INTIMP-03384; No of Pages 6 International Immunopharmacology xxx (2014) xxx–xxx
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
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Lay-Jing Seow ⁎, Hooi-Kheng Beh, Muhammad Ihtisham Umar, Amirin Sadikun, Mohd Zaini Asmawi
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School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia
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Article history: Received 19 May 2014 Received in revised form 14 August 2014 Accepted 15 August 2014 Available online xxxx
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Keywords: Gynura segetum Antioxidant Anti-inflammatory Cotton pellet Pro-inflammatory cytokine Cyclooxygenase
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Anti-inflammatory and antioxidant activities of the methanol extract of Gynura segetum leaf
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Gynura segetum, family Compositae, is a cultivated species and can be found growing in the tropical regions of Indonesia and Malaysia. The plant is known for its use for the treatment of cancer, inflammation, diabetes, hypertension and skin afflictions. In the current study, in vivo anti-inflammatory effect of the methanol extract G. segetum leaf and its antioxidant effect in vitro have been investigated for the first time. The in vitro antioxidant activities of the methanol extract were measured using common methods including total phenolic content; total flavonoid content; scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and β-carotene bleaching assays. The in vivo anti-inflammatory activities were tested using the cotton pellet implanted animal model. The measurement of pro-inflammatory cytokine (TNF-α and IL-1) levels in the blood samples of the rats was carried out by using ELISA kits. The inhibitory activity on cyclooxygenase (COX) enzyme of methanol extract was also evaluated. The methanol extract exhibited good antioxidant activity which is associated with their total phenolic and flavonoid contents. Methanol extract strongly inhibited the granuloma tissue formation in rats and the antiinflammatory potential was mediated through the inhibition of pro-inflammatory cytokines and COX-2 enzyme activities. Taken together, the present study suggests that G. segetum's leaf is a natural source of antioxidants and has potential therapeutic benefits against chronic inflammation. © 2014 Published by Elsevier B.V.
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1. Introduction
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Inflammation is a complex biological response of vascular tissues to invasion by an infectious agent, antigen challenge, physical, chemical or traumatic damage [1]. The complex events and mediators involved in the inflammatory reactions can aggravate many diseases, such as rheumatoid arthritis, chronic asthma, multiple sclerosis, inflammatory bowel disease and psoriasis [2]. Non-steroidal anti-inflammatory drugs (NSAIDs) are often applied for treating several inflammatory diseases, but the side effects of these drugs include aspirin-induced asthma and disturbances of the gastrointestinal (GI) tract [3,4]. Therefore, new drugs that provide symptomatic relief without causing side effects are required. Medicinal plants are believed to be an important source for the discovery of potential anti-inflammatory substances. Several plant extracts and different classes of phytochemicals have been investigated and shown potential anti-inflammatory activity [5–8]. Gynura segetum (family Compositae) has drawn a lot of attention due to its uses in traditional medicine. The plant is known for its traditional use for the treatment of cancer, inflammation, diabetes, hypertension
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⁎ Corresponding author. Tel.: +60 4 6533888x4711; fax: +60 4 6570017. E-mail address: [email protected] (L.-J. Seow).
and skin afflictions [9]. Preliminary phytochemical screening revealed the presence of alkaloids, terpenes, flavonoids, tannins and saponins in leaves of G. segetum [10]. The presence of phenolic compounds in the plants indicates that this plant may be anti-microbial and this agreed with our previous findings [11]. Our previous study demonstrated that the leaf extracts of G. segetum reduced the growth of blood vessels and exhibited the potent antiangiogenic effect in the chick embryo chorioallantoic membrane (CAM) model. Anti-angiogenic activity of G. segetum provided a pharmacological basis for its folkloric use for the treatment of inflammatory diseases and cancer [12]. Preliminary study of anti-inflammatory effects of the leaf extracts of G. segetum was carried out by using a modified version of the chicken chorioallantoic membrane (HET-CAM) assay and the methanol extract showed good anti-inflammatory effects when compared with the anti-inflammatory drug indomethacin [13]. The aim of the present study was to investigate the antiinflammatory effect of methanol extract of G. segetum's leaf by studying the cotton pellet granuloma model in rats, inhibitory effects on proinflammatory cytokine generation and inhibition of cyclooxygenase activity. Evaluation of its antioxidant activity was undertaken to support its anti-inflammatory effects, as free radicals are known mediator of inflammation. Plant phenolic and flavonoid are known to act as powerful antioxidant; hence the total content in the plant extract was also carried out.
http://dx.doi.org/10.1016/j.intimp.2014.08.020 1567-5769/© 2014 Published by Elsevier B.V.
Please cite this article as: Seow L-J, et al, Anti-inflammatory and antioxidant activities of the methanol extract of Gynura segetum leaf, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.08.020
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2. Materials and methods
2.5. Animals
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2.1. Plant material and preparation of extract
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The leaves of G. segetum were collected from Jabatan Pertanian Relau, Penang. A voucher specimen (No. 11013) has been deposited at the herbarium of School of Biological Sciences, Universiti Sains Malaysia. The dried powdered leaves (500 g) were extracted successively with solvents of increasing polarity, namely petroleum ether (60–80 °C), chloroform and methanol by using soxhlet apparatus. Each extract was then filtered and evaporated in a rotary evaporator and stored in a refrigerator until use. The percentages of yield of petroleum ether, chloroform and methanol extracts were 6.2%, 7.4% and 10.4%, respectively. The methanol extract which was previously evaluated [13] for its preliminary anti-inflammatory activity was selected for further investigation for its effects by animal model and mechanisms of action.
Male Sprague Dawley rats weighing between 200 and 250 g were obtained from the Animal Research and Service Centre (ARSC), Universiti Sains Malaysia. The rats were housed in the animal transit room of School of Pharmaceutical Sciences, and maintained on a 12 hour light/dark cycle at room temperature (28 to 30 °C). The rats were allowed on acclimatization period of at least one week before starting the experiment. Animals were allowed free access to commercial food pellets (Gold Coin, Penang, Malaysia) and tap water during the acclimatization period. The experimental protocols and the use of animal were approved by the Animal Ethics Committee USM [No. of Animal Ethics Approval: USM/Animal Ethics Approval/2013/(90)(519)].
