 3Chemistry of Natural Compounds Dept., National Research Center, 12622, Egypt.
Email: matlouba2002@hotmail.com

Received: 19 Jun 2014 Revised and Accepted: 21 Jul 2014

ABSTRACT

Objective: Bromobenzene (BB) is frequently encountered in table-ready foods as contaminant residues. Therefore, the present study is designed to evaluate the petroleum ether extract of Atriplex lindleyi to attenuate the hepato-renal injury induced by BB exposure and study its cytotoxic activity against different human cell line as well as to describe the chemical composition of the petroleum ether extract.

Methods: The phytochemical study of petroleum ether extract was implemented using both GC/MS and column chromatography analysis. The isolated compounds were identified using different spectroscopic analysis.

Hepato-renal assay, rats were intraperitonealy injected bromobenzene at a dose 460 mg/kg BW. The petroleum ether extract as well as Hepaticum were administrated orally twice a week for three consecutive weeks with a dose 150 & 100 mg/kg body weight, respectively. Liver marker enzymes, liver function indices and kidney function tests were estimated.

The cytotoxic activity of, petroleum ether extract was assessed by the mitochondrial dependent reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide).

Results: Forty two compounds as well as sixteen fatty acids were identified in unsaponifiable and saponifiable fractions, respectively. Unsaponifiable fraction constituted of hydrocarbons (73.39% of total unsaponifiable matter), alcoholic (0.88%) and steroidal compounds (2.22%). Furthermore, column chromatography of petroleum ether extract afforded nonsterol tritrepenoids; olean-12-en-3,11-dione (1), β-amyrenone (2), erythrodiol I (3), Lupeol (4) as well as sterol triterpenoids; cholesterol (5) and mixture of β-sitosterol and stigmasterol (6). Compounds 1, 3 and 5 are first reported from Atriplex lindleyi. In addition, GC/MS analysis of the main fraction isolated from column chromatography revealed phytol as a major component.

Drastic changes were observed after BB intoxication in liver function parameters; kidney disorder indices and certain oxidative stress markers. Treatment with petroleum ether extract improved all biochemical parameters under investigation as well as the histopathology of liver and kidney. Petroleum ether extract showed growth inhibition of HepG2 and MCF7 human cells by 44.8 and 29.9%, respectively at 100 µg/ml.

Conclusion: The petroleum ether extract of A. lindleyi contains bioactive compounds exhibiting hepato-renal protection and cytotoxic activity.

Keywords: Atriplex lindleyi, Petroleum ether extract, Oleanane skeleton, Fatty acids, Hepato-renal and cytotoxic activity.

INTRODUCTION

The genus Atriplex belongs to the family Chenopodiaceae (Goosefoot) and includes 225 species. They are valuable sources of vitamins and are used in spring (instead of spinach) as a green leafy vegetable [1]. Several species of Atriplex have been investigated for their chemical constituents. The presence of triterpenes [2-4], saponins [5, 6], alkaloids [7,8], flavonoids [9], carbohydrate [8] and protein [10] have been reported in various Atriplex species. Some triterpene as β-amyrenone, lupeol, sitosterol mixture, β-sitosterol glucoside and 3β, 23-dihydroxy betulin, in addition, 20-hydroxyecdysone have been isolated from Atriplex lindleyi [9,11]. Various species of Atriplex have been used for their important medicinal values such as Atriplex semibacata and A. vestita have been used as an antifungal agent and in the treatment of bronchitis [12]. The total ethanolic extract of A. inflata exhibited antihepatotoxic, liver protection and anti-inflammatory activities [11]. Also, A. inflata extract was found to be effective against Herpes simplex [13]. However, the hexane extract of A. lindleyi showed potent antimicrobial activities against Bacillus subtilis and Pseudomonas aeruginosa [9]. Also, petroleum ether extract of A. inflat had potent antifungal activity against Botrytis cinerea and Fusarium oxysporum [12].

Bromobenzene (BB) has been detected at low frequencies and at low concentrations in samples of food, air, and water [14]. The metabolites of BB are highly hepatotoxic, while secondary metabolites are highly nephrotoxic [15]. The severity and number of people affected by hepatic diseases and renal failure is an Egyptian problem with thousands of people affected annually. In the course of our search for treating these diseases with natural products from Egyptian plants. The work aimed the detailed description of the lipoidal profile as well as hepato-renal protective effect of petroleum ether extract of Atriplex lindleyi aerial parts against BB intoxication in rats and its cytotoxic activity.

MATERIALS AND METHODS

General

Melting points (uncorrected) were determined on an electrothermal melting point apparatus. GC/MS analysis was carried out using a Agilent HP6890 GC/MS spectrophotometer equipped with library software Wiley 138 and NBS 75 under the following conditions: Thermo scientific, TR-5MS (5% phenyl polysil phenylene siloxane), capillary column, 30 m in length, 0.25 mm i.d. and 0.25 µm film thickness, carrier gas, helium at a flow rate of 1ml/min, temperature programmed 60-2900C at a rate of 4°C/min (unsaponifiable matters), 60-280°C at a rate of 5°C/min (fatty acids methyl ester derivatives), chart speed: 0.5 cm/min, ionization voltage 70 eV, detector: mass. NMR spectra was measured on JEOL EX-500 spectrometer (Tokyo, Japan) (500 MHz for 1HNMR and 125 MHz for 13CNMR)) using CDCl3 as internal reference.

Plant Material

The aerial parts of Atriplex lindleyi Moq. Subsp. Inflata, (syn.: Atriplex inflata, Blackiella inflata), common name Lindley’s saltbush, were harvested in March from Road of Suez, Egypt. The plant samples were dried in the shade and conserved for authentication. Voucher specimen (no 954) was authenticated by Prof. S. A. Kawashty, taxonomist in the herbarium of National Research Centre [CAIRC], Department of chemotaxonomy.

Investigation of Lipoidal Matter

The dried powdered aerial parts of Atriplex lindeyl (1 kg) were extracted with petroleum ether (40-60 °C) (SDFCI, India) using a Soxhlet apparatus.

