doi 10.1515/jcim-2013-0023

J Complement Integr Med. 2014; 11(1): 9–18

Ravikumar Aruna, Arumugam Geetha*, Periyanayagam Suguna and Vijayashankar Suganya

Rutin rich Emblica officinalis Geart. fruit extract ameliorates inflammation in the pancreas of rats subjected to alcohol and cerulein administration Abstract Background: The modulating effect of methanolic extract of Emblica officinalis (MEEO) on ethanol (EtOH)and cerulein (Cer)-induced pancreatitis in rats was investigated in this study. Methods: Male albino Wistar rats were divided into four groups. Group 1 and 2 rats served as control and fed normal diet. Group 3 and 4 rats were fed isocalorically adjusted diet containing EtOH (36% of total calories) for 5 weeks and also subjected to intraperitoneal injection of Cer 20 µg/kg b.wt. thrice weekly for the last 3 weeks of the experimental period. In addition, group 2 and 4 rats received 200 mg/kg b.wt. of MEEO from 15th day till the experimental period. Serum levels of lipase (L), amylase (A), cytokines IL-1β, IL-18, caspase-1 and oxidative stress index (OSI) were determined. Levels of fecal trypsin, total collagen, caspase-1, myeloperoxidase (MPO), antioxidants and mRNA expression of caspase-1, IL-1β and IL-18 were determined in the pancreas. Results: HPLC analysis showed the presence of rutin in MEEO. We observed a significant elevation in serum L/A ratio, IL-1β, IL-18, caspase-1, OSI, collagen, MPO activity and the mRNA expression of IL-1β, IL-18 and caspase-1 and significant reduction in fecal trypsin and antioxidant status in EtOH- and Cer-administered rats. The inflammatory markers were found to be reduced and the antioxidant status of pancreas was maintained in MEEOcoadministered rats. Conclusions: The rutin rich nature of E. officinalis can be claimed for its anti-inflammatory and pancreato protective effects.

Keywords: caspase-1, cerulein, cytokines, Emblica officinalis, inflammation

*Corresponding author: Arumugam Geetha, Department of Biochemistry, Bharathi Women’s College – Affiliated to University of Madras, Chennai, Tamilnadu, India, E-mail: [email protected]

Ravikumar Aruna, Periyanayagam Suguna, Vijayashankar Suganya, Department of Biochemistry, Bharathi Women’s College – Affiliated to University of Madras, Chennai, Tamilnadu, India

Introduction Pancreatitis is a major gastrointestinal disease in which the pancreas falls prey to its own, prematurely activated digestive enzymes to cause injury and inflammation due to the formation of cytokines [1]. The development of systemic inflammatory response syndrome is one of the leading events responsible for the mortality of pancreatitis. Chronic pancreatitis is a progressive and destructive necroinflammatory disorder of the pancreas characterized by irreversible fibrosis of the gland with eventual failure of exocrine and endocrine functions [2]. Production of proinflammatory cytokines such as interleukin (IL)-1β, IL18 [3], IL-6 and TNF-α are well correlated with the severity of pancreatitis [4]. IL-1β plays an essential role in the induction of systemic acute phase response and in the release of other members of proinflammatory cytokines [5]. Proinflammatory cytokines act as inflammatory markers or acute phase reactants which are used in the diagnosis of inflammation related disorders. IL-1β is a potent chemokine which is multifunctional, affecting nearly all the cell types in combination with other cytokines [6]. Its activity has been shown elevated in various inflammatory disorders. This cytokine also activate IL-18 and both work together in the process of inflammation [7]. TNF-α plays an important role in stress related inflammatory disorders. It has also been shown to participate in propagating pancreatitis [8]. Excessive ethanol (EtOH) consumption is a common risk factor for both acute and chronic pancreatitis [9]. Evidences obtained from animal models of pancreatitis indicate that alcohol consumption sensitizes the pancreas to inappropriate intrapancreatic activation of digestive enzymes, acinar cell death, inflammation and fibrosis [10]. Acute oral administration of EtOH has been found to stimulate pancreatic secretion and intestinal cholecystokinin (CCK) release [11].

Brought to you by | Georgetown University Authenticated | 10.248.254.158 Download Date | 9/9/14 7:36 PM

10

Aruna et al.: Anti-inflammatory effect of Emblica officinalis

Rodents given a supramaximally stimulating dose of cerulein (Cer) were found to develop pancreatitis with acinar cell injury, pancreatic inflammation and intrapancreatic digestive enzyme activation [12]. Although the CCK analog Cer induces a relatively mild manifestation of pancreatitis, the rapid induction, noninvasiveness, high reproducibility and the remarkable similarity to the histological changes in human pancreatitis have made the Cer-induced pancreatitis animal model the most favored pancreatitis model [13]. Cer increases the production of pancreatic enzymes inside the acinar cells and the metabolite of EtOH decreases the cell membrane stability. This leads to leakage of enzymes within the pancreas resulting in autodigestion of tissues and pancreatitis. There are no much allopathic medicines available for pancreatitis except sompraz-40/omiprazole which could only act as supportive medicines to reduce the severe pain associated with pancreatitis. Medicinal plants and their products with anti-inflammatory effect could be of alternative choice for the prevention and the treatment of pancreatitis. Emblica officinalis Geart. (EO) is one such traditionally used medicinal plant in India which belongs to Euphorbiaceae family. The fruits contain phenolic compounds, tannins, phyllembelic acid, phyllemblin, rutin, curcuminoides and emblicol [14]. The dried fruit has been prescribed in ayurveda for pancreas related disorders. The fruit extract was reported to have hypolipidemic [15], antidiabetic [16], antioxidant [17] and anti-inflammatory activities [18]. The aim of the study was to evaluate whether the methanolic extract of EO (MEEO) has anti-inflammatory effect in rats subjected to experimental pancreatitis.