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2.2. Determination of total phenolic content
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The total phenolic contents of methanol extract of G. segetum's leaf were determined using Folin–Ciocalteu method and using gallic acid as a standard [14]. The concentration of phenolic compounds was calculated according to the following equation that was obtained from the standard gallic acid graph: 2 Absorbance ¼ 0:0105 gallic acid ðμ gÞ þ 0:0056 R ¼ 0:9998 :
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2.3. Determination of total flavonoid content
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2.4. Evaluation of antioxidant activity
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2.4.1. DPPH radical scavenging assay Free radical scavenging activity of methanol extract of G. segetum's leaf was evaluated by using the DPPH (1,1-diphenyl-2-picrylhydrazyl) method described by Kumaran and Karunakaran [16] with minor modifications. The test samples were dissolved in methanol and tested at five concentrations (100, 200,300, 400, 500 μg/mL). A solution of 0.004% DPPH in methanol was prepared and quercetin was used as a positive control. The DPPH radical scavenging activity was calculated using the following formula: ðAo−A1 Þ 100 Scavengingactivityð%Þ ¼ Ao
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where Ao is the absorbance of control (blank, without test sample) and A1 is the absorbance in the presence of the test samples or quercetin.
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2.4.2. Beta-carotene–linoleic acid assay The antioxidant activity of methanol extract of G. segetum's leaf was carried out using the beta-carotene–linoleic acid method adopted from the method of Gursoy et al. [15]. Quercetin was used as positive control while negative control was prepared by adding 0.5 mL of methanol to the mixture. The antioxidant activity was calculated as the following equation: Absorbance after 2 h of incubation 100%: Antioxidant activity ¼ Absorbance before incubation
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Anti-inflammatory effect of methanol extract of G. segetum's leaf was evaluated in three doses i.e. 125, 250 and 500 mg/kg by using cotton pellet granuloma in rats as used by Anosike et al. [1] with minor modifications. Rats were anesthetized by intra-peritoneal administration of pentobarbitone sodium (60 mg/kg). Two pouches were formed (one on either side of the ventral abdominal area of each rat under the loosed skin) and one autoclaved pre-weighted cotton pellet (30 mg) was aseptically implanted subcutaneously in each pouch. The pouches were stitched close thereafter using surgical silk. After 24 h of this implantation of cotton pellets, animals were given G. segetum's leaf extract dose once daily for 7 days through oral gavage. Reference group rats were given indomethacin (5 mg/kg) whereas negative control rats were given 1% Tween 80. On the 8th day, rats were anesthetized with pentobarbitone sodium (60 mg/kg) and 3 mL of blood was collected from each rat through cardiac puncture. The animals were sacrificed by giving an overdose of CO2 inhalation and the cotton pellets were dissected out from each rat. The pellets were dried in an oven at 40 °C until a constant weight was achieved. The weight of each cotton pellet was recorded and the percent inhibition of granuloma tissue formation was calculated by using the following formula: % inhibition of granuloma tissue formation ¼
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Total flavonoid content of methanol extract of G. segetum's leaf was carried out using the method described by Gursoy et al. [15] by the addition of aluminium chloride reagent to test samples (1000 μg/mL). A sample consisting of a 1 mL extract solution with 1 mL methanol without aluminium trichloride was used as control. The concentration of flavonoid compounds was calculated according to the following equation that was obtained from the standard quercetin graph: 2 Absorbance ¼ 0:0205 quercetin ðμ gÞ þ 0:0346 R ¼ 0:9980 :
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2.6. Cotton pellet granuloma assay
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ðTc−TtÞ 100 Tc
where Tc is the weight of cotton pellets of the control group and Tt is the 165 weight of cotton pellets of the treatment group. 2.7. Enzyme-linked immunosorbent assay (ELISA)
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2.7.1. Pro-inflammatory cytokines The blood samples collected from rats on the 8th day of cotton pellet assay were centrifuged to get plasma that was tested for TNF-α and IL-1. The measurement of TNF-α and IL-1 was carried out by using rat TNF-α (Catalog No. CSB-E11987r) and rat IL-1 (Catalog No. CSB-E08064r) ELISA kits, according to the manufacturer's instructions (CUSABIO, China). The concentration of respective cytokine in each sample was calculated by standard curve equation. Percent inhibition of cytokine production was calculated by using the following formula:
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% inhibition of cytokine production ¼
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ðODc−ODtÞ 100 ODc
where ODt is the optical density of the treatment group samples and 178 ODc is the optical density of the control group samples. 2.7.2. Cyclooxygenase assay 179 The ability of the methanol extract to inhibit the cyclooxygenase 180 (COX-1 and COX-2) was determined by using a COX inhibitor screening 181
Please cite this article as: Seow L-J, et al, Anti-inflammatory and antioxidant activities of the methanol extract of Gynura segetum leaf, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.08.020
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3. Results
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3.1. Total phenolic and flavonoid contents
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In the present study, the total phenolic and flavonoid contents of methanol extract were determined using spectrophotometric methods. The phenolic and flavonoid contents in methanol extract were found to be 71.0 ± 0.6 μg/mL gallic acid equivalent and 35.8 ± 0.3 μg/mL quercetin equivalent, respectively.
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3.2. DPPH (1,1-diphenyl-2-picrylhydrazyl) radical-scavenging assay
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The DPPH free radical scavenging activity of methanol extract of G. segetum's leaves was tested at five different concentrations (100, 200, 300, 400 and 500 μg/mL) and the results were summarized in Table 1. Results showed that the scavenging activity was concentrationdependent and statistically significant (p b 0.05).
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3.3. Beta-carotene–linoleic acid assay
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In this study, methanol extract of G. segetum's leaves showed the ability to prevent the bleaching of beta-carotene with inhibition more than 80%. As can be seen from Table 1, at 250 μg/mL the methanol extract exhibited similar antioxidant activity (80.4 ± 0.2%) to the standard quercetin (94.4 ± 0.0%). Inhibition by methanol extract was statistically significant (p b 0.01) when compared with the control.