The solvent was evaporated under vacuum at 40 °C by Buchi Rotavapor R-200 to dryness. Quantitative estimation of the total steroidal and triterpenoidal contents in the petroleum ether extract was performed according to Swift [16].

Isolation of unsaponifiable matter and fatty acids

The unsaponifiable and saponifiable matter as well as methyl ester of fatty acid was prepared as mentioned in [17], then subjected to GC/MS analysis.

The identification of the composition of nonsaponifiable matter and fatty acid methyl esters were performed, depending on the fragmentation pattern and comparing with those of available authentic material such as mixture hydrocarbon, α- and β- amyrin, lupeol, oleanolic acid, urosolic acid, squalene, cholesterol, campesterol, fucosterol, stigmasterol, and β- sitosterol (Sigma-Aldrich Chemie GmbH, Germany) or published data [18-21] and a library database Wiley and NIST [22]. Quantitative determination was carried out based on peak area measurements of the GC chromatograms.

Column Chromatography

Petroleum ether extract (10 g) was chromatographed over activated aluminium oxide (BDH, England) column and eluted with a mixture of solvents with increasing polarity, including n- hexane (SD Fine Chem, Mumbai), diethyl ether (SDFCI, India), chloroform (RFCI, India) and methanol (Fisher Scientific, UK). Fractions (100 ml each) were successively collected and those exhibiting similar TLC profiles, were combined. Further, purification by PTLC [Merck GF254, 0.5 mm, benzene: ethyl acetate (6:2 v/v)] was performed using sulfuric acid (H2SO4) and vanillin/ H2SO4 reagents.

Fraction I was eluted by 100% n- hexane and subjected to GC/MS analysis. Compounds 1-6 were eluted with 10 % ether in hexane responded positively to Salkowski and Lieberman-Büchard reactions for triterpenes [23].

Compound 1: White powder (21.32 mg), m. p. 241-244 °C, isolated from fraction II eluted with 10% ether in hexane. MS (70 eV), m/z (rel. int) 438 [M]+ (C30H46O2) (30), 423 (35), 410 (50), 273 (65), 232 (100), 217 (40), 135 (25), 69 (32) and 55 (46). 1H-NMR (CDCl3, 500 MHz), δ 2.39 (1H, s, H-9), 5.60 (1H, s, H-12), 1.04(3H, s, H-23), 1.07(3H, s, H-24), 1.24(3H, s, H-25), 1.14 (3H, s, H-26), 1.34 (3H, s, H-27), 0.84 (3H, s, H-28), 0.90 (3H, s, H-29), 0.87 (3H, s, H-30). 13C-NMR (CDCl3, 125 MHz), δ 40.1 (C-1), 34.5 (C-2), 217.1 (C-3), 47.8 (C-4), 55.3 (C-5), 18.7 (C-6), 31.9 (C-7), 45.1 (C-8), 61.0 (C-9), 35.6 (C-10), 199.6 (C-11), 127.9 (C-12), 171.1(C-13), 43.5 (C-14), 26.4 (C-15), 26.3 (C-16), 32.2 (C-17), 47.6 (C-18), 45.1 (C-19), 30.9 (C-20), 34.3 (C-21), 36.1 (C-22), 22.0 (C-23), 26.4 (C-24), 15.7 (C-25), 18.5 (C-26), 23.4 (C-27), 28.4 (C-28), 23.5 (C-29), 32.9 (C-30).

Compound 2: White crystals (52.4 mg), m. p. (168 °C) [24], isolated from fraction II eluted with 10% ether in hexane. MS (70 eV), m/z (rel. int) 424 [M]+ (C30H48O) (16), 409 (8), 355 (20), 327 (16), 281(40), 218(100), 205(24), 203(59), 189(20), 163(14), 133 (24), 119 (22) and 55(60). 1H-NMR (CDCl3, 500 MHz), δ 2.33 (1H, m, H-9), 5.27 (1H, dd, J= 3.7, 3.4 Hz, H-12), 1.04(3H, s, H-23), 1.07(3H, s, H-24), 1.01(3H, s, H-25), 0.99 (3H, s, H-26), 1.24 (3H, s, H-27), 0.84 (3H, s, H-28), 0.90 (3H, s, H-29), 0.86 (3H, s, H-30). 13C-NMR (CDCl3, 125 MHz), δ 39.8 (C-1), 34.5 (C-2), 217.2 (C-3), 47.6 (C-4), 55.3 (C-5), 18.8 (C-6), 33.2 (C-7), 40.1 (C-8), 47.7 (C-9), 36.6 (C-10), 23.7 (C-11), 122.4 (C-12), 144.1(C-13), 42.5 (C-14), 26.2 (C-15), 26.9 (C-16), 32.5 (C-17), 47.2 (C-18), 47.1 (C-19), 31.0 (C-20), 34.6 (C-21), 36.9 (C-22), 26.4 (C-23), 22.1 (C-24), 15.6 (C-25), 16.8 (C-26), 25.9 (C-27), 28.6 (C-28), 23.7 (C-29), 32.8 (C-30).

Compound 3: White crystals (19.3 mg) of m.p. 228 °C isolated from fraction eluted with 10% ether in hexane. MS (70 eV), m/z (rel. int) 442 [M]+ (C30H50O2) (8), 409 (9), 234 (28), 216 (10), 207 (11), 204 (26), 203 (100), 189 (13), 107 (9), 95 (9), 81 (15) and 69 (17). 1H-NMR (CDCl3, 500 MHz), δ 3.17 (1H, dd, J= 11.2, 5.2 Hz, H-3), 5.11 (1H, t, J = 3.1Hz, H-12), 3.53 (1H, d, J= 10.8Hz, Hb-28), 3.20 (1H, d, J= 10.8Hz, Ha-28), 0.91 (3H, s, H-23), 0.77(3H, s, H-24), 0.86 (3H, s, H-25), 0.87 (3H, s, H-26), 0.99 (3H, s, H-27), 0.92 (3H, s, H-29), 1.14 (3H, s, H-30). 13C-NMR (CDCl3, 125 MHz), δ 38.6 (C-1), 27.3 (C-2), 79.1 (C-3), 38.8 (C-4), 55.1 (C-5), 18.4 (C-6), 32.6 (C-7), 39.8 (C-8), 46.7 (C-9), 36.9 (C-10), 23.6 (C-11), 122.7 (C-12), 144.2 (C-13), 41.8 (C-14), 25.6 (C-15), 22.3 (C-16), 36.9 (C-17), 42.6 (C-18), 46.6 (C-19), 30.9 (C-20), 34.1 (C-21), 31.1 (C-22), 28.1 (C-23), 15.5 (C-24), 15.6 (C-25), 16.8 (C-26), 25.9 (C-27), 56.5 (C-28), 23.6 (C-29), 33.2 (C-30).

