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Hepatoprotective evaluation and isolation of the major secondary metabolites from the ethyl acetate extract of liquid culture filtrate of Chaetomium globosum
Biomedicine & Pharmacotherapy 97 (2018) 174–180
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Original article
Hepatoprotective evaluation and isolation of the major secondary metabolites from the ethyl acetate extract of liquid culture filtrate of Chaetomium globosum
MARK
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Nagwa E. Awada, Hanaa A. Kassemb, Manal A. Hamedc, , Amal M. El-Fekya, Mohamed A.A. El-Naggard a
Pharmacognosy Department, National Research Center, Dokki, Cairo, Egypt Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt TTherapeutic Chemistry Department, National Research Centre, 33 El- Buhouth St., Dokki, Cairo, Egypt. ID: 60014618 d Plant pathology Department, National Research Center, Dokki, Cairo, Egypt b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Chaetomium globosum Fungi Hepatoprotective Volatile compounds
The aim of the present study was to evaluate the hepatoprotective activity of ethyl acetate extract of the liquid culture filtrate of Chaetomium globosum fungus (family Chaetomiaceae). Rats were intraperitoneally injected by CCl4 (0.5 ml/kg) twice a week for six consecutive weeks. Treatment tacks (250 mg/kg) place at the same time of CCl4 induction and with the same duration. The evaluation was done through determination of liver function indices; aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total serum protein content. In addition, the oxidative stress markers; hepatic glutathione content (GSH), hepatic malondialdehyde (MDA), hepatic superoxide dismutase (SOD), and hepatic total protein were estimated. Moreover, the liver architectures were also examined. Isolation and identification of the main secondary metabolites were identified. Seven volatile compounds were identified from the plain chloroform fraction where, 1Cyclopentyl-2,2-dimethyl-1-propanol (54.63%) was presented as the major compound. Eleven compounds were also identified from the fraction eluted by chloroform: methanol (85:15). 1,5,5-Trimethyl-6-methylene-1-cyclohexene (25.79%) and Norbornan-2-one (26.84%) are presented as the major compounds of this fraction. In conclusion, the extract recorded hepatoprotective effect by ameliorating the biochemical parameters under investigation. The liver histopathological pictures confirmed our results.
1. Introduction
andantimicrobial activity on different bacteria and fungi [5]. The aim of the present study is to evaluate the ethyl acetate extract of the liquid culture filtrate of Chaetomium globosum fungus as antioxident and hepatoprotective agent on liver injury induced by CCl4 in rats.
Endophytes are considered as one of the essential interesting topics in the field of natural product chemistry and one of the major clades of life [1]. Chaetomium globosum, the type species of the genusChaetomium belonging to Phylum scomycota. Class Sordariomycetes(FamilyChaetomiaceae), spread all over the worldin soil, water, plant material and other cellulosic substances [2]. This fungusproduces diverse groups of secondary metabolites with several biological activities such as antimicrobial, immunomodulatory, and anticancer [3], where ithas also been proven to be an essential source of diverse bioactive constituentssuch ascytoglobosins, azaphilones, chaetoviridins, pyrones, orsellides and globosumones [4]. Prenisatin, chrysophanol, chrysazin,chaetoviridin A and B were isolated from the ethyl acetate extract of the liquid culture filtrate of Chaetomiumglobosum, where recorded remarkable antioxidant
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2. Material and methods 2.1. Fungal isolation During autumn and winter growing season of 2014/2015, a surveystudy was conducted at the major cucumber (CucumissativusL.) growingareas under plastic greenhouse conditions. Samples of cucumber plants exhibiting damping-off, root-rot, stem-rot and wilt symptoms were collected and immediately transferred to Plant Pathology laboratory for isolation procedures. The pathogens were
Corresponding author at: Therapeutic Chemistry Department, National Research Centre, 33 El- Buhouth St., Dokki, Cairo, Egypt. ID: 60014618. E-mail address: [email protected] (M.A. Hamed).
http://dx.doi.org/10.1016/j.biopha.2017.10.120 Received 29 August 2017; Received in revised form 11 October 2017; Accepted 21 October 2017 0753-3322/ © 2017 Published by Elsevier Masson SAS.
Biomedicine & Pharmacotherapy 97 (2018) 174–180
N.E. Awad et al.
Table 1 GC/MS analysis of the fraction 1 eluted with 100% chloroform. No
RT
Mol. Formula
Mol. weight
BP
Compound
%
1
15.17
C5H12O2
104
43
3-Ethoxy propanol
2.59
2
27.60
C10H20 O
156
57
1-Cyclopentyl-2,2-dimethyl-1-propanol
54.63
3
30.23
C12H24
168
41
Dodecene
3.15
4
34.71
C18H32 O2
280
55
17-Octadecynoic acid
4.44
5
42.62
C19H40 O
284
57
2-Nonadecanol
4.19
6
56.99
C19H32 O2
292
43
Methyl octadeca-9-ene-12-ynoate
1.74
7
62.02
C22H46 O
326
55
1-Docosanol
5.07
Structure
isolated by tissue segment method [6]. The root pieces were cultured onto the surface of sterilized Petriplates containing freshly preparing Potato Dextrose Agar (PDA) medium. After 10 days of incubation at 25 ± 1 °C, the frequency occurrence (%) of isolated fungi was recorded. The fungal hyphal tip of developed colonies around the root pieces were transferred onto PDA medium and incubated for10 days at 25 ± 1oC for further studies [6].
done, then evaporated under reduced pressure and subjected to TLC examination using toluene: ethyl acetate (8:2). Similar fractions were collected together. Characterization of the compounds were carried out by GC/MS analysis (Shimadzu GC/MS −QP5050A, 70 eV) for of the volatile constituents.