Materials and methods Chemicals and reagents AxyPrep multisource total RNA miniprep kit was purchased from Axygen Biosciences, CA, USA, and cDNA reverse transcription kit was purchased from Applied Biosystems, CA, USA. ELISA kit for IL-1β was purchased from Abcam, Cambridge, USA and IL-18 from Invitrogen, Bangalore, India. All other chemicals and reagents used were of analytical grade.

Centre (PARC/2011/995). The pulp of the fresh fruits of EO was dried and macerated into homogenous powder. The methanolic extract was prepared by soaking 10 g of homogenous powder in 100 mL of 70% methanol for 7 days with continuous swirling, and the resulting extract was filtered and evaporated to dryness using a vacuum evaporator.

HPLC analysis The amount of rutin in MEEO was determined by HPLC analysis (LCGC AGLIENT). The stationary phase was octadecylsilyl silica gel, and mobile phase was a linear gradient with methanol, water and phosphoric acid (100:100:1). The flow rate of sample was 1.5 mL/min with the injection volume of 20 μl. Standard rutin was used as the reference compound. The UV spectra were monitored at 270 nm.

Experimental protocol Male albino Wistar rats weighing 175–200 g were kept in an air-conditioned room where temperature and artificial light were controlled (20°C, 24 h circadian cycle: 12 h light, 12 h dark). All animals had free access to food and drinking water. After the acclimatization period, they were divided randomly into four groups. Group 1 and 2 rats fed normal diet; group 3 and 4 rats received isocalorically adjusted diet containing EtOH (36% of total calories) for 5 weeks and injected Cer (i/p) at the dose of 20 µg/kg b.wt. thrice weekly for the last 3 weeks of the experimental period [19]. In addition, group 2 and 4 rats received 200 mg/kg b.wt. of MEEO from 15th day till the experimental period. This study was conducted according to the guidelines given by the Institutional Animal Ethics Committee (XIII/ VELS/COL/24A/CPCSEA/IAEC/23.9.11). At the end of experimental period, the rats were subjected to ketamine hydrochloride (30 mg/kg b.wt.) and killed by cervical decapitation; immediately the blood was collected and the plasma/serum separated was stored at 4°C until analyses.

Biochemical investigations

Preparation of MEEO

Preparation of tissue homogenate and fecal suspension

The fruits of EO were purchased from the local market, and it was duly authenticated at Plant Anatomy Research

Pancreas was removed immediately, carefully washed and homogenized in 0.1 M Tris–HCl buffer; pH 7.4 and

Brought to you by | Georgetown University Authenticated | 10.248.254.158 Download Date | 9/9/14 7:36 PM

Aruna et al.: Anti-inflammatory effect of Emblica officinalis

centrifuged at low speed to remove any cell debris. The supernatant was used for the determination of caspase-1, total collagen, lipid peroxides, reduced glutathione (GSH) and antioxidant enzymes. The feces weighing 4.5 to 5.5 g was dissolved in 0.5% sodium bicarbonate (1/10 dilution) and this suspension was again diluted with 0.5% sodium bicarbonate (1/100 dilution).

Determination of serum lipase activity The activity of lipase (EC: 3.1.1.1) in serum was measured by the method of Lowry and Tinsley [20]. The serum was added in 25 mL olive oil/triton X 100 emulsion as substrate to initiate the lipolysis reaction. The liberated free fatty acids were assayed with the 0.3 mL subsamples of reaction mixture taken at predetermined time intervals at 715 nm and expressed as IU/L.

11

developed was measured at 450 nm, and the stop solution changes the color from blue to yellow. The activity of IL-1β was expressed as pg/mL.

Assay of IL-18 The serum sample and standards were pipetted into antibody immobilized wells. The assay was carried out as per the instruction of kit manual (KRC2341). Biotinylated secondary antibody was added after the incubation. After the removal of excess secondary antibody, streptavidin– peroxidase was added. Then, the substrate solution was added to bound enzyme to produce color. The intensity of this color was measured at 450 nm. The activity of IL-18 was expressed as pg/mL.

Assay of caspase-1 Determination of serum amylase activity Method of Gomori [21] was used to determine the activity of amylase (EC: 3.2.1.1). The method was based on the activity of enzyme on substrate starch and the measurement of maltose liberated by using lugol’s iodine solution.

Assay of tissue total collagen and fecal trypsin The total collagen content was determined by the method of Woessner and Taplin [22]. After digesting the tissue sample, the total collagen content was determined in terms of hydroxyproline a characteristic imino acid of collagen. Fecal trypsin was measured according to the method of Charney and Tomarelli [23].

