|Year : 2016 | Volume
| Issue : 47 | Page : 446-453
Antihepatotoxic effect and metabolite profiling of Panicum turgidum extract via UPLC-qTOF-MS
Mohamed A Farag1, Ahlam M El Fishawy1, Sayed A El-Toumy2, Khadiga F Amer3, Ahmed M Mansour4, Hala E Taha3
1 Pharmacognosy Department, College of Pharmacy, Cairo University, Cairo, Egypt
2 Chemistry of Tannins Department, National Research Center, Dokki, Cairo, Egypt
3 Aromatic and Medicinal Plants Department, Desert Research Center, Cairo, Egypt
4 Pharmacology and Toxicology Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt
|Date of Submission||09-Jan-2016|
|Date of Decision||10-Feb-2016|
|Date of Web Publication||30-Sep-2016|
Mohamed A Farag
Pharmacognosy Department, College of Pharmacy, Cairo University, Cairo
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Panicum turgidum, desert grass, has not reported any detailed phytochemical or biological study as yet Objective: To establish P. turgidum secondary metabolite profile and to assess its antihepatotoxic effect Materials and Methods: Ultra-performance liquid chromatography (UPLC) coupled to quadrupole high-resolution time of flight mass spectrometry (qTOF-MS) was used for large-scale secondary metabolites profiling in P. turgidum extract, alongside assessing median lethal dose (LD50) and hepatoprotective effect against carbon tetrachloride (CCl 4 ) intoxication Results: A total of 39 metabolites were identified with flavonoids as the major class present as O/C-glycosides of luteolin, apigenin, isorhamnetin and naringenin, most of which are first time to be reported in Panicum sp. Antihepatotoxic effect of P. turgidum crude extract was revealed via improving several biochemical marker levels and mitigation against oxidative stress in the serum and liver tissues, compared with CCl4 intoxicated group and further confirmed by histopathological examination. Conclusion: This study reveals that P. turgidum, enriched in C-flavonoids, presents a novel source of safe antihepatotoxic agents and further demonstrates the efficacy of UPLC-MS metabolomics in the field of natural products drug discovery.
Abbreviations used: UPLC: Ultra-performance liquid chromatography (UPLC), LD50: median lethal dose, MDA: malondialdehyde, GSH: glutathione reductase, CAT: catalase, SOD: superoxide dismutase, ALT: alanine aminotransferase, AST: aspartate aminotransferase, ALP: alkaline phosphatase, TG: triglycerides.
Keywords: Antihepatotoxic, flavonoids, Panicum turgidum, toxicity, UPLC MS analysis
|How to cite this article:|
Farag MA, El Fishawy AM, El-Toumy SA, Amer KF, Mansour AM, Taha HE. Antihepatotoxic effect and metabolite profiling of Panicum turgidum extract via UPLC-qTOF-MS. Phcog Mag 2016;12, Suppl S4:446-53
|How to cite this URL:|
Farag MA, El Fishawy AM, El-Toumy SA, Amer KF, Mansour AM, Taha HE. Antihepatotoxic effect and metabolite profiling of Panicum turgidum extract via UPLC-qTOF-MS. Phcog Mag [serial online] 2016 [cited 2019 Dec 10];12, Suppl S4:446-53. Available from: http://www.phcog.com/text.asp?2016/12/47/446/191455
- UPLC coupled to qTOF-MS was used for large scale secondary metabolites profiling in P. turgidum.
- A total of 39 metabolites were identified with flavonoids amounting as the major metabolite class.
- Anti-hepatotoxic effect of P. turgidum extract was revealed via several biochemical markers and histopathological examination.
- This study reveals that P. turgidum, enriched in C-flavonoids, present a novel source of antihepatotoxic agents.
| Introduction|| |
Panicum turgidum Forssk. (fam. Poaceae), known as desert grass by natives in Egypt, is a common perennial bunchgrass from Mauritania and Senegal eastwards through the Sahara and Sahel to Sudan, Ethiopia, Eritrea, and Somalia and through northern Africa and western Asia to Pakistan and India,  and very common in the Mediterranean and Red Sea coastal regions of Egypt.  Previous studies on Panicum genus revealed the presence of steroidal saponins,  phenolics including caffeic acid, ferulic acid, p-coumaric acid, p-hydroxybenzoic acid, protocatechuic acid,  quercetin-3-O-rhamnoside, and rutin flavonoids.  With regard to biological effect of its members, investigation of antioxidant activity,  diuretic effect,  antidiabetic activity,  and antiinflammatory and antipyretic properties  has been reported. Albeit, no previous detailed study of P. turgidum either on its phytochemical composition or its biological effect has been reported.