Compound 4: Colorless crystals (40 mg), m. p. 212-214 °C. MS (70 eV), m/z (rel. int) 426 [M]+ (C30H50O) (22), 411 (15), 408 (30), 393 (35), 384 (15), 220 (80), 218 (90), 207 (25), 189 (38), 139 (70) and 55 (100). 1H-NMR (CDCl3, 500 MHz), 3.18 (1H, dd, J = 11.2, 5.2 Hz, H-3), 2.34 (1H, m, H-19), 0.79 (3H, s, Me-23), 0.83 (3H, s, Me-24), 0.94 (3H, s, Me-25), 0.96 (3H, s, Me-26), 1.03 (3H, s, Me-27), 0.75 (3H, s, Me-28), δ 4.66 (1H, br s, Ha-29), 4.55 (1H, br s, Hb-29), 1.66 (3H, s, Me-30).

Compound 5: Colorless needles (47.2 mg), m. p. 148-149 °C [25]. MS (70 eV), m/z (rel. int) 386 [M]+ (C27H46O) (22), 371(7), 368(9), 353(4), 273(25), 255(42), 231(15), 213 (20), 147 (39) and 55(100).

Compound 6: White needles (76 mg), m. p. 136-142 °C [26]. MS (70 eV), m/z (rel. int) 414 [M]+ (C29H50O) (26), 412 (18), 399 (7), 329 (3), 314 (7), 301(9), 300 (5), 281(4), 273 (15), 271(25), 255(21), 231 (12), 213 (15) and 55(100).

Hepatorenal toxicity studies

In vivo study

Animals

Male Wistar albino rats (100-120g) were selected for this study. They were obtained from the Animal House, National Research Center, Egypt. All animals were kept in the controlled environment of air and temperature with the access of water and diet.

Ethics

Anesthetic procedures and handling with animals were complied with the ethical guidelines of the Medical Ethical Committee of the National Research Centre in Egypt and performed for being sure that the animals do not suffer at any stage of the experiment.

Toxicity study

Animals were subdivided into 4 subgroups (8 rats each). All groups received one oral dose of 100, 300, 500 and 1000 mg of petroleum ether extract/kg body weight. After 24 hours, there were no dead animals; representing the safety of the extract.

Doses

The administration regimens was twice a week for three consecutive weeks. The petroleum ether extract was administrated orally with a dose 150 mg/kg body weight. The dose was selected according the toxicity study. The selected dose was confirmed by [27].

Bromobenzene was intraperitonealy administrated at a dose 460mg/kg BW (1:10 w/v corn oil) [15]. Hepaticum as a reference herbal drug (active constituent; silymarin) was orally given at a dose 100mg/kg BW [5].

Experimental design

Six animals groups (6 rats each) were classified as follows:

Group 1: Normal healthy rats. Group 2: orally administered with plant extracts. Group 3: i.p. injected with bromobenzene. Group 4: forced with petroleum ether extract and BB at the same time and for the same duration. Group 5: forced with hepaticum as reference drug and BB as group 4. Group 6: orally received Hepaticum drug only.

Sample preparations

Serum sample: Blood collected from each animal by puncture the sub-lingual vein in a clean and dry test tube, left 10 min to clot and centrifuged at 3000 g for 10 min at 4oC for serum separation. The separated serum was stored at -80 oC for further determinations of liver and kidney functions tests and serum protein.

Liver tissues were homogenized in normal physiological saline solution (0.9% NaCl) (1:9 w/v). The homogenate was centrifuged at 3000 rpm for 10 min at 4oC. The supernatant was used for estimation of hepatic antioxidant and marker enzymes.

Biochemical assays

Cell organelle marker enzymes; hepatic succinate dehydrogenase [28], lactate dehydrogenase [28], acid phosphatase [30], glucose-6-phosphatase [31], 5`-nucleotidase [32], were carried out. Liver function indices; serum aspartate & alanine aminotransferase [33], alkaline phosphatase [34], gamma glutamyl transferase [35], and bilirubin were estimated. Kidney function tests; serum cratinine [36], urea [37] and total protein [38] were done. Antioxidant parameters; liver glutathione [39], lipid peroxides [40] and superoxide dismutase [41] were estimated.

Histopathological analysis

Liver and kidney sections of all groups were stained with haematoxyline & eosin as well as Masson's Trichome to detect changes in cells and different degree of fibrosis [42].

Cytotoxic Assay

Cytotoxic effect was accomplished on human cell line (HePG2 – MCF7 – HT29 and A549). Cell viability was assessed by the mitochondrial dependent reduction of yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) to purple formazan [43].

Procedure: All the following procedures were performed in a sterile area using a Laminar flow cabinet Biosafety class II level (Baker, SG403INT, Sanford, ME, USA). The method was carried out according to Thabrew [44]. Cells were batch cultured for 10 days, then seeded at concentration of 10x103 cells/well in fresh complete growth medium in 96-Well Microtiter plastic plates at 37 °C for 24 h under 5% CO2 using a Water jacket CO2 incubator (Sheldon, TC2323, Cornelius, OR, USA). Media was aspirated, fresh medium (without serum) was added and cells were incubated either alone (negative control) or with different concentrations of sample to give a final concentration of (100, 50, 25, 12.5 and 6.25 µg/ml).