Assay procedure for IL-1β The assay was performed according to the manufacturer’s instructions (ab100767). Standards or serum samples were pipette into the wells precoated with IL-1β antibody, and IL-1β present in a sample is bound to the well by the immobilized antibody. The wells were washed, and biotinylated anti-rat IL-1β antibody was added. The wells were washed again to remove unbound biotinylated antibody and added HRP-conjugated streptavidin to the wells. The wells were washed again, and TMB substrate solution was added to the wells. The amount of IL-1β present in the sample was proportional to the intensity of the color developed. The intensity of the color

According to the method of Thornberry [24], caspase-1 (EC: 3.4.22.36) activity was determined colorimetrically in prepared serum or pancreatic extract, as the enzyme source. Briefly, the pancreas was homogenized in a lysis buffer (25 mM HEPES [pH 7.5], 1 mM EDTA, 10 μg of aprotinin/mL, 10 μg of leupeptin/mL, 2 mM dithiothreitol) at 5 mL/100 mg of pancreas tissue. Extracts were centrifuged at 15,000  g for 30 min at 4°C, and the supernatant was centrifuged again at 200,000  g for 1 h at 4°C. The cytosol was used for caspase-1 activity measurements. The assay in undiluted serum or pancreas extract was performed as per the kit manufacturer instruction. The plates were read at 405 nm.

Determination of myeloperoxidase activity Myeloperoxidase (MPO) activity in the pancreatic tissue was measured according to the method of Bradley [25]. In 0.5% hexadecyltrimethyl ammonium bromide in 50 mM potassium phosphate buffer (pH 6) pre-weighed tissue was homogenized (1:10 w/v) before sonication in an ice bath for 20 s. Three freeze/thaw cycles were performed followed by sonication (20 s in icebath). The samples were centrifuged at 17,000  g (5 min, 4°C) and the enzyme activity was assayed by mixing 0.1 mL of supernatant and 2.9 mL of 10 mM potassium phosphate buffer (pH 6) containing 0.167 g/L o-dianisidine dihydrochloride and 0.0005% hydrogen peroxide (H2O2). The change in absorbance was measured using UV visible spectrophotometer at 460 nm for 4 min. The enzyme activity was expressed as units/mg protein.

Brought to you by | Georgetown University Authenticated | 10.248.254.158 Download Date | 9/9/14 7:36 PM

12

Aruna et al.: Anti-inflammatory effect of Emblica officinalis

Estimation of lipid peroxides and oxidative stress index The level of lipid peroxides (LPO) in plasma was determined by measuring thiobarbituric acid-reacting substances (TBARS) [26]. The value was expressed as nM/mL plasma. FOX 2 method with minor modifications was used to measure the peroxide content (LHP) in plasma [27]. The FOX 2 test system is based on oxidation of ferrous ion to ferric ion by various types of peroxides contained within samples, to produce a colored ferricxylenol orange complex whose absorbance was measured at 560 nm. According to the method of Miller [28] total antioxidant capacity (TAC) was determined. The decolorization of the assay mixture containing 2,2′-azino bis 3-ethyl benzo-thiazoline-6-sulfonate and the sample was monitored by measuring the absorbance at 734 nm and the percentage inhibition was calculated and plotted as a function of concentration of antioxidants and of trolox for the standard reference data. The ratio of total peroxides to TAC was calculated as oxidative stress index (OSI).

Estimation of glutathione and antioxidant enzymes Glutathione (GSH) level was determined by the method of Moron [29]. Equal volume of ice cold 5% trichloroacetic acid was added to the aliquots of homogenate and the precipitated proteins were removed by centrifugation. The supernatant was added to equal volume of 0.2 M phosphate buffer, pH 8 and measured at 412 nm. Glutathione peroxidase (GPx) was assayed by the method of Flohe and Gunzler [30]. The activity of GPx was expressed as nM of GSH oxidized/min/mg protein. Superoxide dismutase (SOD) activity was measured according to the method of Kakkar [31]. The inhibition of reduction of nitroblue tetrazolium to blue colored formazan in the presence of phenazine methosulfate and NADH was measured at 560 nm using n-butanol as blank. The enzyme activity was expressed as units/mg protein. In the presence of catalase (CAT) decomposition of H2O2 was kinetically measured at 240 nm [32]. CAT activity was

Table 1

defined as the amount of enzyme required to decompose 1 µM of H2O2/min. The enzyme activity was expressed as µM of H2O2 consumed/min/mg protein. RT-PCR analysis Total RNA from frozen tissues was extracted (AxyPrep multisource total RNA miniprep) and quantified. Reverse transcription reactions were performed with total RNA according to the cDNA reverse transcription kit. For real-time PCR, ABI PRISM Sequence Detection System 7700 (Applied Biosystems, USA) was employed. The primer sequences used for real-time PCR analysis are shown in Table 1. PCR conditions were as follows: 40 cycles of 95°C for 20 s (denaturation), 55°C for 30 s (annealing) and 60°C for 30 s (extension). Ct values obtained were used to quantify mRNA expression.

Statistical analysis Data were analyzed by using a commercially available statistics software package (SPSS for window V. 10). The statistical significance of mean values between different groups was determined by applying one-way analysis of variance (ANOVA) with post hoc Bonferroni test, and the p-value < 0.05 was considered as significant.

Results The effect of MEEO on EtOH- and Cer-induced changes in rats was assessed by biochemical parameters, and the following results were obtained.

Fractionation of MEEO From HPLC-UV chromatogram of MEEO, the rutin level was determined. It showed the peak at 270 nm with the retention time of 8.207 min. The UV spectra of this peak together with the analysis of retention time of standard

Primer sequences for RT-PCR analysis.