In addition, complete secondary metabolites profile in plants belonging to Panicum genus is still lacking, with most studies based on a targeted type of analysis. An essential purpose of the scientific discipline of metabolomics is the study of the biochemical composition of living organisms in a rather holistic manner. The current progress in plant metabolomics techniques , has made it possible to profile plant crude extracts in a rather untargeted manner. Metabolomics makes the use of hyphenated techniques that rely on chromatographic separation of metabolites coupled to mass spectrometry (MS) to analyze complex mixtures. Considering flavonoid known antihepatotoxic effects  and in search for other resources of active hepatoprotective agents, P. turgidum was assessed herein and results from this study provides a new evidence for its possible use as an adjunct therapy for alleviating acute and chronic toxicity effects in liver diseases, that is, Hepatitis C, a worldwide problem that is also endemic in Egypt.
| Materials and Methods|| |
Plant material and extraction process
The panic grass (Panicum turgidum) used in this study was grown wild in Wadi-Habes, 18 km Mersa-Matroh, Egypt. The plant was collected on April 2012 and identified by Dr. Attia Mohamed, Desert Research Center, Egypt (herbarium specimen no., 5/3/2015-B, Fac. of Pharmacy, Cairo Univ.). Freeze-dried aerial parts of the plant were ground with a pestle in a mortar using liquid nitrogen. The powder (20 mg) was then homogenized with 4 ml 70% ethanol containing 10 μg/ml umbelliferone (internal standard) using an ultrasonic bath for 30 min. Extract was then vortexed and centrifuged at 10,000 g for 10 min to remove plant debris and filtered through 22 μm Millipore filter and stored at −20°C for high-resolution Ultra-performance liquid chromatography (UPLC)-PDA-MS (UPLC/ photodiode array detection/MS) analysis. For biological assays, dried, powdered aerial parts of the plant were extracted with 70% ethanol till exhaustion.
Adult male Sprague-Dawley rats weighing 200-250 g, aged from 10 to 16 weeks, were purchased from the Lab. animal colony, Ministry of Health and Population, Helwan, Cairo, Egypt and maintained under standard laboratory conditions at the animal center, College of Pharmacy, Al-Azhar Univ., Cairo, Egypt. Rats were fed with basal diet pellets, supplied with water ad libitum, and kept in a temperature-controlled environment (20−22°C) and 40-60% relative humidity with an alternating 12-h light-dark cycle. The experiments were conducted in accordance with the ethical guidelines for investigations in laboratory animals and comply with the Guidelines for the Care and Use of Laboratory Animals.
Chemicals and drugs
Acetonitrile and formic acid (LCMS grade) were obtained from J.T. Baker (Deventer, Netherlands), milliQ water was used for LC analysis. Umbelliferone (P98%) (St. Louis, MO, USA). Silymarin, Ellman's reagent, thiobarbituric acid (TBA), reduced glutathione reductase (GSH), catalase (CAT), 1, 1, 3, 3-tetraethoxypropane, pyrogallol, trichloroacetic acid (TCA) and EDTA-Na 2 were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA), while (CCl 4 ) was obtained from Loba-chemie Co. (Mumbai, Maharashtra, India). Neutral formalin, HCl, n-butanol, o-phosphoric acid, potassium chloride, sodium chloride, sodium dibasic phosphate were obtained from El-Nasr Pharm. Chem. Co. (Cairo, Egypt), anhydrous potassium hydrogen phosphate and potassium dihydrogen phosphate were purchased from Fluka Lab. Chem. and Anal. Reagents Co. (Steinheim, Germany), and H 2 O 2 was provided from Fisher scientific (Fair lawn, NJ, USA.).
Analysis of plant extracts using UPLC/PDA/MS
UPLC/PDA/high resolution TOF-MS
Chromatographic separations were performed using  an Acquity UPLC system (Waters) equipped with a HSS T3 column (100- 1.0 mm, particle size 1.8 μm; Waters). The following elution binary gradient was used at a flow rate of 150 μL min -1 :0-1 min, isocratic 95% A (water/formic acid, 99.9/0.1 v/v), 5% B (acetonitrile/formic acid, 99.9/0.1 v/v); 1-16 min, linear from 5-95% B; 16-18 min, isocratic 95% B; 18-20 min, isocratic 5% B. Eluted compounds were detected from m/z 100 to 1000 using a MicroTOF-Q hybrid quadrupole time-of-flight mass spectrometer (BrukerDaltonics) equipped with an Apollo-II electrospray ion source in negative ion modes using the following instrument settings: nebulizer gas, nitrogen, 1.6 bar; dry gas, nitrogen, 6 l min-1, 190-C; capillary, 5500 V (+4000 V); end plate offset, 500 V; funnel 1 RF, 200 Vpp.