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DPPH scavenging activity (percentage inhibition of test sample)
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3.5.2. Cyclooxygenase assay The inhibitory activity on COX enzyme of methanol extract of G. segetum was evaluated using COX inhibitor screening assay kit (Cayman Catalog No. 560131). Aspirin and celecoxib at 200 μg/mL were tested as non-selective COX inhibitor and selective COX-2 inhibitor references, respectively. The inhibitory activity of methanol extract was performed at 200 and 50 μg/mL and the percentage inhibition of the COX-1 and COX-2 was determined. As shown in Fig. 3, methanol extract and positive control (aspirin and celecoxib) at 200 μg/mL showed significant inhibition as compared with the control (p b 0.001). Aspirin abolished COX-1 activity. Inhibition values of COX-1 by methanol extract at 200 and 50 μg/mL were
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14.6 ± 0.4* 86.7 ± 0.3***
20.1 ± 0.4** 91.8 ± 0.1***
25.6 ± 1.0** 94.3 ± 0.1***
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Beta-carotene linoleic acid activity (percentage inhibition of test sample)
Methanol extract Quercetin
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3.5.1. Pro-inflammatory cytokines The measurement of TNF-α and IL-1 levels in the plasma samples of the rats implanted with cotton pellets was carried out by using rat TNFα and rat IL-1 ELISA kits (CUSABIO, China). The estimation of TNF-α and IL-1 levels demonstrated a significant decrease in circulating proinflammatory cytokine levels in the methanol extract treated group as compared to the control group. The percentage inhibition of TNF-α and IL-1 in rat plasma was significantly (p b 0.001) decreased in a dose-dependent manner as shown in Fig. 2. Indomethacin (5 mg/kg) treated group significantly lowered the TNF-α level by 77.4 ± 3.7% (p b 0.001). Methanol extract 500 mg/kg treated group exhibited a comparable inhibition with indomethacin reducing TNF-α levels by 72.2 ± 3.5%. Methanol extract in doses of 250 and 125 mg/kg showed significant reduction of TNF-α level by 62.9 ± 5.5% and 26.1 ± 1.4%, respectively compared to the control group (p b 0.001). Indomethacin (5 mg/kg) significantly inhibited the production of IL1 by 89.2 ± 1.9% (p b 0.001). The IL-1 inhibitory activity of methanol extract (500 mg/kg) was similar to indomethacin, with 91.8 ± 1.8% inhibition. A lower concentration of methanol extract (250 mg/kg) was also effective (p b 0.001) at inhibiting IL-1 production whereas the lowest concentration studied (125 mg/kg) was ineffective.
Table 1 The inhibitory effect of Gynura segetum leaves on DPPH scavenging activity and beta-carotene–linoleic acid activity. The results are shown in percent inhibition with respect to the control group. Data is presented as mean ± SD (n = 6). *, ** and *** represent the statistical significance between the treatment groups and the control with p b 0.05, p b 0.01 and p b 0.001 respectively.
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3.5. Enzyme-linked immunosorbent assay (ELISA)
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The results were expressed as means ± SD. Statistical significances of the raw data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey Post Hoc Multiple Comparison tests (SPSS version 20). p values less than 0.05 were considered to be significant.
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The anti-inflammatory effect of methanol extract of G. segetum's leaves was studied at the doses of 125, 250 and 500 mg/kg by using cotton pellet granuloma in rats and the results are summarized in Fig. 1. The methanol extract significantly (p b 0.001) and dose-dependently inhibited the formation of granuloma tissues compared with the control group. At the doses of 125, 250 and 500 mg/kg, methanol extract inhibited granuloma tissue formation by 17.1, 39.7 and 47.2%, respectively. The standard drug indomethacin showed 53.4% reduction in the weight of the granuloma tissues at 5 mg/kg oral dose.
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3.4. Cotton pellet granuloma assay
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assay kit (Cayman Chemical Company, Michigan, USA). By blocking cyclooxygenase you block prostaglandin production. Cyclooxygenase catalyzes the first step in the biosynthesis of arachidonic acid to PGH2. PGF2α produced from PGH2 by reduction with stannous chloride was quantified by using enzyme immunoassay according to the manufacturer's instructions. A standard curve was plotted and the level of prostaglandin was estimated in treatment and control wells by linear regression using Microsoft Excel Software (Version 2010). Percent inhibition of the COX enzymes was determined by comparing the extract loaded wells with the positive and negative controls.
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80.4 ± 0.3*** 94.4 ± 0.1***
83.4 ± 0.3*** 96.2 ± 0.2***
89.5 ± 0.6*** 98.7 ± 0.1***
Please cite this article as: Seow L-J, et al, Anti-inflammatory and antioxidant activities of the methanol extract of Gynura segetum leaf, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.08.020
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respectively. A lower concentration of methanol extract (50 μg/mL) did 267 not significantly inhibit COX-2 compared with the control group. 268 4. Discussion
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55.0 ± 0.6% and 1.8 ± 1.0%, respectively. Methanol extract (200 μg/mL) was more effective than celecoxib (200 μg/mL) as an inhibitor of COX-2. The extract and celecoxib inhibited COX-2 by 86.8 ± 1.5 and 43.3 ± 1.7%,
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Fig. 1. Inhibitory effect of Gynura segetum extract on granuloma tissue formation in rats. A shows images of cotton pellets dissected out from the rats on the 8th day of treatment with graded doses of methanol extract. B represents weights of dried cotton pellets that were taken out from the rats treated with extract (ME 125, 250 and 500 mg/kg), standard drug indomethacin (5 mg/kg) and control. The results are represented as mean ± SD (n = 6). *** shows statistical significance of the treatment groups with p b 0.001 when compared with control.
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Fig. 3. Inhibitory effect of standard drugs and methanol extract of Gynura segetum on the activities of cyclooxygenase (COX-1 and COX-2) enzymes in an in vitro colorimetric assay. The standard drugs (aspirin for COX-1; celecoxib for COX-2) were taken in the concentration of 200 μg/mL whereas the extract (ME) was tested in the concentrations of 50 and 200 μg/mL. The results are presented as mean ± SD (n = 3). *** presents a statistical significance of treatment groups with p b 0.001 when compared with control.
Fig. 2. Inhibitory effect of methanol extract of Gynura segetum on the levels of tumor necrosis factor alpha (TNF- ) and interleukin-1 (IL-1) in the plasma samples of rats treated with 125, 250 and 500 mg/kg of extract (treatment groups) and 5 mg/kg of indomethacin (reference group). The results are presented as mean ± SD (n = 6). *** shows statistical difference between the treatment groups and control at a significance of p b 0.001.
