Anti-inflammatory activity of roots of Cichorium intybus due to its inhibitory effect on various cytokines and antioxidant activity.

BACKGROUND: Cichorium intybus L. commonly known as chicory is one of the important medicinal plants commonly used in Ayurvedic system of medicine. It is commonly used for the treatment of diseases involving a khapa and pitta doshas. Traditionally, C. intybus is used for the treatment of inflammatory conditions, but there are only few in vitro studies reporting the anti-inflammatory activity of roots of chicory.
OBJECTIVE: Evaluation of anti-inflammatory activity of roots of chicory and mechanisms involved in it using in vivo models of inflammation.
MATERIALS AND METHODS: Albino Wistar rats of either sex weighing 150-200 g were used. Ethanolic and aqueous extracts of roots of chicory were prepared with the help of Soxhlet's apparatus. The anti-inflammatory activity was studied using carrageenan-induced paw edema method and cotton pellet granuloma method. Levels of cytokines such as tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and IL-1 and activity of antioxidant enzymes such as catalase (CAT) and glutathione peroxidase (GPx) were estimated.
RESULTS: Chicory roots demonstrated significant dose-dependent decrease in paw edema in carrageenan-induced paw edema method. Chicory roots diminished the serum TNF-α, IL-6, and IL-1 levels. They also significantly attenuated the malonylaldehyde levels and increased the activities of CAT and GPx in paw tissue. Similarly, chicory roots demonstrated a significant decrease in granuloma formation in cotton pellet induced granuloma method.
CONCLUSION: Chicory roots possess anti-inflammatory activity, and this might be due to the inhibition of various cytokines, antioxidant effects, and their free radical scavenging activity.

INTRODUCTION

Cichorium intybus L. commonly known as chicory in English and kasani in Sanskrit belongs to the family Asteraceae. It is a biennial herb, erect, glandular with a tuberous taproot and rosette leaves.[1] Chicory is a root vegetable whose green leafy part is often used in cooking or salads. Its roots are the earliest known and commonly used raw materials for manufacturing of coffee substitutes. Around 90% of the coffee consumed in India contains Chicory.[2] It is one of the important medicinal plants commonly used in Ayurvedic system of medicine. According to Ayurvedic description, chicory has the properties of tikta (rasa), laghu and rooksha (gunas) and shita (virya). It is commonly used for the treatment of diseases involving khapa and pitta doshas.[3] In Ayurveda, it is mainly used for the treatment of liver disorders, menstrual disorders, fever, and inflammatory swellings.[4] Roots of C. intybus are a rich source of dietary fibers as they contain a high amount of inulin and oligosaccharides. They are reported to have bifidogenic property due to their rich inulin content.[5] These fibers also reported to possess anticarcinogenic and diuretic activities.[6] The whole plant has been reported to have hepatoprotective,[7] anti-diabetic,[8] antibacterial,[9] antioxidant,[10] and cardioprotective properties.[11] It is also reported that inulin-type fructans present in chicory roots regulate appetite and glucose metabolism through glucagon-like peptide-1.[12] However, there are very few in vitro studies reporting the anti-inflammatory activity of roots of the chicory.[13] Hence, this study was conducted for the evaluation of anti-inflammatory activity of roots of chicory and various mechanisms involved in it using in vivo acute and sub-acute models of inflammation.

MATERIALS AND METHODS

Plant material and extraction

The roots of C. intybus were collected from local fields from Aligarh and shade dried. The roots were identified and authenticated by Botanist, Department of Botany, Aligarh Muslim University and a voucher specimen was submitted. Roots were dried and powdered with the help of a mechanical grinder and 100 g of root powder was extracted separately with 300 ml of distilled water and ethanol for aqueous and ethanolic extract, respectively, with the help of Soxhlet's apparatus. The extracts were collected in Petri dishes and evaporated till dryness in an incubator. The yield obtained was 12.4% and 15.25% for aqueous and ethanolic extract, respectively. The extracts were sealed with aluminum foil and stored at 4°C for further experimental work.

Drugs and chemicals

Aspirin (Reckitt Benckiser, India), Propylene glycol (BDH, Mumbai), and Carrageenan (Sigma Chemicals, USA) were used in the study. The other chemicals used were of analytical grades manufactured by Merck Laboratories (Mumbai, India), Rat tumor necrosis factor alpha (TNF-α) Elisa Kit (Biomolecular Integrations), Rat interleukin 6 (IL-6) Elisa Kit (Koma Biotech, Korea), Rat IL-1α Elisa Kit (Boster Biological Technology, Ltd.).