MicrOTOF-Q: Precursor ions were selected in Q1 with an isolation width of ±2 D and fragmented in the collision cell applying collision energies in the range of 10-30 eV. Product ions were detected using the following parameter settings: collision RF 150/400 Vpp (timing 50/50); transfer time, 70ls; pre pulse storage, 5ls; pulser frequency, 10 kHz; spectra rate, 1.5 Hz. For Collision-induced dissociation (CID) of in-source fragment ions (pseudo-MS3),in-source CID energy was increased from 0 to 100 V. Ion Trap MS: MSn mass spectra were also obtained from a LCQ Deca XP MAX system (ThermoElectron, San Jose, USA) equipped with an ESI source (electrospray voltage 4.0 kV, sheath gas: nitrogen; capillary temperature: 275°C) in negative ionization modes. The Ion Trap MS system is coupled with the exact Waters UPLC setup and using same elution gradient. The MS n spectra were recorded by using the following conditions: collision-induced dissociation energy of 30 eV and an isolation width of ±2 D in a data dependent, negative ionization mode.
Identification of metabolites
UPLC-MS files were converted to netcdf file format using the File Converter tool in BrukerDaltoniks software and further processed using AMDIS software.  Metabolites were characterized by their UV-vis spectra (220-600 nm), retention times, mass spectra, and searching the phytochemical dictionary of natural products database and reference literature.
Acute and subchronic toxicity
Lethality test (LD 50 ) was estimated in mice orally, according to OECD guidelines No. 420. In a preliminary test, four groups each of 5 animals received the following different doses of the tested extract suspended in the vehicle (3% v/v Tween 80): 5, 50, 300 and 2000 mg/ kg. B. wt. Animals were observed for 24 h for signs of toxicity and number of deaths. From these results, extra doses of 5, 7.5, and 15 g/ kg b. wt. of the tested extract were administered. Control animals only received the vehicle and were kept under the same conditions without any treatments. Signs of toxicity and number of deaths per dose in 24 h were recorded and the LD 50 was calculated as the geometric mean of the dose that results in 100% mortality, which causes no lethality. Further, 10 mature rats of both sex (200-250 g) were divided into two groups, for a chronic type toxicity study, each made up of 5 animals. In the first group, control rats received the vehicle, whereas second group was orally administered P. turgidum ethanol extract (500 mg/ kg b. wt.). Extracts were administered to animals orally using gastric intubation for a period of 4 weeks.
A total of 40 rats were divided into four groups, ten animals each, and the schedule of treatment was adopted as follows: Group 1: rats were given corn oil (2 ml/ kg b. wt., s c) twice a week for two consecutive weeks and served as negative control group. Group 2: rats were injected CCl 4 (2 ml/ kg b. wt., s.c.) diluted in corn oil (50 % v/v) twice a week for two consecutive weeks. Group 3 and 4: rats were treated with silymarin orally (50 mg/ kg b. wt.) and alcohol extract of P. turgidum orally (500 mg/ kg b. wt.), respectively, daily for three consecutive weeks and injected CCl 4 (2 ml/ kg b. wt., s.c.) diluted in corn oil (50 % v/v) twice a week for two consecutive weeks (starting from the 2 nd week of study). Plant extract and silymarin drug were each administered orally to the animals by gastric intubation for 3 weeks following the module described by Dutta et al., 2013. 
Serum and tissue preparation
Blood samples were collected from retro-orbital venous plexus under light ether anesthesia in nonheparinized tubes and the serum was separated for measuring different biochemical parameters. Animals were later killed and the liver sample was divided into two portions: first was formalin fixed for histopathological examination and the second part was homogenized for the determination of liver malondialdehyde (MDA) and glutathione reductase (GSA) levels as well as catalase (CAT), and superoxide dismutase (SOD) enzymatic activities. Kidney samples for the chronic toxicity module were also maintained for histopathological examination
Serum was carefully separated into clean dry Eppendorf tubes using a Pasteur pipette and used for the determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), urea and creatinine,  total bilirubin and direct bilirubin, total proteins, albumin, HDL-cholesterol, total cholesterol, and triglycerides,  using colorimetric Spectrum Diagnostics and Diamond kits (Cairo, Egypt). The liver homogenate was centrifuged at 5000 rpm for 10 min and the resulting supernatant was used for estimation of lipid peroxides MDA determination, GSH, CAT, and SOD activities. 