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4.1. Antioxidant activities of methanol extract of G. segetum's leaves
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Phenolic compounds are a large class of plant secondary metabolites, which are constituted in a large number of heterogeneous structures that range from simple molecules (e.g. phenolic acids) to highly polymerized compounds (e.g. flavonoid) [17]. Flavonoids are the most widely distributed phenolic phytoconstituents that are known to be the most potent antioxidants from herbs. The antioxidant activity of flavonoids is related to the presence of a number of phenolic hydroxyl groups which are attached to the ring structures [17]. The antioxidant activity of phenolic compounds are mainly because of their redox potential, which allows them to act as reducing agents, hydrogen donators, singlet oxygen quenchers and metal chelators [18]. Therefore, it would be valuable to determine the total phenolic and flavonoid contents of the plant extracts in order to estimate the antioxidant potential of these extracts. Antioxidants affect the process of lipid oxidation at different stages due to the differences in their mode of action [19]. Therefore, it is crucial to implement more than one antioxidant method specific to their chemical properties to evaluate the antioxidant activity of a natural product. In this study, the antioxidant activities of methanol extract of G. segetum's leaves have been determined by two different methods: DPPH radical-scavenging assay based on electron transfer reaction and beta-carotene–linoleic acid based on hydrogen atom transfer reaction [20]. To the best of our knowledge, this is the first report of the antioxidant activity of G. segetum leaf extract. The free radical scavenging DPPH assay is widely used to evaluate antioxidant activity of plants, due to its stability, simplicity, sensitivity and reproducibility [21]. DPPH is a stable free radical and presents the ability of accepting an electron or hydrogen radical [22]. In this assay, the antioxidants were able to reduce the purple-colored stable radical DPPH to the yellow-colored DPPH-H (diphenyl-picrylhydrazine) [16, 23,24]. The degree of color change is proportional to the concentration and potency of the antioxidants in plant extracts [25]. In the present study, methanol extract showed significant scavenging ability and this
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Please cite this article as: Seow L-J, et al, Anti-inflammatory and antioxidant activities of the methanol extract of Gynura segetum leaf, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.08.020
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5. Conclusions
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In conclusion, the results suggest that G. segetum's leaf is a natural source of antioxidants and could have potential therapeutic benefits against chronic inflammation. These findings provide a scientific basis for the traditional use of this plant for treating inflammatory disorders. The methanol extract of G. segetum leaf requires further study in order to identify the active constituents and clarify its mechanism of action.
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Alternative medicine for the treatment of inflammation is getting more popular [5]. Plant based drugs have become the focus of current research due to the great therapeutic potential without many of the side effects associated with synthetic drugs [1]. G. segetum is traditionally used in inflammatory disorders, but to the best of our knowledge, there is a lack of scientific data about the use of this plant in inflammation. The results from our previous study suggested that the antiinflammatory activity of methanol extract may be attributed to reduction of inflammatory cytokine expression and ROS production in monocytes and macrophages [13,28]. Pro-inflammatory macrophages participate in the formation of granulomas, inflammatory lesions that may be associated with and/or reflect chronic inflammation [29]. The activated pro-inflammatory macrophages contribute to the exacerbation of inflammation by producing and releasing more than 100 substances, including pro-inflammatory cytokines such as IL-1β and prostaglandins [30]. However, there is no evidence that the granuloma formed in response to SDS (sodium dodecyl sulfate) irritation in our previous study predominantly consists of macrophages [13,28]. Therefore, in this study, the anti-inflammatory effect of the methanol extract was investigated using the cotton pellet granuloma assay. Cotton pellet granuloma assay is a reliable in vivo model for studying chronic inflammation and the proliferative phase of inflammation [31, 32]. Inflammatory events occurring during the implant of cotton pellet involve the proliferation of macrophages, neutrophils and fibroblasts [33]. Non-steroidal anti-inflammatory drugs such as indomethacin produce the inhibitory effect in cotton pellet granuloma assay by inhibiting granulocyte infiltration to foreign body (cotton pellet), preventing the generation of collagen fibers and suppression of mucopolysaccharide [1]. Methanol extract significantly reduced the formation of the cotton pellet granuloma compared to the control group, suggesting that methanol extract is a natural anti-proliferative agent of granulation tissue formation and has the potential to treat chronic inflammation. A wide variety of mediators (e.g. nitric oxide, eicosanoids and proinflammatory cytokines) have been demonstrated to be critically involved in the development of inflammatory diseases [34]. Most of the phytochemicals that are known to exert anti-inflammatory effects
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4.2. Anti-inflammatory activity of methanol extract of G. segetum's leaves
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have been studied extensively for their potential to inhibit proinflammatory cytokines [35,36]. Methanol extract of G. segetum leaf has been shown to possess good anti-inflammatory potential, but its mechanism of action has not been evaluated. Thus, the potential inhibitory effects of methanol extract on cytokines (TNF-α and IL-1) and cyclooxygenase enzyme were investigated. Tumor necrosis factor α (TNF-α) is a pleiotropic inflammatory cytokine produced by the immune system that suppresses tumor cell proliferation and a key mediator of inflammation [37,38]. Interleukin1 (IL-1) is a prototypical pro-inflammatory cytokine that stimulates both local and systemic responses, also known as endogenous pyrogen, leukocyte endogenous mediator, mononuclear cell factor and lymphocyte activating factor [39]. Treatment with methanol extract produced a significant reduction in the concentrations of TNF-α and IL-1 in the blood samples of the rats used in the cotton pellet assay as compared to the control. The inhibition of TNF-α and IL-1 levels in macrophages suggests that methanol extract exerts its anti-inflammatory effects by suppressing the ability of macrophages to produce pro-inflammatory mediators. Both TNF-α and IL-1 synergistically are involved in chronic inflammatory reactions through induction of COX-2 expression in activated immune cells [38]. PGE2 is produced by COX-2 at sites of inflammation, whereas the constitutive isoform, COX-1, is relevant in the production of prostaglandins that regulate normal cellular processes [34]. Nonsteroidal anti-inflammatory drugs (NSAIDs) act by inhibiting COX-1 and COX-2 enzymes, thereby reducing the production of prostaglandins, resulting in their anti-inflammatory effects. The major adverse effect of the non-selective NSAIDs resulting from inhibition of COX-1 is injury to the upper gastrointestinal tract (ulcers and bleeding). COX-2 selective inhibitors are associated with fewer upper gastrointestinal tract disturbances [41]. Selective inhibition of COX-2 has been suggested to be an approach for treating inflammatory disorders [42]. Consequently, the main purpose of developing COX-2 selective inhibitors is not to increase potency, but to increase therapeutic safety. The present study found that the methanol extract was comparatively more effective against COX-2 (86.8% inhibition) than COX-1 (55.0%) and comparatively more effective than celecoxib (selective COX-2 inhibitor) as an inhibitor of COX-2. These results suggest that the reduction of COX-2 activity is another potential mechanism responsible for the anti-inflammatory activity of methanol extract of G. segetum.