Cells HePG2 & HT29 were suspended in Roswell Park Memorial Institute 1640 medium and MCF7 & A549 were suspended in Dulbecco’s modified Eagles Medium, supplemented with 1% antibiotic-antimycotic mixture (10,000U/ml Potassium Penicillin, 10,000µg/ml Streptomycin Sulfate and 25µg/ml Amphotericin B) and 1% L-glutamine (all purchased from Lonza, Belgium) in 96-well flat bottom microplate at 37 °C under 5% CO2. After 48 h of incubation, the medium was aspirated, 40ul MTT salt (2.5μg/ml) were added to each well and incubated for a further four hours at 37°C under 5% CO2.

To stop the reaction and dissolving the formed crystals, 200μL of 10% Sodium Dodecyl Sulphate (SDS) in deionized water was added to each well and incubated overnight at 37°C. A positive control which composed of 100µg/ml of Annona cherimolia leaves extract was used as a known cytotoxic natural agent which gives 100% lethality under the same conditions. The absorbance was then measured using a microplate multi-well reader (Bio-Rad Laboratories Inc., model 3350, Hercules, California, USA) at 595nm and a reference wavelength of 620nm. Adriamycin ® (Doxorubicin) 10 mg vials (Pharmacia, Sweden) was used as the reference drug.

Statistical analysis

For hepato-renal assay: All data were expressed as mean ± S.D. of six rats in each group. Statistical analysis was carried out by one-way analysis of variance (ANOVA), Costat Software Computer Program.

For cytotoxicity assay: All values were expressed as the mean ± SEM of the three replicates for each treatment. Data were subjected to paired- samples SPSS Statistical Software Package (version 8.0). P<0.005 was regarded as significant.

RESULTS AND DISCUSSION

The yield of petroleum ether extract from Atriplex Lindleyi aerial parts was 6.57%. It showed positive Liebermann-Bürchard and Salkowski tests indicating the presence of triterpenes and sterols. The total steroidal and triterpenoidal contents were quantitatively estimated and were found 2.8%w/w as stigmasterol and 3.55%w/w as α-amyrin, respectively. Saponification of lipoidal matter afforded 74.80 % of unsaponifiable matter and 18.36 % of the fatty acids. GC/MS analysis of unsaponifiable matter led to identify 42 compounds summarized in Table (1), accounting for 84.27%. The unsaponifiable matter was composed of hydrocarbons (73.39% of total unsaponifiable matter), alcoholic hydrocarbons (0.88 %) and sterols (2.22%). While, GC/MS analysis of fatty acid methyl ester derivatives revealed 16 fatty acids presented in Table (2). The saturated fatty acids represent 85.10 % of total identified fatty acid, whereas unsaturated fatty acids represent 14.90%. The Cerotic acid (20.30%) and palmitic acid (15.70 %) were major fatty acids. GC/MS analysis of unsaponifiable matter didn’t reveal triterpenes compounds that led us to fractionate the pet. ether extract over column chromatography of alumina oxide. The main fraction I (1.34 g) eluted with 100% n-hexane and further subjected to GC/MS. The GC/MS analysis led to identify 22 compounds representing 87.09% and illustrated in Table (3). Phytol (26.17%) and 2,6-bis (1,1-dimethylethyl)-4-methyl phenol (18.23%) represent the main components of fraction I. Whilst, phytol wasn’t detected in unsaponifiable matter. In addition, four nonsterol tritrepenoids; 12-oleanene- 3,11-dione (1), 12-oleanene-3-one (2), olean-12-ene-3β, 28β-diol (3), 5α-lup-20(29)-en-3β-ol (4) as well as three sterol tritrepenoids; cholesterol (5), mixture of β-sitosterol (24-ethylcholest-5-en-3β-ol) and stigmasterol (24-ethylcholesta-5,22-diene-3β-ol )(6) were isolated and identified by comparing their physical and spectroscopic data with those reported in the literature and by comparison with authenticated samples.

Compound 1 was obtained as white powder of m. p. 241~245 °C and the electron impact mass (EI-MS) [M]+ peak at m/z 438 corresponding to the molecular formula C30H46O2. Mass spectrum showed prominent peaks at m/z 423[M-15]+, 273 [C16H24O]+, 135[C16H24O]+, 232[C16H24O]+ retro-Diels-Alder (RDA) fragments characteristic for Δ 12-oleane series with diketones [45] and are confirmed by the appearance of ketone groups at δ 217.1 & 199.6 in 13C NMR. The intense peak at m/z 273 indicated the presence of a carbonyl group in rings A-C and that no other groups were present in these rings. Further, the appearance of two singlet signals at δ 5.60 & 2.39 in 1H NMR and signal at 127.9 (C-12) & 60.0 (C-9) in 13C NMR prove that the one ketone was present at C-11. Comparing the characteristic features of compound 1 with that of the reported of compound 12-oleanene-3,11-dione [46-48] emphasized that compound 1 is 12-oleanene-3,11-dione and is recorded here for the first time isolated from Atriplex species.

Compound 2 was isolated as white crystals of m. p. 168 °C and (EI-MS) [M]+ peak at m/z 424 corresponding to the molecular formula C30H48O. The spectroscopic analysis of this compound was closely similar to the reported for β- amyrenone (12-oleanene-3-one) isolated previously from Atriplex inflate [11]. The 1H NMR spectra revealed the presence of a doublet of doublet signal at 5.31 with J=3. 7, 3.4 Hz for olefinic proton and eight methyl groups H-23, 24, 25, 26, 27, 29, 28, 30 appeared in δ 1.04, 1.07, 1.01, 0.99, 1.24, 0.84, 0.90, 0.86, respectively, together with mass fragments at m/z 409 [M-CH3]+, 218[RDA], 203 [218-CH3]+, 205 [C14H21O]+ and 189 [218-CH2CH3]+ implied the compound 2 is typical for pentacyclic triterpene with Δ 12-oleane skeleton. Further, the 13C NMR spectrum showed characterstic signal at δ 217.2 for carbonyl group of C-3. The mass spectra, physical and NMR spectral data are in agreement with those reported in the literatures of β- amyrenone [18,49].