Autopsy samples were taken from the liver and kidney of rats in different groups of both models and fixed in 10% neutral buffered formalin for 24 h and decalcification was done in formic acid. Washing was performed with water, and then serial dilution of alcohol (methyl, ethyl, and absolute ethyl) was used for tissue dehydration. Specimens were cleared in xylene and embedded in paraffin at 56°C in a hot air oven for 24 h. Paraffin bees wax tissue blocks of 4-μm thickness were prepared for sectioning by sledge microtone. The obtained tissue sections were collected on glass slides, de-paraffinized, and stained by hematoxylin and eosin stain for examination under light electric microscope. 
Statistical analysis of data
All data are presented as mean ± SE. Statistical analysis was performed using GraphPadprisim version 5 (GraphPad, San Diego, CA). Group differences were analyzed using one-way analysis of variance (ANOVA) followed by Tukey-Kramer for multiple comparison tests. The difference was considered significant when P ≤ 0.05.
| Results|| |
Chemical constituents of Panicum turgidum Forssk were analyzed by reversed phase UPLC/PDA/ESI-qTOF-MS, using a gradient mobile phase consisting of acetonitrile and aqueous formic acid that allowed for a comprehensive elution of plant analysts, that is, flavonoids and fatty acids within 17 min (ca. 1000 s). MS base peak intensity chromatogram of P. turgidum is presented in [Figure 1]. The identities, retention times, Ultraviolet (UV) characteristics, and observed molecular and fragment ions for 39 identified peaks are presented in [Table 1].
|Table 1: Peak assignments of metabolites in P. turgidum 70% ethanol extract using UPLC– PDA-MS in negative ionization mode|
Click here to view
P. turgidum chromatogram showed different subclasses of flavonoids (structures of the main nucleus are shown in [Figure 2]). UV spectra revealed the presence of flavone, flavonol, and flavanone structures.  In MS analysis, the nature of the sugars could be revealed by elimination of the sugar residue, that is, 176 amu (hexuronic acid), 162 amu (hexose: glucose or galactose), 132 amu (pentose),
and 146 amu (rhamnose).
|Figure 1: Representative UPLC– MS trace of P. turgidum methanol extract characterized by two main regions: (220– 420 s) with peaks principally due to flavonoid conjugates and a region (420– 940) for fatty acids. Peak numbers follow those listed in Table 1 for metabolite identification using UPLC– UV– MS|
Click here to view
|Figure 2: Flavonoids subclasses detected in P. turgidum extract, with selected compound(s) discussed in the text|
Click here to view
Interpretation of the UV and MS spectra led to the identification of 15 flavonoid conjugates of C- and O-glycosides. Luteolin conjugates were detected in peaks (1, 6, and 9), all of which showed the neutral loss of 120 amu (0,2 cross-ring cleavage), 90 amu (0,3 cross-ring cleavage), and 18 amu (loss of H 2 O), which are characteristic of C-hexosides.  In detail, peak 1 (609.1441, M-H) exhibited fragment ions at m/z 591 (M + -18), 519 (M + - 90), 489 (M + -120), 399 (M + aglycone + 113), and 369 (M + +83) that is typical of di-C-glycosyl flavonoids  and identified as luteolin-6, 8-di-C-hexoside. Peak 6 with an m/z 447.0921, C 21 H 19 O 11 , showed a neutral loss of 120, 90, and 18 amu indicative of a mono C- hexoside; the high intensity of m/z 357 (M + -90) and absence of (M + -4H 2 O) fragment suggested that sugar attachment occurs at the 8-position  and was assigned as luteolin-8-C-hexoside. Peak 9, m/z609.1434 with molecular formula C 27 H 29 O 16 , showed neutral loss of 162 (characteristic for O-hexoside); presence of m/z 357 (M + -90) and absence of (M + -4 H 2 O) suggested similarly that sugar substitution occurs at the 8-position  and was identified as luteolin-8-C-hexosyl-O-hexoside.