2.2. Fungal identification
2.5.1. Acute toxicity Male Wistar albino rats (120–140 g) were obtained from the Animal House, National Research Center, Egypt and kept in controlled environment of air and temperature with access of water and diet. One oral dose of 5, 10, 50 and 100 mg of the ethyl acetate extract body weight. Rats were observed after 24 h of administration and along the following fourteen days. Dead rats were counted and the mortality rate was calculated. Lethal dose that killed 50% of animals (LD50) was monitored. The lethal dose that killed 50% of rats (LD50) along fourteen days was 100 mg/kg b.wt. Therefore, the selected dose is 20 mg/kg b.wt representing 1/5 of the lethal dose.
2.5. Biological study
Isolated fungi were identified at the Plant PathologyDepartment, National Research Centre (NRC), Egypt, and confirmed byFungal Taxonomy Department, Plant Pathology Research Institute, Agricultural Research Centre, Giza, Egypt according to the morphological and culture characters using the methods previously described by Barnett and Hunter [7], and Ramirez [8]. Stock cultures were maintained on PDA slants and kept in a refrigerator at 5 °C for further studies. 2.3. Preparation of the ethyl acetate extract of liquid culture filtrate of C. globosum
2.5.2. Doses and route of administration Administration regimen was twice a week for six consecutive weeks. CCl4 (0.5 ml/kg) was suspended in olive oil (1:9 v/v) and injected intraperitoneally [9]. The ethyl acetate extract was administrated orally at a dose 20 mg/kg. Silymarin; a reference herbal drug was orally administered at a dose 100 mg/kg [10]. Blood samples and slices of liver tissues were taken from the eviscerated animals and kept for the biochemical assays. Handling of the rats was obeyed the ethical guidelines of Medical Ethical Committee of the National Research Centre in Egypt (Approval no: 134, 2011).
Twenty liters of the liquid culture filtrate of C. globosum have been extracted by ethyl acetate. The extract has been concentrated in a rotary evaporator at 45 °C under reduced pressure. 2.4. Identification of the volatile secondary metabolites from the ethyl acetate extract The dried ethyl acetate extract of the liquid culture filtrate of C. globosum was dissolved in the least amount of ethyl acetate, loaded onto a silica gel column (120 cm height × 2.5 cm i.d.) containing 175 g activated silica (70–230 mesh; E. Merck, Darmstadt, Germany). The elution was done using the organic solvents; chloroform and chloroform: methanol (85:15). Collection of the eluted fractions were
2.5.3. Experimental design Forty eight rats were divided into six groups of eight rats each. Group 1 served as normal healthy control rats orally vehicle with 0.5 ml 175
Biomedicine & Pharmacotherapy 97 (2018) 174–180
N.E. Awad et al.
Table 2 GC/MS analysis of the fraction eluted by chloroform: methanol (85:15). No
RT
Mol. Formula
Mol. weight
B.P.
Compound
%
1
7.38
C10H16
136
121
1,5,5-Trimethyl-6-methylene-1-cyclohexene
25.79
2
9.87
C10H16O
152
81
Norbornan-2-one
26.84
3
10.40
C10H18O
154
95
1,7,7-Trimethyl bicyclo heptan-2-ol
4.64
4
12.88
C10H16O2
168
84
11-Oxa-dispiro undecan-1-ol
7.77
5
14.16
C12H20O2
196
95
1-Bornyl acetate
4.88
6
15.57
C12H20O2
196
93
1-Menthen-8-yl acetate
3.89
7
16.23
C15H24
204
93
Humulene
1.60
8
19.21
C10H18O2
206
161
3-Isopropenyl-4-methoxy phenyl propanol
3.75
9
19.44
C15H26O
222
109
Veridiflorol
1.41
10
23.97
C22H44
308
55
1-Docosene
2.72
11
28.27
C20H42 O
298
43
1-Eicosanol
1.51
176
Structure
Biomedicine & Pharmacotherapy 97 (2018) 174–180
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Table 3 Effect of treatment with the ethyl acetate extract of the liquid culture filtrate of Chaetomium globosum on the oxidative stress markers. Groups
Oxidative stress markers Hepatic glutathione content Mean ± SD
Control Control + C. globosum (ethyl acetate) Control +silymarin CCl4 CCl4 + C.globosum (ethyl acetate) CCl4 +silymarin
Hepatic malondialdehyde level
% Change
% Improvement
11.60bc ± 0.37 10.82c ± 1.16
— −6.72
— —
10.45c ± 0.51 5.73e ± 0.24 9.17d ± 0.57
−9.91 −50.60 −20.94
10.75c ± 0.28
−7.32
Mean ± SD
Hepatic superoxide dismutase level
% Change
% Improvement
0.47de ± 0.03 0.44def ± 0.11
−6.38
— —
— — 29.65
0.42def ± 0.08 2.03a ± 0.18 1.10b ± 0.10
−10.63 +333.91 +134.04
43.36
0.48de ± 0.06
+2.12
Mean ± SD
% Change
% Improvement
283.58d ± 60.72 122.31e ± 5.21
— −56.87
— —
— — 197.87
259.04d ± 20.4 541.71a ± 125.86 372.70b ± 31.88
−8.65 +91.02 +31.43
— — 59.59
329.78
306.12cd ± 65.18
+7.94
83.07
Values are mean ± SD of eight rats in each group. Values are expressed as μmol/mg protein. Statistical analysis are done using one way analysis of variance (ANOVA) using Co Stat Computer program accompanied with least significance level (LSD) between groups at p < 0.05. Unshared superscript letters are significant values between groups at p < 0.0001. Table 4 Effect of treatment with the ethyl acetate extract of the liquid culture filtrate of Chaetomium globosum on hepatic total protein level. Groups
Mean ± SD
% Change
% Improvement
Control Control + C.globosum (ethyl acetate) Control +silymarin CCl4 CCl4 + C.globosum (ethyl acetate) CCl4 +silymarin
39.09a ± 1.04 32.27c ± 4.54
— −17.44
— —
30.68cd ± 0.78 10.79h ± 1.19 20.59fg ± 0.45
−21.51 −72.39 −47.32
— — 25.07
22.33ef ± 1.06
−42.87
29.52
Table 6 Effect of treatment with the ethyl acetate extract of the liquid culture filtrate of Chaetomium globosum on serum total protein. Groups
Mean ± SD
% Change
% Improvement
Control Control + C.globosum (ethyl acetate) Control +silymarin CCl4 CCl4 + C.globosum (ethyl acetate) CCl4 +silymarin
6.52cde ± 0.47 6.32cde ± 0.14
— −3.06
— —
6.97bc ± 0.82 8.46a ± 0.37 6.47cde ± 0.14
+6.90 +29.75 −0.76
— — 30.52
7.54b ± 0.44
+15.64
14.11
Values are mean ± SD of eight rats in each group. Values are expressed as mg/g liver tissue. Statistical analysis are done using one way analysis of variance (ANOVA) using Co Stat Computer program accompanied with least significance level (LSD) between groups at p < 0.05. Unshared superscript letters are significant values between groups at p < 0.0001.