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result suggests that the methanol extract contained phytochemical constituents that are capable of donating hydrogen to a free radical to remove the extra electron [24,25]. Peroxidation inhibition by hydrogen atom donation is the underlying basis of beta-carotene–linoleic acid assay [14]. In this test, betacarotene underwent the rapid discoloration in the absence of an antioxidant. This is because of the coupled oxidation of beta and linoleic acid, which generates free radicals [16]. The oxidation of linoleic acid generates peroxyl free radicals due to the abstraction of a hydrogen atom from diallylic methylene groups of linoleic acid [26]. The free radical will then attack the highly unsaturated beta-carotene molecules. This process leads to the loss of conjugation in the beta-carotene molecule and this is accompanied by the loss of the characteristic orange color of carotenoids; this process was monitored spectrophometrically [27]. The presence of antioxidants in the plant extracts will minimize the oxidation of beta-carotene by neutralizing the linoleate free radical and other free radicals formed in system, and the orange color was retained for a long time [15]. As reported by Safaei-Ghomi et al. [14], the compound containing hydrogen atom in the allylic and or benzylic positions will show better activity because of relatively easy abstraction of atomic hydrogen from the functional groups by peroxy radicals formed in the test circumstance. In this study, methanol extract showed pronounced ability to prevent the bleaching of beta-carotene (N80% inhibition). Therefore, the high antioxidant activity of the methanol extract may be a consequence of the presence of allylic/benzylic groups.
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Acknowledgment
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The authors acknowledge the financial support given by the Universiti Sains Malaysia (USM). The authors also thank the School of Pharmaceutical Sciences, USM for providing the required facilities and excellent technical assistance. The first author was supported by MyBrain 15 (MyPhD) scholarship sponsored by the Ministry of Higher Education (MOHE) Malaysia.
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Please cite this article as: Seow L-J, et al, Anti-inflammatory and antioxidant activities of the methanol extract of Gynura segetum leaf, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.08.020
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Please cite this article as: Seow L-J, et al, Anti-inflammatory and antioxidant activities of the methanol extract of Gynura segetum leaf, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.08.020
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Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
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Lay-Jing Seow ⁎, Hooi-Kheng Beh, Muhammad Ihtisham Umar, Amirin Sadikun, Mohd Zaini Asmawi
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School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia
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Article history: Received 19 May 2014 Received in revised form 14 August 2014 Accepted 15 August 2014 Available online xxxx
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Keywords: Gynura segetum Antioxidant Anti-inflammatory Cotton pellet Pro-inflammatory cytokine Cyclooxygenase
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Anti-inflammatory and antioxidant activities of the methanol extract of Gynura segetum leaf
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Gynura segetum, family Compositae, is a cultivated species and can be found growing in the tropical regions of Indonesia and Malaysia. The plant is known for its use for the treatment of cancer, inflammation, diabetes, hypertension and skin afflictions. In the current study, in vivo anti-inflammatory effect of the methanol extract G. segetum leaf and its antioxidant effect in vitro have been investigated for the first time. The in vitro antioxidant activities of the methanol extract were measured using common methods including total phenolic content; total flavonoid content; scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and β-carotene bleaching assays. The in vivo anti-inflammatory activities were tested using the cotton pellet implanted animal model. The measurement of pro-inflammatory cytokine (TNF-α and IL-1) levels in the blood samples of the rats was carried out by using ELISA kits. The inhibitory activity on cyclooxygenase (COX) enzyme of methanol extract was also evaluated. The methanol extract exhibited good antioxidant activity which is associated with their total phenolic and flavonoid contents. Methanol extract strongly inhibited the granuloma tissue formation in rats and the antiinflammatory potential was mediated through the inhibition of pro-inflammatory cytokines and COX-2 enzyme activities. Taken together, the present study suggests that G. segetum's leaf is a natural source of antioxidants and has potential therapeutic benefits against chronic inflammation. © 2014 Published by Elsevier B.V.
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1. Introduction
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Inflammation is a complex biological response of vascular tissues to invasion by an infectious agent, antigen challenge, physical, chemical or traumatic damage [1]. The complex events and mediators involved in the inflammatory reactions can aggravate many diseases, such as rheumatoid arthritis, chronic asthma, multiple sclerosis, inflammatory bowel disease and psoriasis [2]. Non-steroidal anti-inflammatory drugs (NSAIDs) are often applied for treating several inflammatory diseases, but the side effects of these drugs include aspirin-induced asthma and disturbances of the gastrointestinal (GI) tract [3,4]. Therefore, new drugs that provide symptomatic relief without causing side effects are required. Medicinal plants are believed to be an important source for the discovery of potential anti-inflammatory substances. Several plant extracts and different classes of phytochemicals have been investigated and shown potential anti-inflammatory activity [5–8]. Gynura segetum (family Compositae) has drawn a lot of attention due to its uses in traditional medicine. The plant is known for its traditional use for the treatment of cancer, inflammation, diabetes, hypertension
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⁎ Corresponding author. Tel.: +60 4 6533888x4711; fax: +60 4 6570017. E-mail address: [email protected] (L.-J. Seow).
and skin afflictions [9]. Preliminary phytochemical screening revealed the presence of alkaloids, terpenes, flavonoids, tannins and saponins in leaves of G. segetum [10]. The presence of phenolic compounds in the plants indicates that this plant may be anti-microbial and this agreed with our previous findings [11]. Our previous study demonstrated that the leaf extracts of G. segetum reduced the growth of blood vessels and exhibited the potent antiangiogenic effect in the chick embryo chorioallantoic membrane (CAM) model. Anti-angiogenic activity of G. segetum provided a pharmacological basis for its folkloric use for the treatment of inflammatory diseases and cancer [12]. Preliminary study of anti-inflammatory effects of the leaf extracts of G. segetum was carried out by using a modified version of the chicken chorioallantoic membrane (HET-CAM) assay and the methanol extract showed good anti-inflammatory effects when compared with the anti-inflammatory drug indomethacin [13]. The aim of the present study was to investigate the antiinflammatory effect of methanol extract of G. segetum's leaf by studying the cotton pellet granuloma model in rats, inhibitory effects on proinflammatory cytokine generation and inhibition of cyclooxygenase activity. Evaluation of its antioxidant activity was undertaken to support its anti-inflammatory effects, as free radicals are known mediator of inflammation. Plant phenolic and flavonoid are known to act as powerful antioxidant; hence the total content in the plant extract was also carried out.
http://dx.doi.org/10.1016/j.intimp.2014.08.020 1567-5769/© 2014 Published by Elsevier B.V.