Another flavone structure was detected in apigenin conjugates (peaks 2, 3, 4, and 5). Peak 2 (431.1903, M-H) showed typical loss of 120 and 90 amu of C- hexosides, with high intense m/z 341 (M + -90) compared to m/z 413 (M + -18) fragment and absence of (M + - 4H 2 O) fragment denoting 8-C-hexoside. The presence of minor fragment at m/z 269 of apigenin aglycone  in peak 2 led to its identification as apigenin-8-C-hexoside. Peaks 3 and 4 (593.1493, M-H) showed the same molecular formula, C 27 H 29 O 15 , and typical fragmentation pattern of C-hexoside, (M + -18), (M + -90), (M + -120). In contrast, peak 3 showed additional fragments of 383 (M + +113 amu) and 353 (M + +83, [Table 1]) typical fragmentation pattern of flavone di-C-hexoside, whereas peak 4 showed loss of 162 amu characteristic of O-hexoside and identifying peaks 3 and 4 as apigenin-6,8-di-C-hexoside and apigenin-8-C-hexosyl-O-hexoside, respectively. Peak 5 with exact mass m/z 563.1394, C 26 H 27 O 14 was tentatively identified as apigenin-C-hexosyl-C-pentoside showing neutral losses of 90 and 60 amu, characteristic of a C-pentoside  in addition to the loss of 120 and 90 amu due to C-hexoside. Another flavone structure was detected in peak 12, (623.1593, M-H) showing fragmentation pattern of di-C-hexoside [Table 1] and identified as diosmetin-6,8-di-C-hexoside. This is the first report of C-flavonoids in this genus.
The second most abundant subclass of flavonoids, flavonol conjugates, was detected in peaks (7, 10, 14, 15, 20, and 24; [Table 1]), all of which shared the pattern of 3-O-glycosylation as revealed from its band 1 λmax values.  Isorhamnetin,m/z 315.05 , was detected as aglycone in peaks 7, 10, 15, 20, and 24 following the neutral elimination of attached O-sugar residues and identified as isorhamnetin-3-O-rutinoside (7), isorhamnetin-O-pentosyl-O-rhamnoglucoside (10), isorhamnetin-3-O-glucuronide (15) and isorhamnetin-3-O-hexoside (20), peak 24 (m/z 329.0657) showed the loss of 15 amu (-CH 3 ) and was tentatively assigned as isorhamnetin bearing an extra methyl group. Formation of flavonol aglycone radical ions in MS/MS with high-collision energies is a well-known phenomenon  that was observed in peaks 10, 15, and 24, whereas aglycone ions of isorhamnetin gave, in addition to the deprotonated aglycone ion (m/z 315.0412), a mass signal at m/z 314.0353. Another detected flavonol, (m/z 607.1652, C 28 H 31 O 15 ) in peak 14 showed the neutral loss of 162 amu for O-hexoside followed by loss of 146 (O-rhamnoside) and 15 amu (-CH 3 ) to yield fragment ions m/z 445, m/ z 299 (kaempferide), and m/z 285, respectively, and identified as kaempferide-3-O-rhamnoglucoside. UV detection also confirmed the presence of flavanone in peaks (13, 16, 19, and 22; [Table 1]) with a UV maximum at 282 nm. Naringenin, a flavanone with m/z 271.06 was found as aglycone in peaks 13, 16, and 22. Peak 13 (433.1122, M-H) and 16 (491.2129, M-H) both showed neutral losses of 162 amu for O-hexoside, peak 16 exhibited fragment ions m/z at 448 (-42 amu, acetyl gp.) assigned as naringenin-O-acetyl hexoside, whereas 13 was identified as naringenin-O-hexoside. Peak 22 exhibited an (M-H) of 481.1372, C 22 H 24 O 12 and with fragment ion at m/z 271 of naringenin, whereas the other detected flavanone "eriodictyol" was identified in peak 19 from its (M-H) at m/z 287.0546 and fragment ion at m/z 151.002. 
Oxygenated fatty acids
In the second half of the chromatographic run (350-900 s), MS spectra of several unsaturated fatty acids, that is, linolenic acid (36), linoleic (37), oleic (39), and also palmitic acid (38), were identified, as evident from high-resolution mass at 277.2162, 279.2321, 281.24714, and 255.2327 with predicted molecular formulae of C 18 H 29 O 2 , C 18 H 31 O 2 , C 18 H 33 O 2 , and C 16 H 31 O 2 , respectively. MS also revealed other hydroxy fatty acids with predicted molecular formulas of C 18 H 29 O 3 and C 18 H 31 O 3 and (M-H) of 293.2111 and 295.2276, respectively, in peaks (29-33). A mass difference of 2 amu between peaks is indicative of an extra double bond and was tentatively identified as hydroxyoctadecatrienoic and hydroxyoctadecadienoic acid isomers. Similarly, peaks 23 and 26 MS spectra showed a mass weight of 327.2169 and 329.2326 with predicted molecular formula C 18 H 31 O 5 and C 18 H 33 O 5 , respectively, and were tentatively identified as hydroxyoctadecenedioic acid and hydroxyoctadecanedioic acid, respectively. Hydroxyhexadecanoic acid was identified in peak 34 with (M-H) 271.227, showing an extra hydroxy group (16 amu) than in palmitic acid.