Values are mean ± SD of eight rats in each group. Values are expressed as mg/L. Statistical analysis are done using one way analysis of variance (ANOVA) using Co Stat Computer program accompanied with least significance level (LSD) between groups at p < 0.05. Unshared superscript letters are significant values between groups at p < 0.0001.
normal physiological saline and intraperitoneally received 0.5 ml olive oil. Group 2 was normal healthy rats received the ethyl acetate extract. Group 3 was CCl4 treated rats. Group 4 was injected with CCl4 and treated with the ethyl acetate extract at the same time and for the same duration. Group 5 was normal healthy rats received the standard drug (Silymarin). Group 6 was injected with CCl4 and treated with Silymarin.
2.5.4. Biochemical assays Hepatic glutathione content (GSH) was estimated by the method of Moron et al. [11], malondialdehyde (MDA) was determined by the method of and Aust [12]. Superoxide dismutase (SOD) was estimated by method of Nishikimi et al. [13]. Hepatic aspartate and alanine amintransferases (AST & ALT) were measured by the method of Reitman and Frankel [14]. Hepatic alkaline phosphatase (ALP) was estimated by the method of Belfield and Goldberg [15] and total protein
Table 5 Effect of treatment with the ethyl acetate extract of the liquid culture filtrate of Chaetomium globosum on the liver function indices. Groups
Liver function indices AST Mean ± SD
Control Control + C.globosum (ethyl acetate) Control +silymarin CCl4 CCl4 + C.globosum (ethyl acetate) CCl4 +silymarin
ALT % Change
% Improvement
3.04cde ± 0.08 2.96e ± 0.03
— −2.63
— —
1.65f ± 0.06 4.17a ± 0.06 3.21b ± 0.11
−45.72 +37.17 +5.59
2.96bc ± 0.10
−2.63
ALP
Mean ± SD
% Change
% Improvement
1.76c ± 0.05 1.86bc ± 0.03
— +5.68
— —
— — 31.57
1.52d ± 0.04 2.68a ± 0.42 1.78c ± 0.07
−13.63 +52.27 +1.13
39.80
1.85bc ± 0.06
+5.11
Mean ± SD
% Change
% Improvement
96.91d ± 3.39 124.34c ± 5.09
— +28.30
— —
— — 51.13
80.57b ± 5.36 194.42a ± 13.73 73.03e ± 6.83
−16.55 +100.61 −24.33
— — 125.26
47.15
104.12d ± 2.37
+7.43
93.17
Values are mean ± SD of eight rats in each group. Values are expressed as Unit/L. Statistical analysis are done using one way analysis of variance (ANOVA) using Co Stat Computer program accompanied with least significance level (LSD) between groups at p < 0.05. Unshared superscript letters are significant values between groups at p < 0.0001.
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Fig 1. Structure of the isolated compounds from the ethyl acetate extract of the liquid culture filtrate of Chaetomium globosum.