Gene

Forward

Reverse

Caspase-1 NM_012762.2 IL-1β NM_031512.2 IL-18 NM_019165.1 β-Actin NM_031144.3

5′-TATGGAAAAGGCACGAGACC-3′ 5′-CAGGAAGGCAGTGTCACTCA-3′ 5′-GAGGACTGGCTGTGACCCTA-3′ 5′-GAGAAGATTTGGCACCACAC-3′

5′-CAGCTGATGGACCTGACTGA-3′ 5′-AAAGAAGGTGCTTGGGTCCT-3′ 5′-ATCCCCATTTTCATCCTTCC-3′ 5′-CATCACAATGCCAGTGGTAC-3′

Brought to you by | Georgetown University Authenticated | 10.248.254.158 Download Date | 9/9/14 7:36 PM

Aruna et al.: Anti-inflammatory effect of Emblica officinalis

(A) [mV] 400 Rutin Rutin

Table 2 Levels of serum lipase/amylase ratio, fecal trypsin and pancreatic total collagen.

Voltage

300

Groups

Lipase/ amylase ratio

200 100 0 0

5

10 Time

(B) [mV] 600

15

20 [min]

Total collagen Trypsin (mg/min/100 (μg of collagen/ mg protein) mg feces)

Normal control 1.23  0.16 MEEO control 1.29  0.14NS EtOH þ Cer 5.5  0.82a EtOH þ Cer þ MEEO 2.7  0.29a

12.5  1.64 12.1  1.29NS 8.33  0.96a 11.6  1.65a

4.26  0.49 4.3  0.44NS 18.5  2.74a 4.9  0.63a

Values are expressed as mean  SD for six animals in each group. Control vs MEEO control, control vs EtOH þ Cer, EtOH þ Cer þ MEEO vs EtOH þ Cer. ap ¼ 0.000, NS ¼ nonsignificant.

500 400 300 200

Rats coadministered with MEEO showed a significant increase in the level of fecal trypsin.

Rutin

Voltage

13

100 0 0

5

15

10

20

Time

25 [min]

Figure 1 HPLC-UV finger print of rutin. (A) HPLC-UV finger print of standard rutin. (B) HPLC-UV finger print of rutin in methanolic fruit extract of E. officinalis

compound indicated the presence of rutin (Figure 1). The amount of rutin present in MEEO was 533 µg/g dry mass.

Effect of MEEO on serum L/A ratio, fecal trypsin and pancreatic total collagen Table 2 shows the levels of L/A ratio, trypsin and total collagen. In EtOH- and Cer-administered rats, the serum L/A ratio and pancreatic collagen content were found to be elevated. Rats coadministered with MEEO were found to reduce the levels of L/A ratio and collagen significantly. The level of fecal trypsin in rats administered with EtOH and Cer was found significantly decreased. Table 3

Effect of MEEO on inflammatory markers Table 3 shows the levels of serum IL-1β, IL-18, caspase-1 and pancreatic MPO, caspase-1. Elevated level of these inflammatory markers was observed in EtOH- and Ceradministered rats when compared to MEEO-coadministered rats. A nonsignificant reduction was seen in rats received normal diet and MEEO. Figures 2–4 show the upregulated mRNA expression levels of caspase-1, IL-1β and IL-18, respectively, in EtOH and Cer treated rats which were significantly down-regulated by MEEO coadministration.

Effect of MEEO on oxidative stress Table 4 shows the levels of plasma LPO, LHP, TAC and OSI. The levels of LPO, LHP and OSI were found to be elevated in EtOH- and Cer-administered rats whereas TAC was found to be decreased. MEEO-coadministered rats

Levels of serum IL-1β, IL-18, caspase-1 and pancreatic MPO, caspase-1 in experimental animals.

Groups

Normal control MEEO control EtOH þ Cer EtOH þ Cer þ MEEO

IL-1β (pg/mL)

12.1  1.34 11.5  1.69NS 24.7  3.11a 13.6  1.8a

IL-18 (pg/mL)

169.7  21.72 167.2  25.08NS 250.3  33.29a 192.4  22.13a

Caspase-1 Serum (pg/mL)

Pancreas (pM/mg protein)

10.6  1.1 10.1  1.38NS 80.2  10.27a 18.5  2.65a

10.4  1.45 10.2  1.09NS 93.1  11.92a 24.6  3.57a

MPO (units/mg protein)

1.91  0.23 1.69  0.18NS 3.62  0.49a 2.06  0.25a

Values are expressed as mean  SD for six animals in each group. Control vs MEEO control, control vs EtOH þ Cer, EtOH þ Cer þ MEEO vs EtOH þ Cer. a p ¼ 0.000, NS ¼ nonsignificant.

Brought to you by | Georgetown University Authenticated | 10.248.254.158 Download Date | 9/9/14 7:36 PM

Relative gene expression of caspese-1 mRNA

14

Aruna et al.: Anti-inflammatory effect of Emblica officinalis

Caspase-1mRNA gene expression 1.60E–02 a

1.40E–02 1.20E–02 1.00E–02 8.00E–03 6.00E–03 4.00E–03 2.00E–03

NS

a

0.00E+00 Normal control

MEEO control

EtOH + Cer + MEEO

EtOH + Cer

Groups

Figure 2 Alterations in mRNA expression of caspase-1 in EtOH þ Cer and MEEO treated rats. Data were analyzed by one-way analysis of variance (ANOVA) followed by post hoc Bonferroni. Values are means  SD of six rats. a p ¼ 0.000 for control vs EtOH þ Cer, EtOH þ Cer þ MEEO vs EtOH þ Cer; p ¼ 1.000 for control vs MEEO control (not significant)