Table 1: GC/MS analysis of unsaponifiable matter isolated from the aerial parts of Atriplex lindleyi

CompoundsRt%

BP

m/z

MWt

m/z

Main fragments (m/z)
  1. Hydrocarbon

n-Undecane

 12.13 0.27 57 156 71, 85, 99, 113, 127,140
5-Methylundecane 14.22 0.35 57 170 71, 44, 85, 112, 69
4-Methylundecane 14.32 0.28 43 170 71, 41, 57, 85, 126, 127, 98
2-Methylundecane 14.42 0.59 43 170 57, 71, 41, 85, 99, 126,127
3-Methylundecane 14.59 0.60 57 170 71, 85, 43, 99, 113, 141
1-Methyl, 3-pentylcyclohexane 15.44 0.68 97 168 55, 69, 81, 96, 125, 139
Cyclododecane 15.53 0.50 69 182 55, 83, 97, 111,168, 139
n-Dodecane 16.10 4.69 57 170 43, 71, 85, 99, 113, 127
2, 6- Dimethylundecane 16.31 1.31 57 184 43,71, 98, 85, 113, 141
1-Hexylcyclohexane 17.40 0.35 83 168 82, 55, 41, 67, 97
1-Tetradecene 22.51 1.49 55 196 41, 43, 83, 57, 69, 97, 168
n-Tetradecane 23.01 0.17 57 198 43, 71, 85, 99, 113
7-Hexadecene 29.00 2.40 55 224 43, 69, 83, 97, 111,140
n-Hexadecane 29.08 0.19 57 226 43, 71, 85, 99, 127, 113
1-Octadecene 34.30 2.81 57 252 43, 55, 83, 97, 69, 111, 125
Octadecane 34.36 0.19 57 254 43, 71, 85, 99, 113, 127
Cubitene 36.24 1.45 67 272 121,107,133,257,189,215,177, 161, 229
Dolabradiene 37.32 1.16 93 272 81, 107, 121, 135, 67, 147, 257, 243, 215, 201
5-Eicosene 39.28 2.66 55 280 41, 97, 83, 69, 111, 125, 139, 154, 252
n-Heneicosane 41.52 0.42 57 296 71, 43, 85, 99, 113, 127, 141, 169
1-Docosene 44.06 2.67 55 308 57, 43, 97, 83, 69, 111, 125
n-Tricosane 46.11 1.46 57 324 71, 85, 99, 113, 127, 141
Cyclotertacosane 48.11 2.13 83 336 97, 55, 69, 43, 111, 210,308
n -Pentacosane 50.12 7.17 57 352 71, 85, 99, 113, 127, 141
1-Hexacosene 52.20 1.83 57 364 83, 97, 69, 55, 111, 125, 139
5- Methyl, 5-ethyltetracosane 53.11 0.19 57 380 71, 85, 99, 113, 365, 337
n -Heptacosane 53.56 5.90 57 380 71, 85, 99, 113, 127
n -Octacosane 55.38 1.55 57 394 71, 85, 99, 113, 127
Squalene 55.55 0.54 69 410 81, 95, 123, 137, 55, 109, 121
5-Methyl, 5-ethylhexacosane 56.40 1.14 57 408 71, 85, 99, 365, 393
n -Nonacosane 57.27 14.34 57 408 43, 71, 85, 99, 113
n -Triacontane 58.57 1.62 57 422 71, 43, 85, 99, 113
n -Hentriacontane 60.38 10.29 57 436 71,85, 99, 113,43

Alcoholic Hydrocarbon:

3-Nonacosanol

 58.34 0.48 59 424 57,97, 83, 41, 111, 125, 407
3-Triacontanol 61.53 0.40 59 452 57, 97, 83, 41, 111, 125, 435

Ester:

Tributyl acetylcitrate

 45.38 0.61 185 402 259, 129, 57, 157, 329, 213, 273, 301

sterols:

Cholesterol

 58.16 1.55 43 386 371, 355, 341, 313, 301, 267, 147, 97
β- Sitosterol 59.45 0.67 55 414 399, 396, 381, 329, 303, 273, 213, 161, 145, 136, 119, 107, 95

Miscellaneous

1,1- Dioxidetetrahydrothiophene

 18.41 3.06 41 120 56, 55, 60, 64, 76, 90
2,6-Di-(t-butyl)-4-hydroxy-4-methyl-2,5- cyclohexadien-1-one 25.30 2.32 165 236 180, 57, 137, 193, 205, 221
2,6-bis(1,1-dimethylethyl)-4-methyl phenol 26.27 0.69 205 220 57, 204, 145, 177, 105, 189
7,9-Di-tert-butyl-1oxaspiro[4.5] deca-6, 9-diene-2, 8-di-one 32.42 1.10 57 276 205, 175, 217, 189, 232, 261

Rt = Retention time BP= Base Peak MWt= Molecular Weight

was obtained as white crystals of m. p. 228 °C and EI-MS revealed a molecular ion peak at m/z 442 corresponding to the molecular formula C30H50O2. The mass spectrum of compound 3 displayed retro-Diels-Alder fragment peak at m/z 234, characteristic for erythrodiol [50] analog with other fragments at m/z 411 ([M]+- CH2OH), 203, 189, 175, 133, indicating a C-12/C-13 double bond that suggested an oleanane structure substituted by two hydroxyl groups (OH), one located at A/B rings and one D/E rings.

These data confirmed by the presence resonances of tertiary (C-3) and secondary alcoholic carbon (C-28) at 79.1 & 56.5 in 13C NMR and also appeared as a double doublet signal at 3.17 (J= 11.2, 5.2 Hz) and two doublet signals at 3.20 (J= 10.8Hz) and 3.53 (J= 10.8Hz), respectively, in 1H NMR. Further, seven tertiary methyl groups resonating at δ 0.91, 0.77, 0.86, 0.87, 0.99, 0.92 and 1.14, an olefinic proton resonating at δ 5.11 (t, J=3.1 Hz) were observed in the 1H NMR spectrum of 3. These spectral data agreed with those published for erythrodiol [51].