hydrogen peroxide (H2O2) and converts it into H2O by catalase and glutathione peroxidase [24]. In agreement with this explanation, we observed a significant increase in SOD [25]. In the present study, inhibition in GSH level during CCl4 toxicity may be due to the decreased availability of GSH resulted during the enhanced lipid peroxidation processes. Abubakar et al. [26] found a relation between its depletion and disruption of hepatocytes plasma membrane due to the peroxidation process that affects plasma membrane integrity. The elevation of plasma AST and ALT activities could be regarded as an index of damage of the liver parenchymal cells [21–23]. Bilgin et al. [20] attributed the increase in serum enzyme levels to the damage of the structural integrity of liver which lead to enzymes release into the circulation; a process of cytolysis. Abubakar et al. [26] mentioned that ALP is a marker enzyme for the plasma membrane and endoplasmic reticulum. Therefore, significant increase in the serum ALP following CCl4 treatment may be due to disruption of plasma membrane [26]. Romero et al. [27] and Sharma and Shukla [28] showed that CCl4 intoxication induced changes in the process of protein synthesis. Hence, increase in serum total protein content can be deemed as a useful index of cellular dysfunction as clearly shown in our studies where serum protein showed significant increase, while its content in the liver is significantly decreased due to cellular damage and leakage into the blood stream (Fig. 1). Regarding to the histopathological figures of liver, rats treated with the ethyl acetate extract of the liquid culture filtrate of C. globosum showed normal structure of the hepatocytes as compared with control rats (Fig. 2a, b). CCl4 rats showed distorted and loss lobular hepatic architecture and severe fibrosis (Fig. 2c, d). CCl4 treated rats with the ethyl acetate extract of the liquid culture filtrate of Chaetomium globosum recorded improvement of the hepatic cells with more or less like normal structure (Fig. 2e, f). CCl4 sylimarin treated rats showed preserved lobular hepatic architecture and mild steatotic changes (Fig. 2g, h). It is obviously that natural products therapy has emerged as proficient approach with good values in treating different diseases. Wagner and Elmadf [29] and Gross Arya et al. [30] confirmed the role of terpenoids and phenolics compounds as antioxidants. Terpenes and steroidal compounds act as direct antioxidants through free radical scavenging mechanisms and/or as indirect antioxidants by enhancing the antioxidant status (enzymatic and non-enzymatic) [31]. Also, phenolics mediate antioxidant enzyme expression which play an important role in the protection of liver against adverse effects related to mutagenesis and oxidative [32]. Many fungi and some bacteria as soon as they enter the stationary phase of growth, begin to synthesize and excrete compounds with diversity of chemical structures such as poly ketides and terpenoidal compounds. Terpenes and steroids are synthesized by microorganisms from the isoprene unit to form several terpenes with various chain lengths [33]. Administration the most biologically active extracts of the fungi
was assayed by the method of Bradford [16]. 2.6. Histopathological analysis Hepatic tissues slices were fixed in 10% paraformaldehyde and embedded in paraffin wax blocks. Sections of 5 μm thick were stained with hematoxylin & eosin (H & E), then examined under light microscope for determination of pathological changes [17]. 2.7. Statistical analysis and calculations All data were expressed as mean ± S.D. of eight rats in each group. Statistical analysis was carried out by one-way analysis of variance (ANOVA), Costat Software Computer Program. Significance values between groups were at P < 0.05.
• % changes = [(mean treated- mean of control group)/mean of control] × 100. • % of improvement = [(mean treated- mean of CCl4 group)/mean of control] × 100.
3. Results and discussion The isolate was identified as C. globosum based on the criteria of Kirk et al. [18], where the percentage of the dried ethyl acetate extract of liquid culture of C. globosum was 0.04%.The eluted fraction from 100% chloroform that undergo to GC/MS analysis recorded seven compounds, amounting 75.81% of the total fraction. 1-Cyclopentyl-2,2dimethyl-1-propanol (54.63%) was presented as the major compound in the fraction (Table 1).The fraction eluted from chloroform: methanol (85:15) was subjected to GC/MS analysis and eleven compounds were identified amounting 84.8% of the total fraction. 1,5,5-Trimethyl-6methylene-1-cyclohexene (25.79%) and Norbornan-2-one (26.84%) are presented as the major compounds of this fraction (Table 2). Liver injury by CCl4 is a classical system of xenobiotic- induced hepatotoxicity [19] and is commonly used for screening the anti-hepatotoxic, antifibrotic and hepatoprotective activity of drugs [20]. The effect of CCl4 on rat livers enzymes and oxidative stress markers have been illustrated in Table 3–6. Concerning with the oxidative stress markers in the present study and in agreement with the results of Motawi et al. [21] and El-Gengaihi et al. [22], we observed a significant elevation in MDA and SOD, while GSH was significantly decrease in CCl4 rats. Gowri Shankar et al. [23] postulated the role of the reactive metabolite trichloromethyl radical (CCl3−) that has been formed from the metabolic conversion of CCl4 by cytochrome P-450. This free radical initiate the peroxidation of membrane poly-unsaturated fatty acids (PUFA), generates PUFA radicals, covalently binds to membrane lipids and proteins, generates ROS and enhances MDA level. Superoxide dismutase, as one of the most important enzymatic antioxidants, transforms O2− to the more stable 178
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Fig. 2. photomicrographs of liver section of control (a) and control rats treated with extract (b) showed preserved (intact) lobular hepatic architecture and normal morphological appearance (H & E,×100), liver section of CCl4 group (c, d) showed distorted and loss lobular hepatic architecture and formation of micro and macro regenerating nodules (black arrows), mild to moderate ballooning of hepatocytes (red arrow)moderate infiltration by lymphocytes to portal tract (yellow arrow) (H & E, ×100), liver section of CCl4 rates treated with extract showed preserved (intact) lobular hepatic architecture and with mild lobular inflammation (black arrows), mild infiltration by lymphocytes to portal tract (red arrow) and fibrous tissue (yellow arrow) (H & E, ×100) and liver section of CCl4 rats treated with sylimarin showed intact (preserved) lobular hepatic architecture and mild steatotic changes (black arrow), with mild blood vessel congestion(red arrow) (H & E, ×100).
liver function enzymes, improving the oxidative stress markers and enhancing the liver histopathological pictures.