Relative gene expression of IL-1β mRNA

IL-18 mRNA gene expression 1.60E–02

a

1.40E–02 1.20E–02 1.00E–02 8.00E–03 6.00E–03

NS

4.00E–03

a

2.00E–03 0.00E+00 Normal control

MEEO control

EtOH + Cer

Groups

EtOH + Cer + MEEO

Relative gene expression of IL-18 mRNA

Figure 3 mRNA expression of IL-1β in pancreas of experimental rats. Data were analyzed by one-way ANOVA followed by post hoc Bonferroni. Values are means  SD of six rats. ap ¼ 0.000 for control vs EtOH þ Cer, EtOH þ Cer þ MEEO vs EtOH þ Cer; p ¼ 1.000 for control vs MEEO control (not significant)

IL-18 mRNA gene expression 1.80E–03 1.60E–03 1.40E–03 1.20E–03 1.00E–03 8.00E–04 6.00E–04 4.00E–04 2.00E–04 0.00E+00

a

NS

a

Normal control MEEO control

EtOH + Cer

EtOH + Cer + MEEO

Groups Figure 4 mRNA expression of IL-18 in pancreas of experimental rats. Data were analyzed by one-way ANOVA followed by post hoc Bonferroni. Values are means  SD of six rats. ap ¼ 0.000 for control vs EtOH þ Cer, EtOH þ Cer þ MEEO vs EtOH þ HFD; p ¼ 1.000 for control vs MEEO control (not significant)

Brought to you by | Georgetown University Authenticated | 10.248.254.158 Download Date | 9/9/14 7:36 PM

Aruna et al.: Anti-inflammatory effect of Emblica officinalis

Table 4

15

Activity levels of plasma peroxide content, total antioxidant capacity and oxidative stress index in experimental animals.

Groups Normal control MEEO control EtOH þ Cer EtOH þ Cer þ MEEO

TBARS (nM/mL)

Peroxides (mM/mL)

TAC (mmol trolex eq./L)

OSI

0.13  0.02 0.14  0.01NS 0.41  0.05a 0.23  0.03a

180.2  20.9 176.5  25.59NS 283.7  37.45a 187.4  20.24a

341.3  49.83 337.1  43.49NS 199.8  27.37a 329.6  34.61a

0.53  0.06 0.52  0.05NS 1.42  0.21a 0.57  0.08a

Values are expressed as mean  SD for six animals in each group. Control vs MEEO control, control vs EtOH þ Cer, EtOH þ Cer þ MEEO vs EtOH þ Cer. a p ¼ 0.000, NS ¼ nonsignificant.

showed reduced level of LPO, LHP and OSI, with significant elevation of TAC.

Effect of MEEO on antioxidants Table 5 shows the levels of GSH, GPx, SOD and CAT in the pancreas of experimental rats. The level of these antioxidants in EtOH and Cer treated rats was found to be decreased significantly, and MEEO-coadministered rats showed less alteration in their levels.

Discussion The EtOH–Cer model is a well-accepted model of experimental pancreatitis and hence used in the present study. Cer, an analog of CCK acting through the CCK receptors, leads to prematuration of trypsinogen, followed by lysosomal degradation in acinar cells and marked interstitial edema. Along with Cer, EtOH increases the severity of pancreatitis in rats. According to the WHO survey, 80% of the populations living in the developing countries rely almost exclusively on traditional medicine for the primary health care needs [33]. Many investigations have shown that the fruits of EO have wide range of biological activity such as antioxidant and anti-

Table 5

inflammatory activities. The active phytochemicals of the EO include phenols, tannins and flavonoids which provide health benefits in human. The result of HPLCUV analysis showed that MEEO contains a rich amount of rutin. The presence of rutin in EO may be attributed for its anti-inflammatory and antioxidant properties. Rutin was found to possess various pharmacological activities including anti-inflammatory [34] and vasoactive properties [35]. Rutin is also a potent scavenger of hydroxyl and superoxide radicals [36]. L/A ratio has been proposed as a clinical marker for pancreatitis. When the pancreas is diseased or inflamed, amylase and more predominantly lipase are released into the blood circulation [37]. In this investigation the L/A ratio was found to be less than 2 in control rats and greater than 4 in EtOH- and Cer-administered rats. The increased level of L/A ratio in EtOH and Cer received rats might be due to the release of these enzymes to the circulation from injured pancreas. The pathological mechanism is due to a disruption of pancreatic acini or due to alteration in normal exocytosis process, with the secretion of the zymogen contents at the basolateral side of the acinar cells [38]. The pancreatic enzymes are therefore released into the interstitial space and later reabsorbed directly or through the lymphatics into the blood stream and subsequently reaches the general circulation. MEEO coadministration was found to decrease the L/A ratio

Activity levels of antioxidant enzymes and glutathione in pancreatic tissue of the experimental animals.

Groups

Normal control MEEO control EtOH þ Cer EtOH þ Cer þ MEEO

GSH (mg/g protein)

GPx (nM of GSHoxidized/ min/mg protein)

SOD (units/mg protein)

CAT (µmol H2O2 consumed/ min/mg protein)

12.39  1.7 12.01  1.38NS 6.18  0.88a 11.93  1.23a

1.48  0.18 1.40  0.15NS 0.47  0.06a 1.31  0.19a

14.2  1.87 14  1.6NS 8.3  1.25a 12.7  1.38a

101.6  10.46 98.9  14.54NS 73.7  8.92a 97.8  13.11a

Values are expressed as mean  SD for six animals in each group. Control vs MEEO control, control vs EtOH þ Cer, EtOH þ Cer þ MEEO vs EtOH þ Cer. a p ¼ 0.000, NS ¼ nonsignificant.