Compound 4

was isolated as white crystals of m. p. 212-214 °C and (EI-MS) [M]+ peak at m/z 426 corresponding to the molecular formula C30H50O. The 1H NMR spectrum showed seven tertiary methyl singlet signals and one secondary hydroxyl group as a doublet of doublets at δ 3.18.

Also, it showed two olefinic protons at δ 4.68 and 4.56 representing the exocyclic double bond.

The physical and spectral data of this compound were closely similar to the reported data of lupeol in the literature [52] and was confirmed by co-TLC with authentic samples. Lupeol was isolated previously from Atriplex inflate [11].

Table 2: Fatty acid methyl ester derivatives identified by GC/MS from aerial parts of Atriplex lindleyi

Fatty AcidRRTBp m/z

Mwt

m/z

Relative percentage of total fatty acids
14:0

Saturated Fatty acids:

Myristic acid (Tetradecanoic acid)

 0.873 74 242 2.24
16:0 Palmitic acid (Hexadecanoic acid) 1.000 74 270 15.70
18:0 Stearic acid (Octadecanoic acid) 1.115 74 298 1.87
13:0 Tridecanedioic acid 1.190 98 284 4.54
20:0 Arachidic acid (Eicosanoic acid) 1.221 74 326 1.51
22:0 Behenic acid (Docosanoic acid) 1.319 74 354 2.30
23:0 Tricosylic acid (Tricosanoic acid) 1.364 74 368 1.51
24:0 Lignoceric acid (Tetracosanoic acid) 1.384 74 382 8.82
25:0 Pentacosylic acid (Pentacosanoic acid) 1.409 74 396 2.47
26:0 Cerotic acid (Hexacosanoic acid) 1.451 74 410 20.30
26:0 24-Methylhexacosanoic acid 1.493 74 424 2.47
28:0 Montanic acid (Octacosanoic acid) 1.535 74 438 12.33
32:0 Dotriacontanoic acid 1.635 74 494 9.04
Total 85.10
18:1

Unsaturated Fatty Acids:

Oleic acid (9- Octadecenoic acid)

 1.002 55 296 2.53
18:2 Linoleic (9,11-Octadecadienoic acid) 1.098 67 294 4.44
18:3 Linolenic acid (9,12, 15-Octadecadienoic acid) 1.103 79 292 7.93
Total 14.90

RRt = Relative Retention time BP= Base Peak MWt= Molecular Weight


Table 3: GC/MS analysis of fraction I isolated from column chromatography of pet. ether extract of Atriplex lindleyi

CompoundsRt%

BP

m/z

MWt

m/z

Main fragments (m/z)
1,1- Dioxidetetrahydrothiophene 18.25 7.03 41 120 56, 55, 60, 64, 76, 90
1,1- Diethoxypentane 23.04 1.39 103 160 75, 83, 55, 129, 89
2,6-di(t-butyl)-4-hydroxy-4-methyl-2,5-cyclohexadien-1-one 25.31 1.26 165 236 57, 180, 137, 123, 205, 221, 193
2,6-bis(1,1-dimethylethyl)-4-methylphenol 26.40 18.23 205 220 145, 177, 105, 161, 189
5, 6, 7, 7a- Tetrahydro 4, 4, 7a-tri -2-(4H)- benzofuranone 28.31 1.53 111 180 109, 137, 67, 55, 124, 152, 165
1-Hexadecene 28.58 0.95 43 224 55, 69, 83, 97,111
7,9- Benzo-1,5-dithiecane 34.06 0.61 104 210 103, 105, 211, 135, 149, 212
1-Octadecene 34.28 1.83 57 252 55, 83, 97, 69, 111, 125
6, 10, 14-Trimethyl-2-pentadecanone 35.57 1.33 58 268 71, 85, 95, 109, 124, 210
5-Eicosene 39.26 1.79 55 280 69, 83, 97,111
n-Heneicosane 41.50 0.54 57 296 71, 85, 99, 113, 252
Phytol 42.25 26.17 71 296 123, 81, 57, 81, 278
1-Decosene 43.59 1.05 55 308 43, 57, 69, 83, 97, 111
n-Docosane 44.02 0.62 57 310 43, 71, 85, 99, 113
n-Tricosane 46.10 1.21 57 324 71, 43, 85, 99, 113
4,8,12,16-Tetramethylheptadecan-4-olide 47.46 0.95 99 324 43, 55, 69, 114, 126, 83, 151, 166, 196
n-Tetracosane 48.12 1.39 57 338 57, 43, 71, 85, 99, 113
Dioctyl hexanedioate 48.20 0.77 129 370 57, 70, 112, 147, 241, 259
n-Pentacosane 50.10 3.83 57 352 43, 71, 43, 85, 99, 113
n-Hexacosane 52.02 1.49 57 366 43, 71, 85, 99, 113
n-Heptacosane 53.51 4.77 57 380 43, 71, 85, 99, 113
n-Nonacosane 57.18 8.35 57 408 43, 71, 85, 99, 113

Rt = Retention time BP= Base Peak MWt= Molecular Weight

Compound 5 was obtained as colorless needles of m. p. 148-149 °C and its mass spectrum gave one molecule ion (M+) at m/z 386 corresponding to C27H46O and other prominent peaks at m/z 371 (M+- CH3), 368 (M+- HOH), 353 (M+- (CH3+HOH), 273 (M+- side chain), 255 (M+- side chain+HOH), 231 (M+- (side chain+ 42)), 213 (M+-(side chain+ 42+HOH)). These data are typical to Δ 5-cholestane skeleton together with direct comparison (Co-TLC, m.p. and MS) proved to be cholesterol that was also identified in GC/MS analysis. Compound 5 is first reported from Atriplex lindleyi, however, it was identified previously in Atriplex halimus [8], Schanginia aegyptiaca and Salsola tetrandra (Chenopodiaceae) [7].