attenuated the increased levels of the serum enzymes, improved the oxidative stress markers and hepatic architecture after injury with CCl4 causing a subsequent recovery towards normalization. This suggested that these extracts are able to condition the hepatocytes, so as to accelerate regeneration of parenchyma cells, thus protecting against membrane fragility and decrease leakage of the enzymes into circulation. Therefore, these tested extracts perform as silymarin drug in the protection of the plasma membrane of hepatocytes. Also, they are protecting liver cells directly by stabilizing the membrane permeability [34]. In conclusion, the ethyl acetate extract of the liquid culture filtrate of Chaetomium globosum is considered as hepatoprotective and antioxidant agent. It performed the action by attenuating the elevation of
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Original article
Hepatoprotective evaluation and isolation of the major secondary metabolites from the ethyl acetate extract of liquid culture filtrate of Chaetomium globosum
MARK
⁎
Nagwa E. Awada, Hanaa A. Kassemb, Manal A. Hamedc, , Amal M. El-Fekya, Mohamed A.A. El-Naggard a
Pharmacognosy Department, National Research Center, Dokki, Cairo, Egypt Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt TTherapeutic Chemistry Department, National Research Centre, 33 El- Buhouth St., Dokki, Cairo, Egypt. ID: 60014618 d Plant pathology Department, National Research Center, Dokki, Cairo, Egypt b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Chaetomium globosum Fungi Hepatoprotective Volatile compounds
The aim of the present study was to evaluate the hepatoprotective activity of ethyl acetate extract of the liquid culture filtrate of Chaetomium globosum fungus (family Chaetomiaceae). Rats were intraperitoneally injected by CCl4 (0.5 ml/kg) twice a week for six consecutive weeks. Treatment tacks (250 mg/kg) place at the same time of CCl4 induction and with the same duration. The evaluation was done through determination of liver function indices; aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total serum protein content. In addition, the oxidative stress markers; hepatic glutathione content (GSH), hepatic malondialdehyde (MDA), hepatic superoxide dismutase (SOD), and hepatic total protein were estimated. Moreover, the liver architectures were also examined. Isolation and identification of the main secondary metabolites were identified. Seven volatile compounds were identified from the plain chloroform fraction where, 1Cyclopentyl-2,2-dimethyl-1-propanol (54.63%) was presented as the major compound. Eleven compounds were also identified from the fraction eluted by chloroform: methanol (85:15). 1,5,5-Trimethyl-6-methylene-1-cyclohexene (25.79%) and Norbornan-2-one (26.84%) are presented as the major compounds of this fraction. In conclusion, the extract recorded hepatoprotective effect by ameliorating the biochemical parameters under investigation. The liver histopathological pictures confirmed our results.
1. Introduction
andantimicrobial activity on different bacteria and fungi [5]. The aim of the present study is to evaluate the ethyl acetate extract of the liquid culture filtrate of Chaetomium globosum fungus as antioxident and hepatoprotective agent on liver injury induced by CCl4 in rats.
Endophytes are considered as one of the essential interesting topics in the field of natural product chemistry and one of the major clades of life [1]. Chaetomium globosum, the type species of the genusChaetomium belonging to Phylum scomycota. Class Sordariomycetes(FamilyChaetomiaceae), spread all over the worldin soil, water, plant material and other cellulosic substances [2]. This fungusproduces diverse groups of secondary metabolites with several biological activities such as antimicrobial, immunomodulatory, and anticancer [3], where ithas also been proven to be an essential source of diverse bioactive constituentssuch ascytoglobosins, azaphilones, chaetoviridins, pyrones, orsellides and globosumones [4]. Prenisatin, chrysophanol, chrysazin,chaetoviridin A and B were isolated from the ethyl acetate extract of the liquid culture filtrate of Chaetomiumglobosum, where recorded remarkable antioxidant
⁎
2. Material and methods 2.1. Fungal isolation During autumn and winter growing season of 2014/2015, a surveystudy was conducted at the major cucumber (CucumissativusL.) growingareas under plastic greenhouse conditions. Samples of cucumber plants exhibiting damping-off, root-rot, stem-rot and wilt symptoms were collected and immediately transferred to Plant Pathology laboratory for isolation procedures. The pathogens were
Corresponding author at: Therapeutic Chemistry Department, National Research Centre, 33 El- Buhouth St., Dokki, Cairo, Egypt. ID: 60014618. E-mail address: [email protected] (M.A. Hamed).
http://dx.doi.org/10.1016/j.biopha.2017.10.120 Received 29 August 2017; Received in revised form 11 October 2017; Accepted 21 October 2017 0753-3322/ © 2017 Published by Elsevier Masson SAS.
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Table 1 GC/MS analysis of the fraction 1 eluted with 100% chloroform. No
RT
Mol. Formula
Mol. weight
BP
Compound
%
1
15.17
C5H12O2
104
43
3-Ethoxy propanol
2.59
2
27.60
C10H20 O
156
57
1-Cyclopentyl-2,2-dimethyl-1-propanol
54.63
3
30.23
C12H24
168
41
Dodecene
3.15
4
34.71
C18H32 O2
280
55
17-Octadecynoic acid
4.44
5
42.62
C19H40 O
284
57
2-Nonadecanol
4.19
6
56.99
C19H32 O2
292
43
Methyl octadeca-9-ene-12-ynoate
1.74
7
62.02
C22H46 O
326
55
1-Docosanol
5.07
Structure
isolated by tissue segment method [6]. The root pieces were cultured onto the surface of sterilized Petriplates containing freshly preparing Potato Dextrose Agar (PDA) medium. After 10 days of incubation at 25 ± 1 °C, the frequency occurrence (%) of isolated fungi was recorded. The fungal hyphal tip of developed colonies around the root pieces were transferred onto PDA medium and incubated for10 days at 25 ± 1oC for further studies [6].
done, then evaporated under reduced pressure and subjected to TLC examination using toluene: ethyl acetate (8:2). Similar fractions were collected together. Characterization of the compounds were carried out by GC/MS analysis (Shimadzu GC/MS −QP5050A, 70 eV) for of the volatile constituents.