Brought to you by | Georgetown University Authenticated | 10.248.254.158 Download Date | 9/9/14 7:36 PM

16

Aruna et al.: Anti-inflammatory effect of Emblica officinalis

significantly. This shows the curative effect of MEEO on EtOH- and Cer-induced pancreatitis. Evidence of diminished secretion of pancreatic enzymes into the gut can be sought by the estimation of trypsin activity in duodenal juice and feces. A fall in fecal trypsin may occur due to duct obstruction or due to reduced secretion because of acute necrosis or malignant growth in the pancreas. The decreased level of fecal trypsin excretion occurs when the pancreas does not produce enough trypsin and chymotrypsin. Active trypsin appears in the blood stream during pancreatitis [39] but becomes rapidly bound to protein inhibitors such as α1antitrypsin and α2-macroglobulin and hence low concentration in feces was observed. MEEO-coadministered rats markedly reversed this condition, showing the protective role on pancreas. Studies have shown that pancreatic acinar cells activate the mechanisms leading to inflammation and organ destruction in pancreatitis [40]. Pancreatic stellate cells (PSCs) play a crucial role in the initiation and progression of pancreatic fibrogenesis in chronic pancreatitis [41]. When PSCs are stimulated by the profibrogenic mediators, the quiescent cells get converted to myofibroblastlike cells that are highly proliferative and capable of depositing fibrillar collagen in the interstitial spaces [42]. Activated PSC is responsible for the production of extracellular matrix components including collagen and found to play major role in the onset of chronic pancreatitis [43]. When EtOH-fed rats were exposed to Cer administration, it leads to periacinar deposition of collagen. A more profound and long lasting increase in collagen content has been reported in pancreas during pancreatitis [44]. The irreversible inflammatory disease of the pancreas is associated with the replacement of destroyed parenchyma by the extended development of fibrosis. Significant reduction in collagen level was observed in MEEO-coadministered rats, showing the pancreato protective activity. MPO has been implicated in promoting tissue damage in various inflammatory diseases. The predominant physiological activity of MPO is to convert H2O2 and chloride to hypochlorous acid [45] and to promote the formation of reactive oxygen metabolites that damage cells with the liberation of intracellular proteinases. Reactive oxygen species (ROS) down-regulate the proteinase inhibitors, which protect interstitial tissue against autodigestion. The products formed under the influence of MPO have protective properties, however its uncontrolled activity leads to self-damage of the cells. The elevated level of pancreatic MPO in EtOH- and Cer-

administered rats might be to detoxify the free radicals that are being formed but elevated activity might have resulted in tissue injury. The decreased level of MPO in MEEO-coadministered rats might be due to the presence of flavonoids such as rutin. Rutin has been reported to play therapeutic role by its anti-inflammatory and antioxidant properties [46]. Lipid peroxidation is the process in which the free radicals abstract electrons from the phospholipids of cell membranes, initiate and propagate free radical formation that results in cell damage. The enhanced level of LHP, TBARS and OSI might have caused oxidative stressinduced cell damage which is considered to be a common factor in the pathogenesis of pancreatitis. The level of TAC, a reliable biomarker of cellular antioxidant status, was found to be decreased in EtOH- and Cer-administered rats which may be attributed to the oxidative stress and MEEO-coadministration significantly maintained the level of TAC. Cells contain a large number of antioxidants to prevent or repair the damage caused by ROS. The SOD dismutate O2∙ to H2O2, whereas the CAT and peroxidases convert H2O2 into H2O [47]. GSH is an important antioxidant, plays a role in the detoxification of a variety of electrophilic compounds and peroxides through the action of GPx. The decreased level of pancreatic SOD, CAT, GPx and GSH might have lead to accumulation of free radical products and tissue damage. Treatment with MEEO reversed these oxidative changes due to experimental pancreatitis. The antioxidant activity of EO could be due to the presence of rutin which prevents the tissue damage and inhibits the formation of toxic free radicals. Inflammasomes are multi-protein complexes that link recognition of damage-associated molecular patterns by members of the Nod-like receptor family of cytosolic pattern recognition receptors to the activation of caspase-1, process and release of the proinflammatory cytokines IL-1β and IL-18. Caspase-1 (IL-1β converting enzyme) is a cysteine protease [48] that specifically recognizes the aspartic acid residue in the substrates. Caspase-1 seems to be involved in the inflammatory response by cleaving the precursors of IL-1β and IL-18 to their active forms. Inactive procaspase-1 is converted to active form via dimerization, followed by an autocatalytic reaction that generates an active molecule composed of larger and smaller subunits [49]. The elevated levels of serum and pancreatic caspase-1 showed that the inflammatory changes were induced by EtOH and Cer administration. The mRNA expression

Brought to you by | Georgetown University Authenticated | 10.248.254.158 Download Date | 9/9/14 7:36 PM

Aruna et al.: Anti-inflammatory effect of Emblica officinalis

of caspase-1 was found to be up-regulated in EtOH and Cer treated rats due to inflammation. Regulation of the effect of inflammatory caspases at the inflammasome level is an important checkpoint in the control of IL-1β and IL-18 activity. Significant reduction in caspase-1 expression in MEEO-coadministered rats shows the anti-inflammatory role of EO. The results of this investigation clearly show that caspase-1 is down-regulated by MEEO. IL-1β is cleaved to its 17 kDa active form by caspase-1. It is a well-known mediator of inflammation. It stimulates the synthesis and release of inflammatory mediators such as TNF-α, prostaglandins and proinflammatory interleukins [50]. In the present study, there was profound upregulation of the IL-1β mRNA expression, which is reflected in serum level. The anti-inflammatory activity of MEEO might also be due to its effect on IL-1β expression thereby preventing the effect of proinflammatory mediators and reducing the severity of inflammation and tissue damage. It has been demonstrated that IL-18 stimulates the release of TNF-α, chemokines and INF-γ [51]. This cytokine is produced by activated macrophages, which is proteolytically processed to its active form by caspase-1. The increased level of IL-18 is an additional marker to monitor the severity of inflammation during pancreatitis. The decreased level of IL-18 observed in this study shows that MEEO modulates the inflammation induced by EtOH and Cer.