Compound 6 was obtained as colorless needles of m. p 135-140 °C. Two molecular ions given at m/z 414 and 412 analog with other ion peaks at m/z 271, 273 suggesting 6 was a mixture of two compounds. The ion peaks at m/z 271, 273 due to β bond cleavage of side chain leading loss of C10H21 and C10H23, respectively [20]. The fragmentation pattern of compound 6 and direct comparison with authentic samples (Co-TLC, Rf, mp) proved that it was composed of β-sitosterol and stigmasterol with molecular weight 414 and 412, respectively. These compounds were identified as mixture previously from Atriplex inflate [9, 11].

Indeed, most of the pentacyclic triterpenoids obtained from family Chenopodiaceae belongs to the oleanene skeleton [7]. Salt et al (1985) reported that 4-desmethylcholesterols are predominant sterols in the family Chenopodiaceae [57]. Various sterols such as β-sitoterol, cholesterol, stigmasterol and 24ξ- methyl-5α-cholestan-3β-ol were previously reported in Atriplex species [4, 8]. The sterol profiles of Atriplex species were characterized by coexistence of Δ7- and Δ5- sterols in ratios of 0.3:1 to 0.4: 1 and male of Atriplex plants contained a higher proportion of Δ5- sterols than female [58].

Biological activity

Drastic changes in all biochemical parameters under investigation after bromobenzene toxicity were recorded (Tables 4-7).

Treatment with petroleum ether extract of Atriplex lindleyi showed improvement in liver marker enzymes, liver function indices and kidney function tests with variable degrees. In addition, petroleum ether exerts antioxidant activity (Tables 4-7) through its ameliorative effect recorded in the levels of glutathione, lipid peroxide and superoxide dismutase as compared with the reference drug. Hepaticum as an antioxidant flavonoid complex derived from the herb milk thistle (Silybum marianum), has also the ability to scavenge free radicals, chelate metal ions, inhibiting lipid peroxidation and preventing liver glutathione depletion [53].


123
R1 O H, H H, H
R2 O O OH
R3 CH3 CH3 CH2OH

In the oxidative stress process, the free radicals involvement affected the mitochondria, the plasma membrane permeability and the microsomal integrity which lead to leakage of liver enzymes into circulation [54]. The extract role as a free radical scavenger could in turn normalize microsomes, mitochondria and plasma membranes permeability and integrity which lead to restore the hepatic enzymes to normal levels.

BB metabolites are formed in the liver and transported to the kidney, where they exerted the nephrotoxic action [15]. This nephrotoxic action was mediated by the observed disturbance in the urea, creatinine and protein levels. Treatment with Atriplex lindleyi petroleum ether extract improved the kidney function parameters which give an additional support that this extract mops up free radicals generated by BB and induces a healthy state of renal cells, suggesting its role as a renal protective agent.

Lupeol

Liver morphological and histopathological observations (Figure 1 a & b) confirmed our biochemical determinations through reduction in fibrotic and infiltration area as well as collagen distribution. Kidney section also showed minimal infiltration area and low dilatation in Bowman capsules (Figure 2).

24-methylenecholesterol and β-sitosterol were found to decrease significantly the cholesterol level in both serum and liver [59]. Rats pre-treated with lupeol had the serum and liver enzyme levels restored to almost normal. Additionally, treatment with lupeol substantially normalized degenerative alterations in hepatocytes with granular cytoplasm. Lupeol also re-established antioxidant enzyme activities in mouse liver affected by 7, 12- dimethylbez(α) anthracene induced oxidative stress [60].

Table 4: The effect of Atriplex lindleyi petroleum ether extract on liver marker enzymes of healthy, intoxicated and treated rats

ParametersControlControl plant treatedControl drug treated

Intoxicated

(BB)

Intoxicated plant treated

(a)

Intoxicated drug treated

(b)

% of improvement
(a)
Succinate dehydreogenase 245.50±6.18a

240.33±3.14a

(-2.10)

241.33±2.25a

(-1.69)

132.69±11.12c

(-45.95)

206.66±9.97b

(-5.82)

212.55±8.80b

(-13.42)

 30.13
Lactate dehydrogenase 142.03±3.75a

138.23±3.85ab

(-2.67)

136.17±2.78ab

(-4.12)

103.81±9.66c

(-26.91)

133.39±9.04b

(-6.08 )

135.25±6.72ab

(-4.77)

 20.83
Glucose -6- Pase 32.72±1.68a

32.22±1.56a

(-1.53)

32.16±2.13a

(-1.71)

18.66±1.35c

(-42.97)

24.65±2.46b

(-24.66)

26.10±1.71b

(-20.23)

 18.30
Acid phosphatase 5.27±0.74b

5.49±0.46b

(+4.17)

5. 18±0.19b

(-1.70)

8.83±0.98a

(+67.55)

6.16±1.16b

(+16.88)

5.66±1.03b

(+7.40)

 50.66
5`-nucleotidase 188.29±5.40c

190.34±4.65c

(+1.09)

193.66±5.00c

(+2.85)

325.04±9.31a

(+72.63)

230.37±6.73b

(+22.35)

212.83±7.67bc

(+13.03)

 50.28

Table 5: Effect of Atriplex lindleyi petroleum ether extract on liver function indices of healthy, intoxicated and treated rat serum

ParametersControlControl plant treatedControl drug treated

Intoxicated

(BB)

Intoxicated plant treated

(a)

Intoxicated drug treated

(b)

% of improvement
(a)
AST 20.48±1.78d

21.22±3.28d

(+3.61)

21.50±1.04 d

(+4.98)

32.54±2.68a

(+58.88)

28.49±3.60b

(+39.11)

24.73±3.21c

(+20.75)

 17.57
ALT 19.23±1.02d

19.58±1.20d

(+1.82)

19.80±1.04 cd

(+2.96)

26.15±1.73a

(+35.98)

23.93±2.07b

(+24.44)

21.96±0.86c

(+14.19)

 11.54
ALP 7.20±0.87c

7.44±0.78 c

(+3.33)

7.40±0.52c

(+2.77)

14.19±0.86a

(+97.08)

9.15±1.55b

(+27.08)

8.21±0.84bc

(+14.02 )

 70.00
GGT 3.57±0.38c

3.22±0.36c

(-9.80)

3.15±0.18 c

(-11.76)

5.86±0.73a

(+64.14)

4.33±0.94b

(+21.28)