Animals

Albino Wistar rats of either sex weighing (150–200 g) were procured from the Central Animal House, JNMC, Aligarh Muslim University. They were housed in polypropylene cages at ambient temperature (25 ± 2°C), relative humidity (55 ± 5%), and 12-h light-dark cycle. Animals had free access to standard pellet diet and water ad libitum. The study protocol was approved by the Institutional Animal Ethics Committee (Reg. no 401/CPCSEA).

Experimental design

Animals were divided into eight groups of six animals each. Group-1 served as control and was given normal saline 2 ml/kg, group-2 served as standard and was given aspirin (100 mg/kg), groups 3 and 4 were given ethanolic extract of chicory (EEC) at the dose of 300 and 500 mg/kg, respectively, and groups 5 and 6 were given aqueous extract of chicory (AEC) at doses of 300 and 500 mg/kg, respectively.

Carrageenan induced paw edema method

This is one of the most commonly employed methods for the screening of acute inflammation[14]. All the groups were treated with single dose of respective drug, and after 1 h of the administration of the drugs, acute inflammation was produced by sub plantar injection of 0.1 ml of freshly prepared 1% suspension of carrageenan in normal saline in the right hind paw of the rats. The paw was marked at the level of the lateral malleolus and was immersed every time up to this mark. The paw volumes were measured at 0 h, 1 h, 2 h, and 3 h after the carrageenan injection using digital plethysmometer (Orchid Scientific, India).

The percentage inhibition of paw edema at each time interval was calculated using the following formula:

Where,

Vo = Paw volume of test/control group at 0 h

Vt = Paw volume of test/control group at that particular time interval.

At the end of the experiment, rats were anesthetized, and right hind paw tissue was taken and homogenized with cold phosphate buffer solution 4 times of their volume. Then the homogenate was centrifuged, and the supernatant was obtained and stored at − 20°C for malonylaldehyde (MDA) and the antioxidant enzyme activity assays.

Cotton pellet induced granuloma method

This method is commonly used for the screening of sub-acute inflammation[15]. Under anesthesia with aseptic precautions two sterilized cotton pellets (10 mg) were implanted subcutaneously on either side of the lumbar region in each rat. The incisions were sutured by silk 2.0 sutures, and the wounds were sealed with betadine solution to prevent contamination. Bleeding was minimal, and the animals recovered within 5–10 min from the effect of anesthesia. All the groups were treated with drugs daily for 7 days including the day of implantation of pellets. On the 8th day the animals were anesthetized with ether, the cotton pellets were removed and dried at 60°C for 24 h. The dry weight of the granuloma was calculated by noting the difference in the dry weight of the cotton pellets recorded before and after implantation. The incisions were sutured by silk 2.0 sutures and sealed with betadine solution and animals were rehabilitated.

Percent inhibition was calculated using the following formula:

Where,

WC = Weight of the cotton pellets in control animal

WT = Weight of the cotton pellets in drug-treated animals.

Estimation of cytokines tumor necrosis factor alpha, interleukin 6 and interleukin 1 in serum

After completion of the carrageenan-induced paw edema experiment, the rats were anesthetized and blood samples were collected from the orbital sinus. The serum was separated by allowing blood to clot followed by centrifugation and was stored at − 20°C until use. TNF-α, IL-6, and IL-1 from each sample were measured in duplicate with highly sensitive rat TNF-α Elisa Kit (Biomolecular Integrations), rat IL-6 Elisa Kit (Koma Biotech), rat IL-1α Elisa Kit (Boster Biological Technology, Ltd.), respectively, specifically designed for rats, according to manufacturer's instructions.

Determination of tissue lipid peroxidation

Malonylaldehyde from Carrageenan-induced edema foot was evaluated by the thiobarbituric acid reacting substance method.[16] Briefly, at high temperature MDA reacted with thiobarbituric acid at acidic pH resulting in the formation of a red complex TBARS. The absorbance of TBARS was determined at 532 nm.

Determination of antioxidant enzyme activity

The glutathione peroxidase (GPx) enzyme, in the presence of H2O2, reduces H2O2 to water while catalyzing the reaction of glutathione (GSH) turning into oxidized glutathione (GSSG). The GSSG formed is reduced again to GSH by the glutathione reductase reaction using NADPH as the reducing substrate. There is a decrease in absorbance during the oxidation of NADPH to NADP+, and this is measured by a spectrophotometer at 340 nm for the calculation of the GPx activity.[17] The results were given as units/mg protein. Total catalase (CAT) activity estimation was done as follows; the reduction of 10 mM H2O2 20 mM of phosphate buffer (pH 7) was monitored by measuring the absorbance at 240 nm.[18] The activity was calculated using a molar absorption coefficient, and the enzyme activity was defined as nanomoles of dissipating hydrogen peroxide per milligram protein per minute. Protein concentration was measured by Lowry method[19] using bovine serum as a standard. The enzyme activities were expressed as units of enzyme activity per milligram of protein.