17

Conclusions The present investigation, thus, establishes the pancreato protective effect of MEEO in rats administered with EtOH and Cer. MEEO modulates the changes induced by EtOH and Cer by minimizing MPO activity, cytokine production and collagen deposition and also by maintaining the antioxidant status in the glandular organ. The presence of rutin may be accounted for the anti-inflammatory nature of E. officinalis. Acknowledgments: The authors acknowledge the help rendered by Dr P. Jayaraman, Director, National institute of Herbal Science, Plant Anatomy Research Centre, Tambaram, Chennai, for having authenticated the source of test material.

Conflict of interest statement Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Received June 26, 2013; accepted January 13, 2013; previously published online February 7, 2014

References 1. Mews P, Phillips P, Fahmy R. Pancreatic stellate cells respond to inflammatory cytokines: potential role in chronic pancreatitis. Gut 2002;50:535–41. 2. Stevens T, Conwell DL, Zuccaro G. Pathogenesis of chronic pancreatitis: an evidence – based review of past theories and recent developments. Am J Gastroenterol 2004;99:2256–70. 3. Sims JE, Smith DE. The IL-1 family: regulators of immunity. Nat Rev Immunol 2010;10:89–102. 4. Norman JG, Fink GW, Denham W, Yang J, Carter G, Sexton C, et al. Tissue-specific cytokine production during experimental acute pancreatitis. A probable mechanism for distant organ dysfunction. Dig Dis Sci 1997;42:1783–8. 5. Dinarello CA. Interleukin-1 and interleukin-1 antagonism. Blood 1991;77:1625–52. 6. Church LD, Churchman SM, Hawkins PN, McDermott MF. Hereditary auto-inflammatory disorders and biologics. Springer Semin Immunopathol 2006;27:494–8.

7. Dinarello CA. Interleukin 1 and interleukin 18 as mediators of inflammation and the aging process. Am J Clin Nutr 2006;3:447–55. 8. Makhija R, Kingsnorth AN. Cytokine storm in acute pancreatitis. J Hepatobiliary Pancreat Surg 2002;9:401–10. 9. Lerch MM, Albrecht E, Ruthenburger M. Pathophysiology of alcohol-induced pancreatitis. Pancreas 2003;27:291–6. 10. Gukovsky I, Lugea A, Shahsahebi M, Cheng JH, Hong PP, Jung YJ, et al. A rat model reproducing key pathological responses of alcoholic chronic pancreatitis. Am J Physiol Gastrointest Liver Physiol 2008;1:68–79. 11. Luges A, Gong J, Nguyen J. Cholinergic mediation of alcoholinduced experimental pancreatitis. Alcohol Clin Exp Res 2010;34:1768–81. 12. Bhagat L, Singh VP, Song AM, van Acker GJ, Agrawal S, Steer ML, et al. Thermal stress-induced HSP70 mediates protection against intrapancreatic trypsinogen activation and acute pancreatitis in rats. Gastroenterology 2002;122:156–65.

Brought to you by | Georgetown University Authenticated | 10.248.254.158 Download Date | 9/9/14 7:36 PM