Acute and subchronic toxicity
To assess for P. turgidum extract safety margin, acute and chronic toxicity effects were monitored in animals including estimation of LD 50 and following complete blood picture analysis, liver and kidney serum enzymes measurement and followed by histopathological examination after 4 weeks of administration. Concerning the lethality test, treated mice with different doses of P. turgidum extract (up to 15000 mg/ kg b. wt.) were alive during the 24-h observation time, showing no visible signs of acute toxicity. Consequently, according to Hodge and Sterner toxicity scale,  the LD 50 value of the extract is categorized under the nontoxic category. Similarly, Urea and creatinine, biochemical markers, were used for the diagnosis of possible renal damage,  showing no significant difference in (36.4 ± 2.14 and 0.64 ± 0.03 mg/dl, respectively) compared to control animals (41.5 ± 2.16 and 0.70 ± 0.03 mg/dl, respectively) at (P ≤ 0.05) and in agreement with histopathological sections showing normal nephron structure [Figure 4]. Additionally, no significant change was found in kidney weight ratio at a dose of 500 mg/kg b. wt. of Panicum extract (0.6 %) when compared with control (0.6%). Moreover, oral administration of P. turgidum extract for 4 weeks did not show any significant effect on AST (22.0 ± 1.2 U/L), ALT (24.9 ± 2.3 U/L), ALP (71.2 ± 6.3 U/L), total bilirubin (1.2 ± 0.06 mg/dl), and total protein (8.1 ± 0.46 g/dl) serum levels or on liver weight ratio (3.2 ± 0.10 %) as compared to control group (21.3 ± 1.5, 22.1 ± 1.9, 72.3 ± 4.8U/L, 1.1 ± 0.07mg/dl, 8.0 ± 0.09g/dl and 3.1 ± 0.09%, respectively) further confirmed by histopathological examination, showing normal hepatocyte structure [Figure 3]B. Additionally, blood picture parameters in rats post extract administration were found at normal levels compared to control.
|Figure 3: Histology of liver samples from (A) control, (B) treated group with P. turgidum extract for 4 weeks, (C) CCl4-treated, (D) CCl4 intoxicated group and treated with silymarin and (E) CCl4-intoxicated group and treated with P. turgidum extract. The central vein (CV), hepatocytes (h), fatty changes (arrow), inflammatory cells infiltration (m), mild degenerative change and (d) and the portal vein (PV). Hematoxylin– Eosin staining, magnifications: ×40 except E: ×16|
Click here to view
|Figure 4: Histology of kidney samples from (A) control, (B) treated group with P. turgidum extract. (g) The glomeruli and (t) the tubules. Hematoxylin– Eosin staining, magnifications: ×40|
Click here to view
The ability of alcohol extract analyzed using UPLC-MS to protect against CCl 4 hepatotoxic effects was assessed in a rat animal model along with silymarin as a positive drug control. Treatment of animals with the hepatotoxic agent, CCl 4 , resulted in significant (P ≤ 0.05) increase in the liver aminotransferases (AST and ALT) and ALP levels, indicative of hepatocytes damage and decreased serum albumin and total protein. Severe jaundice was also revealed from elevated bilirubin level in addition to elevated serum total cholesterol and triglycerides and accompanied by a decrease in serum HDL-cholesterol and in accordance with. , Additionally, CCl 4 administration led to significant increase in MDA (lipid peroxidation) and reduction of both GSH content and activity which are endogenous protective antioxidants known to protect from free oxygen radicals in hepatic tissue. 
Silymarin, the well characterized antihepatotoxic agent along with P. turgidum extract ameliorated CCl 4 -induced alterations [Table 2]. P. turgidum extract induced significant reduction of ALT, AST, ALP, total bilirubin, direct bilirubin, total cholesterol, and Triglycerides (TG) serum elevated levels concurrent with a significant increase in total proteins, serum albumin level, and HDL-cholesterol in comparison to CCl 4 -treated group. In addition, Panicum extract significantly mitigated the lipid peroxidation activity in the rat liver induced by CCl 4 as manifested by a decrease in MDA levels and restored GSH content and CAT and SOD activities. It should be noted that animals treated with Panicum extract did not improve CCl 4 effect on body weight, but rather decreased liver-body weight ratio as compared to CCl 4 -treated group.