3.82±0.63bc

(+7.00)

 42.85
Total bilirubin 5.30±0.52d

4.77±0.44 d

(-10.00)

4.59±0.55 d

(-13.39)

9.74±0.92a

(+83.77)

8.11±0.62b

(+53.01)

7.22±0.69c

(+36.22)

 30.75

Table 6: The effect of Atriplex lindleyi petroleum ether extract on antioxidant levels of healthy, intoxicated and treated rat liver

ParametersControlControl plant treatedControl drug treated

Intoxicated

(BB)

Intoxicated plant treated

(a)

Intoxicated drug treated

(b)

% of improvement
(a)
Lipid peroxides 0.92±0.05b

0.91±0.06b

(-1.08)

0.86±0.06b

(-6.52)

1.72±0.30a

(+86.95)

1.03±0.11b

(+11.95)

0.97±0.02b

(+5.43)

 75.00
Glutathione 67.93±3.24a

62.54±3.83bc

(-7.93)

63.00±2.36b

(-7.25)

59.47±4.3c

(-12.45)

61.98±1.57bc

(-8.75)

64.82±1.23ab

(-4.57)

 3.29
Superoxide dismutase 16.64±1.48bc

16.94±1.35c

(+1.80 )

16.90±1.05c

(+1.56)

20.70±6.92a

(+24.39)

19.64±2.37b

(+8.02)

18.80±2.80bc

(+12.98)

 6.37

Table 7: Effect of Atriplex lindleyi petroleum ether extract on kidney functions of healthy, intoxicated and treated rats

ParametersControlControl plant treatedControl drug treated

Intoxicated

(BB)

Intoxicated plant treated

(a)

Intoxicated drug treated

(b)

% of improvement
(a)
Urea 21.42±1.45bc

20.13±1.37c

(6.02)

20.16±1.47 c

(5.88)

18.23±0.64d

(14.89)

27.16±1.77a

(26.79)

22.35±1.45b

(4.34)

 41.69
Creatinine 0.59±0.05c

0.66±0.06c

(11.86)

0.57±0.04 c

(3.38)

0.81±0.03a

(37.28)

0.68±0.04b

(15.25)

0.65±0.03b

(10.16)

 22.03
Serum protein 17.03±2.21a

15.97±0.45ab

(6.22)

15.57±0.51 ab

(8.57)

14.68±1.20b

(13.79)

15.16±1.46b

(10.98)

15.83±1.22ab

(7.04)

 2.81

Fig. 1a: Morphological structure of control liver (a), BB intoxicated liver (b), intoxicated liver treated with pet. ether extract (c), intoxicated liver treated with Hepaticum drug (d). b: Heamatoxyline & eosin and Masson's Trichrome stain liver sections of control rat (e, k), control treated with petroleum ether extract of Atriplex lindleyi (f, l), control treated with Hepaticum drug (g, m), BB intoxicated (h, n), BB intoxicated rats treated with pet. ether extract (i, o), BB intoxicated rats treated with drug (j, p). Arrows show normal hepatic cells in normal and normal treated liver (e, f, g, k, l, m). Massive fibrosis with collagen deposition in intoxicated liver (h, n). Less fibrotic tissue and collagen deposition were seen in treated liver with pet. ether extract and Hepaticum drug (i, j, o, p).


Fig. 2: Heamatoxyline & eosin and Masson's Trichrome stain kidney section (400 x) of control rat (a, g), control treated with petroleum ether extract of Atriplex lindleyi (b, h), control treated with Hepaticum drug (c, i), BB intoxicated (d, j), BB intoxicated treated with petroleum ether extract (e,k), BB intoxicated treated with drug (f, l). Arrows show normal glomeruli in normal and normal treated rats (a, b, c, g, h, i). Dilatation in Bowman capsules with massive infiltrations and fibrosis in interstitial space were observed in intoxicated rats (d, j). Normal glomeruli with minimal infiltrations and less fibrosis in interstitial space were seen in intoxicated rats treated with plant extract and Hepaticum drug (e, f, k, l).

Furthermore, the assessment of cytotoxic activity of petroleum ether extract against different human cell line showed growth inhibition on HepG2 and MCF7 human cells to 29.9 and 44.8%, respectively at concentration 100 ppm (Table 8). Capua et al (2010) reported that the hexane fraction of Atriplex confertifolia reduced MCF7 cell viability by less than 20%.[55]. Whereas, Donaldson (2000) found that the hexane fraction of Atriplex canescens showed 48.7% cell inhibition against HeLa cells [56].

Table 8: Cytotoxic activity of petroleum ether extract of Atriplex lindleyi in vitro on different human cell lines

Cell lines Conc. µg/ml % 0f Inhibition ± SEM
Petroleum Ether Extract Doxrubicin
A549 100 13± 1.05* 100± 0.00
50 3.4± 1.76* 75.63± 1.71
25 0.0± 0.003* 53.66± 1.55
HCT116 100 0.0± 0.00* 100± 0.00
50 0.0± 0.02* 65.2± 0.66
HepG2 100 29.9± 1.45* 100± 0.00
50 11.9± 0.81* 97.2± 0.57
25 3.8± 2.10* 60.2± 1.51
12.5 0.0± 0.00* 32.4± 1.02
MCF7 100 44.8± 1.15* 100± 0.00
50 18.7±1.73* 84.3± 2.42
25 6.1± 1.83* 57.6± 1.53
12.5 0.0± 0.001* 32.7± 0.52

Each value represents the percentage of inhibition growth ± SEM (standard error of mean of percentage of inhibition cells of three replicates).

* Significantly different from Doxorubicin value at P˂ 0.005 according to paired-samples t-test.

CONCLUSIONS

Atriplex lindleyi Moq. susp. Inflate is an edible plant commonly distributed in the Egyptian deserts used as food for human and animals. A detailed information on phytoconstituent and hepatorenal protection efficacy as well as cytotoxic activity of petroleum ether extract are presented, suggesting its potential medicinal use as hepato-renal protective agent upon further clinical studies.

CONFLICT OF INTEREST

There are no conflicts of interest

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