18

Aruna et al.: Anti-inflammatory effect of Emblica officinalis

13. Chan YC, Leung PS. Acute pancreatitis: animal models and recent advances in basic research. Pancreas 2007;34:1–14. 14. Yokozawa T, Kim HY, Kim HJ, Okubo T, Chu DC, Juneja LR. Amla (Emblica officinalis Geart.) prevents dyslipidaemia and oxidative stress in the ageing process. Br J Nutr 2007;97:1187–95. 15. Anila L, Vijayalakshmi NR. Flavonoids from Emblica officinalis and Mangifera indica – effectiveness for dyslipidemia. J Ethnopharmacol 2002;79:81–7. 16. Sabu MC, Kuttan R. Anti-diabetic activity of medicinal plants and its relationship with their antioxidant property. J Ethnopharmacol 2002;81:155–60. 17. Bandyopadhyay SK, Pakrashi SC, Pakrashi A. The role of antioxidant activity of Phyllanthus emblica fruit on prevention from indomethacin induced gastric ulcer. J Ethnopharmacol 2000;70:171–6. 18. Asmawi MZ, Kankaanranta H, Moilanen E, Vapaatalo H. Antiinflammatory activities of Emblica officinalis Geartn leaf extracts. J Pharm Pharmacol 1993;45:581–4. 19. Deng X. Chronic alcohol consumption accelerates fibrosis in response to cerulein-induced pancreatitis in rats. Am J Pathol 2005;166:93–6. 20. Lowry RR, Tinsley IJ. Rapid colorimetric determination of free fatty acids. J Am Oil Chem Soc 1976;53:470–2. 21. Gomori G. Assay of serum amylase with small amounts of serum. Am J Clin Pathol 1957;27:714–6. 22. Woessner JF, Taplin CJ. Purification and properties of a small latent matrix metalloproteinase of the rat uterus. J Biol Chem 1988;263:16918–25. 23. Charney J, Tomarelli RM. A colorimetric method for the determination of the proteolytic activity of duodenal juice. J Biol Chem 1947;171:501–5. 24. Thornberry NA, Bull HG, Calaycay JR, Chapman KT, Howard AD, Kostura MJ, et al. A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature 1992;356:768–74. 25. Bradley PP, Priebat DA, Christensen RD, Royhstein G. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J Invest Dermatol 1982;78:206–9. 26. Draper HH, Hadley M. Malondialdehyde determination as index of lipid peroxide. Methods Enzymol 1990;186:421–31. 27. Miyazawa T. Determination of phospholipid hydroperoxides in human blood plasma by a chemiluminescence-HPLC assay. Free Radic Biol Med 1989;7:209–17. 28. Miller NJ, Rice Evans CA, Davis MJ, Gopinathan V, Milner A. A novel method for measuring antioxidant capacity status in premature neonates. Clin Sci 1993;84:407–12. 29. Moran MS, Depierre JW, Mannervik B. Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochem Biophys Acta 1979;582:67–78. 30. Flohe L, Gunzler WA. Assays of glutathione peroxidase. Methods Enzymol 1984;105:114–21. 31. Kakkar P, Das B, Viswanathan PN. A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys 1984;21:130–2. 32. Aebi H. Catalase in vitro. Methods Enzymol 1984;105:121–6.

33. Patel SS, Goyal RK. Emblica officinalis Geart: a comprehensive review on phytochemistry, pharmacology and ethnomedicinal uses. Res J Med Plant 2012;6:6–16. 34. Lindahl M, Tagesson C. Flavonoids as phopholipase A2 inhibitors: importance of their structure for selective inhibition of group II phospholipase A2. Inflammation 1997;21:347–56. 35. Oomah BD, Mazza D. Flavonoids and antioxidative in buckwheat. J Agric Food Chem 1996;44:1746–50. 36. Metodiewa D, Kochman A, Karolczak S. Evidence for antiradical and antioxidant properties of four biologically active N, Ndiethylaminoethyl ethers of flavanone of oximes: a comparison with natural polyphenolic flavonoid (rutin) action. Biochem Mol Biol Int 1997;41:1067–75. 37. Frulloni L, Patrizi F, Bernardoni L, Cavallini G. Pancreatic hyperenzymemia: clinical significance and diagnostic approach. J Pancreas 2005;6:36–51. 38. Scheele G, Adler G, Kern H. Exocytosis occurs at the lateral plasma membrane of the pancreatic acinar cell during supramaximal secretagogue stimulation. Gastroenterology 1987;92:345–53. 39. Mc Gowan GK, Wills MR. The diagnostic value of faecal trypsin estimation in chronic pancreatic disease. J Clin Pathol 1962;15:62–8. 40. Shimizu K. Mechanisms of pancreatic fibrosis and applications to the treatment of chronic pancreatitis. J Gastroenterol 2008;43:823–32. 41. Apte MV, Wilson JS. Mechanisms of pancreatic fibrosis. Dig Dis 2004;22:273–9. 42. Charrier AL, Brigstock DR. Connective tissue growth factor production by activated pancreatic stellate cells in mouse alcoholic chronic pancreatitis. Lab Invest 2010;90:1179–88. 43. Berna MJ, Seiz O, Nast JF. CCK1 and CCK2 receptors are expressed on pancreatic stellate cells and induce collagen production. J Biol Chem 2010;285:385905–14. 44. Perides G, Tao X, West N, Sharma A, Steer ML. A mouse model of ethanol dependent pancreatic fibrosis. Gut 2005;54:1461–7. 45. Klebanoff SJ. Myeloperoxidase: friend and foe. J Leukoc Biol 2005;77:598–625. 46. Rotelli AE, Guardia T, Juarej AO, Rocha NE, Pelzer LE. Comparative study of flavonoids in experimental models of inflammation. Pharmacol Res 2003;48:601–6. 47. Teoh ML, Sun W, Smith BJ. Modulation of reactive oxygen species in pancreatic cancer. Clin Cancer Res 2007;13:7441– 50. 48. Paszkowski AS, Rau B, Mayer JM, Moller P, Beger HG. Therapeutic application of caspase1/interleukin-1β converting enzyme inhibitor decreases the death rate in severe acute experimental pancreatitis. Ann Surg 2002;235:68–76. 49. Martinon F, Burns K, Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol Cell 2002;10:417–26. 50. Dinarello CA, Wolff SM. The role of interleukin-1 in disease. N Engl J Med 1993;328:106–13. 51. Stutz A, Golenbock DT, Eicke L. Inflammasomes: too big to miss. J Clin Invest 2009;119:3502–11.

Brought to you by | Georgetown University Authenticated | 10.248.254.158 Download Date | 9/9/14 7:36 PM

Rutin rich Emblica officinalis Geart. fruit extract ameliorates inflammation in the pancreas of rats subjected to alcohol and cerulein administration.

The modulating effect of methanolic extract of Emblica officinalis (MEEO) on ethanol (EtOH)- and cerulein (Cer)-induced pancreatitis in rats was inves...
898KB Sizes 0 Downloads 0 Views