Please cite this article as: Seow L-J, et al, Anti-inflammatory and antioxidant activities of the methanol extract of Gynura segetum leaf, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.08.020
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2. Materials and methods
2.5. Animals
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2.1. Plant material and preparation of extract
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The leaves of G. segetum were collected from Jabatan Pertanian Relau, Penang. A voucher specimen (No. 11013) has been deposited at the herbarium of School of Biological Sciences, Universiti Sains Malaysia. The dried powdered leaves (500 g) were extracted successively with solvents of increasing polarity, namely petroleum ether (60–80 °C), chloroform and methanol by using soxhlet apparatus. Each extract was then filtered and evaporated in a rotary evaporator and stored in a refrigerator until use. The percentages of yield of petroleum ether, chloroform and methanol extracts were 6.2%, 7.4% and 10.4%, respectively. The methanol extract which was previously evaluated [13] for its preliminary anti-inflammatory activity was selected for further investigation for its effects by animal model and mechanisms of action.
Male Sprague Dawley rats weighing between 200 and 250 g were obtained from the Animal Research and Service Centre (ARSC), Universiti Sains Malaysia. The rats were housed in the animal transit room of School of Pharmaceutical Sciences, and maintained on a 12 hour light/dark cycle at room temperature (28 to 30 °C). The rats were allowed on acclimatization period of at least one week before starting the experiment. Animals were allowed free access to commercial food pellets (Gold Coin, Penang, Malaysia) and tap water during the acclimatization period. The experimental protocols and the use of animal were approved by the Animal Ethics Committee USM [No. of Animal Ethics Approval: USM/Animal Ethics Approval/2013/(90)(519)].
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2.2. Determination of total phenolic content
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The total phenolic contents of methanol extract of G. segetum's leaf were determined using Folin–Ciocalteu method and using gallic acid as a standard [14]. The concentration of phenolic compounds was calculated according to the following equation that was obtained from the standard gallic acid graph: 2 Absorbance ¼ 0:0105 gallic acid ðμ gÞ þ 0:0056 R ¼ 0:9998 :
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2.3. Determination of total flavonoid content
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2.4. Evaluation of antioxidant activity
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2.4.1. DPPH radical scavenging assay Free radical scavenging activity of methanol extract of G. segetum's leaf was evaluated by using the DPPH (1,1-diphenyl-2-picrylhydrazyl) method described by Kumaran and Karunakaran [16] with minor modifications. The test samples were dissolved in methanol and tested at five concentrations (100, 200,300, 400, 500 μg/mL). A solution of 0.004% DPPH in methanol was prepared and quercetin was used as a positive control. The DPPH radical scavenging activity was calculated using the following formula: ðAo−A1 Þ 100 Scavengingactivityð%Þ ¼ Ao
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where Ao is the absorbance of control (blank, without test sample) and A1 is the absorbance in the presence of the test samples or quercetin.
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2.4.2. Beta-carotene–linoleic acid assay The antioxidant activity of methanol extract of G. segetum's leaf was carried out using the beta-carotene–linoleic acid method adopted from the method of Gursoy et al. [15]. Quercetin was used as positive control while negative control was prepared by adding 0.5 mL of methanol to the mixture. The antioxidant activity was calculated as the following equation: Absorbance after 2 h of incubation 100%: Antioxidant activity ¼ Absorbance before incubation
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Anti-inflammatory effect of methanol extract of G. segetum's leaf was evaluated in three doses i.e. 125, 250 and 500 mg/kg by using cotton pellet granuloma in rats as used by Anosike et al. [1] with minor modifications. Rats were anesthetized by intra-peritoneal administration of pentobarbitone sodium (60 mg/kg). Two pouches were formed (one on either side of the ventral abdominal area of each rat under the loosed skin) and one autoclaved pre-weighted cotton pellet (30 mg) was aseptically implanted subcutaneously in each pouch. The pouches were stitched close thereafter using surgical silk. After 24 h of this implantation of cotton pellets, animals were given G. segetum's leaf extract dose once daily for 7 days through oral gavage. Reference group rats were given indomethacin (5 mg/kg) whereas negative control rats were given 1% Tween 80. On the 8th day, rats were anesthetized with pentobarbitone sodium (60 mg/kg) and 3 mL of blood was collected from each rat through cardiac puncture. The animals were sacrificed by giving an overdose of CO2 inhalation and the cotton pellets were dissected out from each rat. The pellets were dried in an oven at 40 °C until a constant weight was achieved. The weight of each cotton pellet was recorded and the percent inhibition of granuloma tissue formation was calculated by using the following formula: % inhibition of granuloma tissue formation ¼
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Total flavonoid content of methanol extract of G. segetum's leaf was carried out using the method described by Gursoy et al. [15] by the addition of aluminium chloride reagent to test samples (1000 μg/mL). A sample consisting of a 1 mL extract solution with 1 mL methanol without aluminium trichloride was used as control. The concentration of flavonoid compounds was calculated according to the following equation that was obtained from the standard quercetin graph: 2 Absorbance ¼ 0:0205 quercetin ðμ gÞ þ 0:0346 R ¼ 0:9980 :
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ðTc−TtÞ 100 Tc
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2.7.1. Pro-inflammatory cytokines The blood samples collected from rats on the 8th day of cotton pellet assay were centrifuged to get plasma that was tested for TNF-α and IL-1. The measurement of TNF-α and IL-1 was carried out by using rat TNF-α (Catalog No. CSB-E11987r) and rat IL-1 (Catalog No. CSB-E08064r) ELISA kits, according to the manufacturer's instructions (CUSABIO, China). The concentration of respective cytokine in each sample was calculated by standard curve equation. Percent inhibition of cytokine production was calculated by using the following formula:
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% inhibition of cytokine production ¼
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ðODc−ODtÞ 100 ODc
where ODt is the optical density of the treatment group samples and 178 ODc is the optical density of the control group samples. 2.7.2. Cyclooxygenase assay 179 The ability of the methanol extract to inhibit the cyclooxygenase 180 (COX-1 and COX-2) was determined by using a COX inhibitor screening 181
Please cite this article as: Seow L-J, et al, Anti-inflammatory and antioxidant activities of the methanol extract of Gynura segetum leaf, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.08.020
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3. Results
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In the present study, the total phenolic and flavonoid contents of methanol extract were determined using spectrophotometric methods. The phenolic and flavonoid contents in methanol extract were found to be 71.0 ± 0.6 μg/mL gallic acid equivalent and 35.8 ± 0.3 μg/mL quercetin equivalent, respectively.