|Table 2: Effect of pretreatment with silymarin and P. turgidum extract on changes in body weight, relative liver weight ratio, serum enzymes, total and direct bilirubin, total proteins, albumin and lipid profile levels and oxidative stress and activities of antioxidant liver enzymes in carbon tetrachloride-treated animals (n = 10)|
Click here to view
Furthermore, improvement in biochemical parameters following P. turgidum treatment was also confirmed from the liver histopathological examination [Figure 3]. Liver section of control group showed no histolopathological alterations, with a normal histological structure of the central vein and surrounding hepatocytes [Figure 3]A. In contrast, administration of CCl 4 revealed centrolobular fatty change as observed in most of the hepatocytes associated with inflammatory cells infiltration in the portal area and dilatation in the portal vein [Figure 3]C. Pretreatment with silymarin during CCl 4 administration showed mild degenerative changes in some hepatocyte cells [Figure 3]D, whereas treatment P. turgidum extract showed dilatation in both central and portal veins associated with mild fatty changes in few hepatocytes [Figure 3]E, which was much less severe than that observed in CCl 4 group.
| Conclusions|| |
The approach utilized herein allowed for the identification of several O- and C-flavonoid conjugates of luteolin, apigenin, isorhamnetin, and naringenin, unprecedented in that genus. As such, these derivatives are major secondary metabolites in other monocots, that is, maize, wheat, and rice. In these cereals, C-glycosides of the simple flavones apigenin and/or luteolin predominate predominate and function as natural antioxidants in these plants,  which explain the mitigation of oxidative stress resulted from P. turgidum extract administration to CCl 4 - intoxicated rats. Enrichment of these antioxidant flavonoids in plant extract is likely to mediate for its potential antihepatotoxic effects.
Standardization of Panicum extract based on these flavonoids could assist in quality control analysis. To the best of our knowledge, this study provides the first metabolite profile not only in P. turgidum but also in the whole genus. Further isolation and examination of these metabolites including unknown peaks will help provide more evidence for its potential use in the treatment of liver disorders. Additionally, further studies are still needed for the adjustment of the protecting clinical dose and revealing the mechanism of action mediating the observed antihepatotoxic effect.
Specializing in metabolomics and natural products chemistry, Dr. Mohamed A. Farag completed his PhD at Texas Tech University, USA, in 2003. Dr. Farag currently works as a full professor at the Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Egypt. In 2009-2010 he held Alexander von Humboldt fellowship, where he explored the possible use of metabolomics in phytomedicines analysis. His research work focuses primarily around applying innovative biochemical technologies (metabolomics) to help answer complex biological questions in medicine, herbal drugs analysis and agriculture.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Brink M, Belay G. Plant resources of tropical Africa. 1. Cereals and pulses. 2006. PORTA Foundation, Wageningen, the Netherlands/Backhuys Publishers Leiden, the Netherlands/CTA, Wageningen, the Netherlands. p. 298.
Abdel-Majeed AA, Rabie SH, Azer SA, Mahmoud AS, Index to Scientific Names of Flora of Egypt. Original illustration collection of the Department of flora and Phytotaxonomy Researches.Agricultural Research Center;2009.p. 89.
Lee ST, Stegelmeier BL, Gardner DR, Vogel KP, Characterization of steroidal sapogenins in Panicum virgatum
L. (switchgrass).J Nat Tox 2004;10:273-81.
Chitindingu K, Ndhlala AR, Chapano C, Benhura MA, Muchuweti M, Phenolic compound content, profiles and antioxidant activities of Amaranthus hybridus
(pigweed), Brachiari abrizantha
(upright brachiaria) and Panicum maximum
(guinea grass).J Food Biochem 2007;31:206-16.
Uppugundla N, Engelberth A, Vandhana SR, Clausen EC, Lay JO, Gidden J, et al.
Switchgrass water extracts: extraction, separation and biological activity of rutin and quercetin.J Agric Food Chem 2009;57:7763-70.
Cogne A, Phytochemical investigation of plants used in African traditional medicine: Dioscoreasylvatica
(Dioscoreaceae), Urginea altissima
(Liliaceae), Jamesbrittenia fodina
and Jamesbrittenia elegantissima
. PhD thesis, Faculty of Science, Lausanne University, Lausanne, Switzerland 2002.
Khare CP, Indian Medicinal Plants: An Illustrated Dictionary.Springer-Verlag, Berlin, Heidelberg. LLc.2007;pp 229-30.
Bassey SA, Okokon JE, Umoh EE, Udobang JA, Antidiabetic activity of ethanolic leaf extract of Panicum maximum
.Inter J Drug Develop Res 2010;2:488-92.
Okokon JE, Anti-inflammatory and antipyretic activities of Panicum maximum
. Afr J Biomed Res2012;14:125-30.