Histological examination

Under deep anesthesia biopsies of the paws were taken 3 h after the injection of Carrageenan. The tissue slices were fixed in 10% neutral-buffered formaldehyde, dehydrated by graded ethanol and were embedded in paraffin. 5 μm thick slices were sectioned and were stained with hematoxylin and eosin. All samples were observed and photographed with Olympus microscopy. Tissue slices were randomly chosen from carrageenan, aspirin, EEC, and AEC-treated (500 mg/kg) groups. The numbers of neutrophils were counted in each scope (400×) and average count from 5 scopes of every tissue slice was counted.

Statistical analysis

All the values are expressed as mean ± standard error of mean (n = 6). Statistical significance was calculated by one-way ANOVA followed by post hoc Dunnett's multiple comparison test. P <0.05 was considered to be statistically significant.

RESULTS

As shown in Table 1, aspirin (100 mg/kg) significantly decreased the paw edema at 1 h (P < 0.001) and 3 h (P < 0.001) after carrageenan injection compared to control and percentage inhibition of edema was 59.58% and 87.06% at 1 h and 3 h, respectively. Both EEC and AEC-treated groups demonstrated dose-dependent decrease in paw edema compared to control the group. EEC showed significant reduction in paw edema both at 1 h and 3 h after carrageenan injection compared to control and percentage inhibition of edema was 34.05% (P < 0.05) and 55.30% (P < 0.001) at 1 h and 3 h, respectively, with 300 mg/kg and was 44.69% (P < 0.001) and 62.36% (P < 0.001) at 1 h and 3 h, respectively, with 500 mg/kg. AEC also showed significant reduction in paw edema both at 1 h and 3 h after carrageenan injection compared to control and percentage inhibition of edema was 12.77% and 31.75% (P < 0.001) at 1 h and 3 h, respectively, with 300 mg/kg and was 21.28% (P < 0.01) and 42.36% (P < 0.001) at 1 h and 3 h, respectively, with 500 mg/kg. Histological examination of paw sections of rats treated with carrageenan revealed a significant increase in infiltration by neutrophils, tissue injury and accumulation of edema fluid [Figure 1a]. Groups treated with chicory root extracts showed a reduction in carrageenan-induced inflammatory response and there was significant decrease in number of neutrophils as compared to the carrageenan-treated group (P < 0.001) [Figures 1b, c, d and 2].

Table 1

Effect of chicory roots on carrageenan-induced paw edema method

Figure 1

Histological examination of paw sections after 3 h of carrageenan injection. (a) Control, (b) Aspirin group, (c) Aqueous extract group, (d) Ethanolic extract group

Figure 2

Effect of chicory roots on neutrophil infiltration in rat paw after carrageenan injection. Values are expressed as mean ± standard error of the mean. *Indicates P < 0.05, ** indicates P < 0.001 when compared to the control group. EEC: Ethanolic extract of chicory roots, AEC: Aqueous extract of chicory roots.

In cotton pellet induced granuloma method, aspirin 100 mg/kg significantly inhibited the granuloma formation by 70.68% (P < 0.001) when compared to control. Whereas EEC significantly inhibited the granuloma formation by 36.69% (P < 0.001) and 55.61% (P < 0.001) compared to control group, at the doses of 300 mg/kg and 500 mg/kg, respectively. AEC significantly decreased granuloma formation by 21.77% (P < 0.05) and 36.82% (P < 0.001) at the doses of 300 mg/kg and 500 mg/kg, respectively, when compared to control [Figure 3].

Figure 3

Effect of chicory roots on dry weight of cotton pellet granu-loma. Values are expressed as mean ± standard error of the mean. *Indicates P < 0.05, ** indicates P < 0.001 when compared to the control group. EEC: Ethanolic extract of chicory roots, AEC: Aqueous extract of chicory roots

As shown in Table 2, there was dose-dependent decrease in all the three serum cytokines (TNF, IL-6, IL-1) in EEC-treated groups at both 300 mg/kg (P < 0.05) and 500 mg/kg (P < 0.001) doses whereas in AEC-treated groups significant increase was seen only at 500 mg/kg (P < 0.05) dose. Furthermore, there was dose-dependent significant decrease in MDA levels in both EEC (P < 0.001) and AEC (P < 0.001) treated groups and also there was dose dependent significant increase in both the antioxidant enzyme activities (CAT and GPx) in all the groups treated with EEC (P < 0.001) and AEC (P < 0.001) [Table 3].