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The DPPH free radical scavenging activity of methanol extract of G. segetum's leaves was tested at five different concentrations (100, 200, 300, 400 and 500 μg/mL) and the results were summarized in Table 1. Results showed that the scavenging activity was concentrationdependent and statistically significant (p b 0.05).
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In this study, methanol extract of G. segetum's leaves showed the ability to prevent the bleaching of beta-carotene with inhibition more than 80%. As can be seen from Table 1, at 250 μg/mL the methanol extract exhibited similar antioxidant activity (80.4 ± 0.2%) to the standard quercetin (94.4 ± 0.0%). Inhibition by methanol extract was statistically significant (p b 0.01) when compared with the control.
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DPPH scavenging activity (percentage inhibition of test sample)
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3.5.2. Cyclooxygenase assay The inhibitory activity on COX enzyme of methanol extract of G. segetum was evaluated using COX inhibitor screening assay kit (Cayman Catalog No. 560131). Aspirin and celecoxib at 200 μg/mL were tested as non-selective COX inhibitor and selective COX-2 inhibitor references, respectively. The inhibitory activity of methanol extract was performed at 200 and 50 μg/mL and the percentage inhibition of the COX-1 and COX-2 was determined. As shown in Fig. 3, methanol extract and positive control (aspirin and celecoxib) at 200 μg/mL showed significant inhibition as compared with the control (p b 0.001). Aspirin abolished COX-1 activity. Inhibition values of COX-1 by methanol extract at 200 and 50 μg/mL were
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14.6 ± 0.4* 86.7 ± 0.3***
20.1 ± 0.4** 91.8 ± 0.1***
25.6 ± 1.0** 94.3 ± 0.1***
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Beta-carotene linoleic acid activity (percentage inhibition of test sample)
Methanol extract Quercetin
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3.5.1. Pro-inflammatory cytokines The measurement of TNF-α and IL-1 levels in the plasma samples of the rats implanted with cotton pellets was carried out by using rat TNFα and rat IL-1 ELISA kits (CUSABIO, China). The estimation of TNF-α and IL-1 levels demonstrated a significant decrease in circulating proinflammatory cytokine levels in the methanol extract treated group as compared to the control group. The percentage inhibition of TNF-α and IL-1 in rat plasma was significantly (p b 0.001) decreased in a dose-dependent manner as shown in Fig. 2. Indomethacin (5 mg/kg) treated group significantly lowered the TNF-α level by 77.4 ± 3.7% (p b 0.001). Methanol extract 500 mg/kg treated group exhibited a comparable inhibition with indomethacin reducing TNF-α levels by 72.2 ± 3.5%. Methanol extract in doses of 250 and 125 mg/kg showed significant reduction of TNF-α level by 62.9 ± 5.5% and 26.1 ± 1.4%, respectively compared to the control group (p b 0.001). Indomethacin (5 mg/kg) significantly inhibited the production of IL1 by 89.2 ± 1.9% (p b 0.001). The IL-1 inhibitory activity of methanol extract (500 mg/kg) was similar to indomethacin, with 91.8 ± 1.8% inhibition. A lower concentration of methanol extract (250 mg/kg) was also effective (p b 0.001) at inhibiting IL-1 production whereas the lowest concentration studied (125 mg/kg) was ineffective.
Table 1 The inhibitory effect of Gynura segetum leaves on DPPH scavenging activity and beta-carotene–linoleic acid activity. The results are shown in percent inhibition with respect to the control group. Data is presented as mean ± SD (n = 6). *, ** and *** represent the statistical significance between the treatment groups and the control with p b 0.05, p b 0.01 and p b 0.001 respectively.
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The results were expressed as means ± SD. Statistical significances of the raw data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey Post Hoc Multiple Comparison tests (SPSS version 20). p values less than 0.05 were considered to be significant.
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The anti-inflammatory effect of methanol extract of G. segetum's leaves was studied at the doses of 125, 250 and 500 mg/kg by using cotton pellet granuloma in rats and the results are summarized in Fig. 1. The methanol extract significantly (p b 0.001) and dose-dependently inhibited the formation of granuloma tissues compared with the control group. At the doses of 125, 250 and 500 mg/kg, methanol extract inhibited granuloma tissue formation by 17.1, 39.7 and 47.2%, respectively. The standard drug indomethacin showed 53.4% reduction in the weight of the granuloma tissues at 5 mg/kg oral dose.
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assay kit (Cayman Chemical Company, Michigan, USA). By blocking cyclooxygenase you block prostaglandin production. Cyclooxygenase catalyzes the first step in the biosynthesis of arachidonic acid to PGH2. PGF2α produced from PGH2 by reduction with stannous chloride was quantified by using enzyme immunoassay according to the manufacturer's instructions. A standard curve was plotted and the level of prostaglandin was estimated in treatment and control wells by linear regression using Microsoft Excel Software (Version 2010). Percent inhibition of the COX enzymes was determined by comparing the extract loaded wells with the positive and negative controls.