Bedair M, Sumner LW, Current and emerging mass-spectrometry technologies for metabolomics.Trends Anal Chem 2008;27:238-50.
Farag MA, Comparative mass spectrometry & nuclear magnetic resonance metabolomic approaches for nutraceuticals quality control analysis: a Brief Review.Recent Pat Biotechnol 2014;8:17-24.
Sandhar HK, Kumar B, Prasher S, Tiwari P, Salhan M, Sharma P, A review of phytochemistry and pharmacology of flavonoids.Intern Pharmaceutica Sciencia 2011;1:25-41.
Farag MA, Wessjohann LA, Metabolome classification of commercial Hypericum perforatum
(St. John′s Wort) preparations via UPLC-qTOF-MS and chemometrics.Planta Med 2012;78:488-96.
Halket JM, Przyborowska A, Stein SE, Mallard WG, Down S, Chalmers RA, Deconvolution gas chromatography/mass spectrometry of urinary organic acids: potential for pattern recognition and automated identification of metabolic disorders. Rapid Commun Mass Spectrom 1999;13:279-84.
Dutta B, Lahkar M, Bibin BA, Lihite M, Hepatoprotective activity of Tamarind indica
and Homalomena aromtica
in rats.Inter J Pharm Pharmaceutical Sci 2013;5:436-38.
Maheshwari N, Clinical Biochemistry.2008 1 st
ed. India Jaypee Brothers Medical Publishers (P) Ltd.216.
Ramakrishman S, Sulochana KN, Manual of Medical Laboratory Techniques.2012;1 st
ed. India Jaypee Brothers Medical Publishers Ltd.
Balahoroðlu R, Dülger H, Özbek H, BayramI, ªekeroðlu M, Protective effects of antioxidants on the experimental liver and kidney toxicity in mice.Eur J Gen Med 2008;5:157-64.
Banchroft JD, Stevens A, Turner DR, Theory and Practice of Histological Techniques.1996 4th edn.New York Churchill Livingstone 766.
Mabry TJ, Markham KR, Thomas MB, The Systematic Identification of Flavonoids. Springer-Verlag, New York. 1970. 354.
Cabrera GM, Mass spectrometry in the structure elucidation of natural products: glycosides.Phytochem Adv Res 2006;1-22.
Djoukeng JD, Arbona V, Argamasilia Rosa, Gomez-Cadenas A, Flavonoid profiling in leaves of Citrus genotypes under different environmental situations.J Agric Food Chem 2008;56:11087-97.
Abad-Garcia B, Garmon-Lobato S, Berrueta LA, Gallo B, VicenteF, New features on the fragmentation and differentiation of C-glycosidic flavone isomers by positive electrospray ionization and triple quadrupole mass spectrometry.Rapid Commun Mass Spectrom 2008;22:1834-42.
Farag MA, Sharaf Eldin MG, Kassem H, Abou El, Fetouh M, Metabolome classification of Brassica napus
L. organs via UPLC-QTOF-PDA-MS and their anti-oxidant potential.Phytochem Anal2013;24:277-87.
Sandu RB, Tarþãu L, Miron A, Zagnat M, Ghiciuc CM, Lupuºoru CE, Experimental researches on acute toxicity of a Bidens tripartita extract in mice: preliminary investigations.Rev Med Chir Soc Med Nat 2012;116:1230-34.
Al-Assaf AH, Preventive effect of corosolic acid on lipid profile against carbon tetrachloride: induced hepatotoxic rats.Pak J Nutr 2013;12:748-52.
El Sohafy SM, Alqasoumi SI, Metwally AM, Omar AA, Amer MM, Abou-Shoer MI, Evaluation of the hepatoprotective activity of some plants belonging to the tribe Cynareae growing in Egypt.J Med Plants Res 2013;7:324-28.
Brazier-Hicks M, Evans KM, Gershater MC, Puschmann H, Steel PG, Edwards R, The C-glycosylation of flavonoids in cereals.J Biol Chem 2009 May [cited 2009 July 3]284:17926-17934. Available from: (http://www.jbc.org
| Authors|| |
Dr. Mohamed A. Farag , Specializing in metabolomics and natural products chemistry, Dr. Mohamed A. Farag completed his PhD at Texas Tech University, USA, in 2003. Dr. Farag currently works as a full professor at the Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Egypt. In 2009-2010 he held Alexander von Humboldt fellowship, where he explored the possible use of metabolomics in phytomedicines analysis. His research work focuses primarily around applying innovative biochemical technologies (metabolomics) to help answer complex biological questions in medicine, herbal drugs analysis and agriculture.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]