2.2. Fungal identification
2.5.1. Acute toxicity Male Wistar albino rats (120–140 g) were obtained from the Animal House, National Research Center, Egypt and kept in controlled environment of air and temperature with access of water and diet. One oral dose of 5, 10, 50 and 100 mg of the ethyl acetate extract body weight. Rats were observed after 24 h of administration and along the following fourteen days. Dead rats were counted and the mortality rate was calculated. Lethal dose that killed 50% of animals (LD50) was monitored. The lethal dose that killed 50% of rats (LD50) along fourteen days was 100 mg/kg b.wt. Therefore, the selected dose is 20 mg/kg b.wt representing 1/5 of the lethal dose.
2.5. Biological study
Isolated fungi were identified at the Plant PathologyDepartment, National Research Centre (NRC), Egypt, and confirmed byFungal Taxonomy Department, Plant Pathology Research Institute, Agricultural Research Centre, Giza, Egypt according to the morphological and culture characters using the methods previously described by Barnett and Hunter [7], and Ramirez [8]. Stock cultures were maintained on PDA slants and kept in a refrigerator at 5 °C for further studies. 2.3. Preparation of the ethyl acetate extract of liquid culture filtrate of C. globosum
2.5.2. Doses and route of administration Administration regimen was twice a week for six consecutive weeks. CCl4 (0.5 ml/kg) was suspended in olive oil (1:9 v/v) and injected intraperitoneally [9]. The ethyl acetate extract was administrated orally at a dose 20 mg/kg. Silymarin; a reference herbal drug was orally administered at a dose 100 mg/kg [10]. Blood samples and slices of liver tissues were taken from the eviscerated animals and kept for the biochemical assays. Handling of the rats was obeyed the ethical guidelines of Medical Ethical Committee of the National Research Centre in Egypt (Approval no: 134, 2011).
Twenty liters of the liquid culture filtrate of C. globosum have been extracted by ethyl acetate. The extract has been concentrated in a rotary evaporator at 45 °C under reduced pressure. 2.4. Identification of the volatile secondary metabolites from the ethyl acetate extract The dried ethyl acetate extract of the liquid culture filtrate of C. globosum was dissolved in the least amount of ethyl acetate, loaded onto a silica gel column (120 cm height × 2.5 cm i.d.) containing 175 g activated silica (70–230 mesh; E. Merck, Darmstadt, Germany). The elution was done using the organic solvents; chloroform and chloroform: methanol (85:15). Collection of the eluted fractions were
2.5.3. Experimental design Forty eight rats were divided into six groups of eight rats each. Group 1 served as normal healthy control rats orally vehicle with 0.5 ml 175
Biomedicine & Pharmacotherapy 97 (2018) 174–180
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Table 2 GC/MS analysis of the fraction eluted by chloroform: methanol (85:15). No
RT
Mol. Formula
Mol. weight
B.P.
Compound
%
1
7.38
C10H16
136
121
1,5,5-Trimethyl-6-methylene-1-cyclohexene
25.79
2
9.87
C10H16O
152
81
Norbornan-2-one
26.84
3
10.40
C10H18O
154
95
1,7,7-Trimethyl bicyclo heptan-2-ol
4.64
4
12.88
C10H16O2
168
84
11-Oxa-dispiro undecan-1-ol
7.77
5
14.16
C12H20O2
196
95
1-Bornyl acetate
4.88
6
15.57
C12H20O2
196
93
1-Menthen-8-yl acetate
3.89
7
16.23
C15H24
204
93
Humulene
1.60
8
19.21
C10H18O2
206
161
3-Isopropenyl-4-methoxy phenyl propanol
3.75
9
19.44
C15H26O
222
109
Veridiflorol
1.41
10
23.97
C22H44
308
55
1-Docosene
2.72
11
28.27
C20H42 O
298
43
1-Eicosanol
1.51
176
Structure
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Table 3 Effect of treatment with the ethyl acetate extract of the liquid culture filtrate of Chaetomium globosum on the oxidative stress markers. Groups
Oxidative stress markers Hepatic glutathione content Mean ± SD
Control Control + C. globosum (ethyl acetate) Control +silymarin CCl4 CCl4 + C.globosum (ethyl acetate) CCl4 +silymarin
Hepatic malondialdehyde level
% Change
% Improvement
11.60bc ± 0.37 10.82c ± 1.16
— −6.72
— —
10.45c ± 0.51 5.73e ± 0.24 9.17d ± 0.57
−9.91 −50.60 −20.94
10.75c ± 0.28
−7.32
Mean ± SD
Hepatic superoxide dismutase level
% Change
% Improvement
0.47de ± 0.03 0.44def ± 0.11
−6.38
— —
— — 29.65
0.42def ± 0.08 2.03a ± 0.18 1.10b ± 0.10
−10.63 +333.91 +134.04
43.36
0.48de ± 0.06
+2.12
Mean ± SD
% Change
% Improvement
283.58d ± 60.72 122.31e ± 5.21
— −56.87
— —
— — 197.87
259.04d ± 20.4 541.71a ± 125.86 372.70b ± 31.88
−8.65 +91.02 +31.43
— — 59.59
329.78
306.12cd ± 65.18
+7.94
83.07
Values are mean ± SD of eight rats in each group. Values are expressed as μmol/mg protein. Statistical analysis are done using one way analysis of variance (ANOVA) using Co Stat Computer program accompanied with least significance level (LSD) between groups at p < 0.05. Unshared superscript letters are significant values between groups at p < 0.0001. Table 4 Effect of treatment with the ethyl acetate extract of the liquid culture filtrate of Chaetomium globosum on hepatic total protein level. Groups
Mean ± SD
% Change
% Improvement
Control Control + C.globosum (ethyl acetate) Control +silymarin CCl4 CCl4 + C.globosum (ethyl acetate) CCl4 +silymarin
39.09a ± 1.04 32.27c ± 4.54
— −17.44
— —
30.68cd ± 0.78 10.79h ± 1.19 20.59fg ± 0.45
−21.51 −72.39 −47.32
— — 25.07
22.33ef ± 1.06
−42.87
29.52
Table 6 Effect of treatment with the ethyl acetate extract of the liquid culture filtrate of Chaetomium globosum on serum total protein. Groups
Mean ± SD
% Change
% Improvement
Control Control + C.globosum (ethyl acetate) Control +silymarin CCl4 CCl4 + C.globosum (ethyl acetate) CCl4 +silymarin
6.52cde ± 0.47 6.32cde ± 0.14
— −3.06
— —
6.97bc ± 0.82 8.46a ± 0.37 6.47cde ± 0.14
+6.90 +29.75 −0.76
— — 30.52
7.54b ± 0.44
+15.64
14.11
Values are mean ± SD of eight rats in each group. Values are expressed as mg/g liver tissue. Statistical analysis are done using one way analysis of variance (ANOVA) using Co Stat Computer program accompanied with least significance level (LSD) between groups at p < 0.05. Unshared superscript letters are significant values between groups at p < 0.0001.