Table 2

Effects of chicory roots on serum cytokines in carrageenan-induced paw edema

Table 3

Effects of chicory on MDA, CAT, and GPx activities in carrageenan-induced paw edema

DISCUSSION

The molecular mechanism of the carrageenan-induced inflammation is well-characterized, and these models of inflammation are standard models of screening for anti-inflammatory activity of various experimental compounds. The early phase of carrageenan edema is related to the production of histamine, leukotrienes, and possibly cyclooxygenase products, while the delayed phase of the carrageenan-induced inflammatory response has been linked to neutrophil infiltration and the production of neutrophil-derived free radicals, such as hydrogen peroxide, superoxide, and OH radicals, as well as to the release of other neutrophil-derived mediators.[20] However, a reduction in paw swelling volume is a good index in determining the protective action of anti-inflammatory agents. In the present study, EEC showed a significant reduction of edema in both the phases of inflammation but the maximum reduction was observed in the second phase of inflammation (62.36%). Aqueous extract showed significant (P < 0.01) reduction of edema mainly in the second phase of inflammation. The effect of chicory roots lasted for 3 h similar to that of aspirin. The anti-inflammatory activity exhibited can be attributed to various phytochemicals like polyphenols, flavonoids, sterols, glycosides, tannins, and terpenoids reported in chicory roots.[2122] Flavonoids present in chicory roots are known to inhibit prostaglandin synthesis which are involved in acute inflammation.[23] Chicory roots are also rich in glycosides, sterols and polyphenols which have been reported to possess anti-inflammatory activity by decreasing various mediators of inflammation such as prostaglandins, NO, TNF-α IL-6, and IL-1.[24] TNF-α is an important inflammatory mediator, inducing immune response by activating T cells and macrophages and stimulating secretion of other inflammatory cytokines such as IL-6 and IL-1.[25] TNF-α is also a mediator in carrageenan-induced inflammatory response and induces further release of kinins and leukotrienes, which are suggested to play an important role in the maintenance inflammatory response.[26] In this study, we found that chicory roots decreased the levels of TNF-α IL-6 and IL-1 in serum after carrageenan injection. Furthermore, there was neutrophil infiltration and generation of neutrophils derived free radicals such as hydroxyl radicals, hydrogen peroxide, and superoxide and release of other neutrophil-derived mediators in carrageenan-induced inflammatory response. In the present study, the histopathology revealed marked decrease in the cellular infiltration by neutrophils and tissue damage. Lipid peroxidation is due to the attack of free radicals on lipids in cell membranes resulting in accumulation of MDA and inflammation. Lipid peroxidation not only serves as a marker of tissue damage in vivo but also has been recognized to be the inducer of inflammatory processes.[27] In this study, chicory roots not only exhibited radical scavenging capacity but also decreased carrageenan-induced lipid peroxidation. Excess reactive oxygen species tend to cause oxidative imbalance of the antioxidant system which may result in oxidative stress and inflammation. Given the importance of the oxidative status in the formation of edema, the anti-inflammatory effect exhibited by the drug in this model might be related to its antioxidant properties.[28] The antioxidant enzymes CAT and GPx plays a crucial role in scavenging H2O2 and hydroperoxide.[29] In this study, there was a significant increase in CAT and GPx activities and the anti-inflammatory effect of chicory roots may be due to elevated intracellular antioxidant enzyme activities and decreased oxidative stress in tissues. The cotton pellet granuloma method has been widely employed to assess the various components of sub-acute inflammation such as transudative, exudative, and proliferative phases. The increase in dry weight of the granuloma measures the proliferative phase due to monocyte infiltration and fibroblast proliferation that take place in chronic inflammation.[30] In this study, both EEC and AEC significantly decreased the dry weight of the granuloma when compared to the control group. The percentage inhibition for the ethanolic and aqueous extracts was highest at the dose of 500 mg/kg, that is, 55.61% and 36.82%, respectively (P < 0.001) [Figure 1]. This anti-inflammatory action may be due to the ability of chicory roots in reducing the number of fibroblasts and inhibiting the synthesis of collagen and mucopolysaccharide, which are natural proliferative agents of granulation tissue formation. The inhibition of production of proinflammatory cytokines, such as IL-1, IL-6 and TNF-α which are powerful chemotactic agents to macrophages and fibroblasts as seen in the study may be responsible for anti-inflammatory effect as discussed earlier.[31] In conclusion, our study suggests that chicory roots possess anti-inflammatory effect and its mechanisms may be attributed to the inhibition of various cytokines and their antioxidant effects due to increase in the activities of antioxidant enzymes and their effect on free radical scavenging.