Values are mean ± SD of eight rats in each group. Values are expressed as mg/L. Statistical analysis are done using one way analysis of variance (ANOVA) using Co Stat Computer program accompanied with least significance level (LSD) between groups at p < 0.05. Unshared superscript letters are significant values between groups at p < 0.0001.
normal physiological saline and intraperitoneally received 0.5 ml olive oil. Group 2 was normal healthy rats received the ethyl acetate extract. Group 3 was CCl4 treated rats. Group 4 was injected with CCl4 and treated with the ethyl acetate extract at the same time and for the same duration. Group 5 was normal healthy rats received the standard drug (Silymarin). Group 6 was injected with CCl4 and treated with Silymarin.
2.5.4. Biochemical assays Hepatic glutathione content (GSH) was estimated by the method of Moron et al. [11], malondialdehyde (MDA) was determined by the method of and Aust [12]. Superoxide dismutase (SOD) was estimated by method of Nishikimi et al. [13]. Hepatic aspartate and alanine amintransferases (AST & ALT) were measured by the method of Reitman and Frankel [14]. Hepatic alkaline phosphatase (ALP) was estimated by the method of Belfield and Goldberg [15] and total protein
Table 5 Effect of treatment with the ethyl acetate extract of the liquid culture filtrate of Chaetomium globosum on the liver function indices. Groups
Liver function indices AST Mean ± SD
Control Control + C.globosum (ethyl acetate) Control +silymarin CCl4 CCl4 + C.globosum (ethyl acetate) CCl4 +silymarin
ALT % Change
% Improvement
3.04cde ± 0.08 2.96e ± 0.03
— −2.63
— —
1.65f ± 0.06 4.17a ± 0.06 3.21b ± 0.11
−45.72 +37.17 +5.59
2.96bc ± 0.10
−2.63
ALP
Mean ± SD
% Change
% Improvement
1.76c ± 0.05 1.86bc ± 0.03
— +5.68
— —
— — 31.57
1.52d ± 0.04 2.68a ± 0.42 1.78c ± 0.07
−13.63 +52.27 +1.13
39.80
1.85bc ± 0.06
+5.11
Mean ± SD
% Change
% Improvement
96.91d ± 3.39 124.34c ± 5.09
— +28.30
— —
— — 51.13
80.57b ± 5.36 194.42a ± 13.73 73.03e ± 6.83
−16.55 +100.61 −24.33
— — 125.26
47.15
104.12d ± 2.37
+7.43
93.17
Values are mean ± SD of eight rats in each group. Values are expressed as Unit/L. Statistical analysis are done using one way analysis of variance (ANOVA) using Co Stat Computer program accompanied with least significance level (LSD) between groups at p < 0.05. Unshared superscript letters are significant values between groups at p < 0.0001.
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Fig 1. Structure of the isolated compounds from the ethyl acetate extract of the liquid culture filtrate of Chaetomium globosum.
hydrogen peroxide (H2O2) and converts it into H2O by catalase and glutathione peroxidase [24]. In agreement with this explanation, we observed a significant increase in SOD [25]. In the present study, inhibition in GSH level during CCl4 toxicity may be due to the decreased availability of GSH resulted during the enhanced lipid peroxidation processes. Abubakar et al. [26] found a relation between its depletion and disruption of hepatocytes plasma membrane due to the peroxidation process that affects plasma membrane integrity. The elevation of plasma AST and ALT activities could be regarded as an index of damage of the liver parenchymal cells [21–23]. Bilgin et al. [20] attributed the increase in serum enzyme levels to the damage of the structural integrity of liver which lead to enzymes release into the circulation; a process of cytolysis. Abubakar et al. [26] mentioned that ALP is a marker enzyme for the plasma membrane and endoplasmic reticulum. Therefore, significant increase in the serum ALP following CCl4 treatment may be due to disruption of plasma membrane [26]. Romero et al. [27] and Sharma and Shukla [28] showed that CCl4 intoxication induced changes in the process of protein synthesis. Hence, increase in serum total protein content can be deemed as a useful index of cellular dysfunction as clearly shown in our studies where serum protein showed significant increase, while its content in the liver is significantly decreased due to cellular damage and leakage into the blood stream (Fig. 1). Regarding to the histopathological figures of liver, rats treated with the ethyl acetate extract of the liquid culture filtrate of C. globosum showed normal structure of the hepatocytes as compared with control rats (Fig. 2a, b). CCl4 rats showed distorted and loss lobular hepatic architecture and severe fibrosis (Fig. 2c, d). CCl4 treated rats with the ethyl acetate extract of the liquid culture filtrate of Chaetomium globosum recorded improvement of the hepatic cells with more or less like normal structure (Fig. 2e, f). CCl4 sylimarin treated rats showed preserved lobular hepatic architecture and mild steatotic changes (Fig. 2g, h). It is obviously that natural products therapy has emerged as proficient approach with good values in treating different diseases. Wagner and Elmadf [29] and Gross Arya et al. [30] confirmed the role of terpenoids and phenolics compounds as antioxidants. Terpenes and steroidal compounds act as direct antioxidants through free radical scavenging mechanisms and/or as indirect antioxidants by enhancing the antioxidant status (enzymatic and non-enzymatic) [31]. Also, phenolics mediate antioxidant enzyme expression which play an important role in the protection of liver against adverse effects related to mutagenesis and oxidative [32]. Many fungi and some bacteria as soon as they enter the stationary phase of growth, begin to synthesize and excrete compounds with diversity of chemical structures such as poly ketides and terpenoidal compounds. Terpenes and steroids are synthesized by microorganisms from the isoprene unit to form several terpenes with various chain lengths [33]. Administration the most biologically active extracts of the fungi
was assayed by the method of Bradford [16]. 2.6. Histopathological analysis Hepatic tissues slices were fixed in 10% paraformaldehyde and embedded in paraffin wax blocks. Sections of 5 μm thick were stained with hematoxylin & eosin (H & E), then examined under light microscope for determination of pathological changes [17]. 2.7. Statistical analysis and calculations All data were expressed as mean ± S.D. of eight rats in each group. Statistical analysis was carried out by one-way analysis of variance (ANOVA), Costat Software Computer Program. Significance values between groups were at P < 0.05.
• % changes = [(mean treated- mean of control group)/mean of control] × 100. • % of improvement = [(mean treated- mean of CCl4 group)/mean of control] × 100.
3. Results and discussion The isolate was identified as C. globosum based on the criteria of Kirk et al. [18], where the percentage of the dried ethyl acetate extract of liquid culture of C. globosum was 0.04%.The eluted fraction from 100% chloroform that undergo to GC/MS analysis recorded seven compounds, amounting 75.81% of the total fraction. 1-Cyclopentyl-2,2dimethyl-1-propanol (54.63%) was presented as the major compound in the fraction (Table 1).The fraction eluted from chloroform: methanol (85:15) was subjected to GC/MS analysis and eleven compounds were identified amounting 84.8% of the total fraction. 1,5,5-Trimethyl-6methylene-1-cyclohexene (25.79%) and Norbornan-2-one (26.84%) are presented as the major compounds of this fraction (Table 2). Liver injury by CCl4 is a classical system of xenobiotic- induced hepatotoxicity [19] and is commonly used for screening the anti-hepatotoxic, antifibrotic and hepatoprotective activity of drugs [20]. The effect of CCl4 on rat livers enzymes and oxidative stress markers have been illustrated in Table 3–6. Concerning with the oxidative stress markers in the present study and in agreement with the results of Motawi et al. [21] and El-Gengaihi et al. [22], we observed a significant elevation in MDA and SOD, while GSH was significantly decrease in CCl4 rats. Gowri Shankar et al. [23] postulated the role of the reactive metabolite trichloromethyl radical (CCl3−) that has been formed from the metabolic conversion of CCl4 by cytochrome P-450. This free radical initiate the peroxidation of membrane poly-unsaturated fatty acids (PUFA), generates PUFA radicals, covalently binds to membrane lipids and proteins, generates ROS and enhances MDA level. Superoxide dismutase, as one of the most important enzymatic antioxidants, transforms O2− to the more stable 178
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Fig. 2. photomicrographs of liver section of control (a) and control rats treated with extract (b) showed preserved (intact) lobular hepatic architecture and normal morphological appearance (H & E,×100), liver section of CCl4 group (c, d) showed distorted and loss lobular hepatic architecture and formation of micro and macro regenerating nodules (black arrows), mild to moderate ballooning of hepatocytes (red arrow)moderate infiltration by lymphocytes to portal tract (yellow arrow) (H & E, ×100), liver section of CCl4 rates treated with extract showed preserved (intact) lobular hepatic architecture and with mild lobular inflammation (black arrows), mild infiltration by lymphocytes to portal tract (red arrow) and fibrous tissue (yellow arrow) (H & E, ×100) and liver section of CCl4 rats treated with sylimarin showed intact (preserved) lobular hepatic architecture and mild steatotic changes (black arrow), with mild blood vessel congestion(red arrow) (H & E, ×100).
liver function enzymes, improving the oxidative stress markers and enhancing the liver histopathological pictures.
attenuated the increased levels of the serum enzymes, improved the oxidative stress markers and hepatic architecture after injury with CCl4 causing a subsequent recovery towards normalization. This suggested that these extracts are able to condition the hepatocytes, so as to accelerate regeneration of parenchyma cells, thus protecting against membrane fragility and decrease leakage of the enzymes into circulation. Therefore, these tested extracts perform as silymarin drug in the protection of the plasma membrane of hepatocytes. Also, they are protecting liver cells directly by stabilizing the membrane permeability [34]. In conclusion, the ethyl acetate extract of the liquid culture filtrate of Chaetomium globosum is considered as hepatoprotective and antioxidant agent. It performed the action by attenuating the elevation of
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