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80.4 ± 0.3*** 94.4 ± 0.1***
83.4 ± 0.3*** 96.2 ± 0.2***
89.5 ± 0.6*** 98.7 ± 0.1***
Please cite this article as: Seow L-J, et al, Anti-inflammatory and antioxidant activities of the methanol extract of Gynura segetum leaf, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.08.020
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55.0 ± 0.6% and 1.8 ± 1.0%, respectively. Methanol extract (200 μg/mL) was more effective than celecoxib (200 μg/mL) as an inhibitor of COX-2. The extract and celecoxib inhibited COX-2 by 86.8 ± 1.5 and 43.3 ± 1.7%,
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Fig. 1. Inhibitory effect of Gynura segetum extract on granuloma tissue formation in rats. A shows images of cotton pellets dissected out from the rats on the 8th day of treatment with graded doses of methanol extract. B represents weights of dried cotton pellets that were taken out from the rats treated with extract (ME 125, 250 and 500 mg/kg), standard drug indomethacin (5 mg/kg) and control. The results are represented as mean ± SD (n = 6). *** shows statistical significance of the treatment groups with p b 0.001 when compared with control.
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Fig. 3. Inhibitory effect of standard drugs and methanol extract of Gynura segetum on the activities of cyclooxygenase (COX-1 and COX-2) enzymes in an in vitro colorimetric assay. The standard drugs (aspirin for COX-1; celecoxib for COX-2) were taken in the concentration of 200 μg/mL whereas the extract (ME) was tested in the concentrations of 50 and 200 μg/mL. The results are presented as mean ± SD (n = 3). *** presents a statistical significance of treatment groups with p b 0.001 when compared with control.
Fig. 2. Inhibitory effect of methanol extract of Gynura segetum on the levels of tumor necrosis factor alpha (TNF- ) and interleukin-1 (IL-1) in the plasma samples of rats treated with 125, 250 and 500 mg/kg of extract (treatment groups) and 5 mg/kg of indomethacin (reference group). The results are presented as mean ± SD (n = 6). *** shows statistical difference between the treatment groups and control at a significance of p b 0.001.
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Phenolic compounds are a large class of plant secondary metabolites, which are constituted in a large number of heterogeneous structures that range from simple molecules (e.g. phenolic acids) to highly polymerized compounds (e.g. flavonoid) [17]. Flavonoids are the most widely distributed phenolic phytoconstituents that are known to be the most potent antioxidants from herbs. The antioxidant activity of flavonoids is related to the presence of a number of phenolic hydroxyl groups which are attached to the ring structures [17]. The antioxidant activity of phenolic compounds are mainly because of their redox potential, which allows them to act as reducing agents, hydrogen donators, singlet oxygen quenchers and metal chelators [18]. Therefore, it would be valuable to determine the total phenolic and flavonoid contents of the plant extracts in order to estimate the antioxidant potential of these extracts. Antioxidants affect the process of lipid oxidation at different stages due to the differences in their mode of action [19]. Therefore, it is crucial to implement more than one antioxidant method specific to their chemical properties to evaluate the antioxidant activity of a natural product. In this study, the antioxidant activities of methanol extract of G. segetum's leaves have been determined by two different methods: DPPH radical-scavenging assay based on electron transfer reaction and beta-carotene–linoleic acid based on hydrogen atom transfer reaction [20]. To the best of our knowledge, this is the first report of the antioxidant activity of G. segetum leaf extract. The free radical scavenging DPPH assay is widely used to evaluate antioxidant activity of plants, due to its stability, simplicity, sensitivity and reproducibility [21]. DPPH is a stable free radical and presents the ability of accepting an electron or hydrogen radical [22]. In this assay, the antioxidants were able to reduce the purple-colored stable radical DPPH to the yellow-colored DPPH-H (diphenyl-picrylhydrazine) [16, 23,24]. The degree of color change is proportional to the concentration and potency of the antioxidants in plant extracts [25]. In the present study, methanol extract showed significant scavenging ability and this
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In conclusion, the results suggest that G. segetum's leaf is a natural source of antioxidants and could have potential therapeutic benefits against chronic inflammation. These findings provide a scientific basis for the traditional use of this plant for treating inflammatory disorders. The methanol extract of G. segetum leaf requires further study in order to identify the active constituents and clarify its mechanism of action.
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Alternative medicine for the treatment of inflammation is getting more popular [5]. Plant based drugs have become the focus of current research due to the great therapeutic potential without many of the side effects associated with synthetic drugs [1]. G. segetum is traditionally used in inflammatory disorders, but to the best of our knowledge, there is a lack of scientific data about the use of this plant in inflammation. The results from our previous study suggested that the antiinflammatory activity of methanol extract may be attributed to reduction of inflammatory cytokine expression and ROS production in monocytes and macrophages [13,28]. Pro-inflammatory macrophages participate in the formation of granulomas, inflammatory lesions that may be associated with and/or reflect chronic inflammation [29]. The activated pro-inflammatory macrophages contribute to the exacerbation of inflammation by producing and releasing more than 100 substances, including pro-inflammatory cytokines such as IL-1β and prostaglandins [30]. However, there is no evidence that the granuloma formed in response to SDS (sodium dodecyl sulfate) irritation in our previous study predominantly consists of macrophages [13,28]. Therefore, in this study, the anti-inflammatory effect of the methanol extract was investigated using the cotton pellet granuloma assay. Cotton pellet granuloma assay is a reliable in vivo model for studying chronic inflammation and the proliferative phase of inflammation [31, 32]. Inflammatory events occurring during the implant of cotton pellet involve the proliferation of macrophages, neutrophils and fibroblasts [33]. Non-steroidal anti-inflammatory drugs such as indomethacin produce the inhibitory effect in cotton pellet granuloma assay by inhibiting granulocyte infiltration to foreign body (cotton pellet), preventing the generation of collagen fibers and suppression of mucopolysaccharide [1]. Methanol extract significantly reduced the formation of the cotton pellet granuloma compared to the control group, suggesting that methanol extract is a natural anti-proliferative agent of granulation tissue formation and has the potential to treat chronic inflammation. A wide variety of mediators (e.g. nitric oxide, eicosanoids and proinflammatory cytokines) have been demonstrated to be critically involved in the development of inflammatory diseases [34]. Most of the phytochemicals that are known to exert anti-inflammatory effects
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have been studied extensively for their potential to inhibit proinflammatory cytokines [35,36]. Methanol extract of G. segetum leaf has been shown to possess good anti-inflammatory potential, but its mechanism of action has not been evaluated. Thus, the potential inhibitory effects of methanol extract on cytokines (TNF-α and IL-1) and cyclooxygenase enzyme were investigated. Tumor necrosis factor α (TNF-α) is a pleiotropic inflammatory cytokine produced by the immune system that suppresses tumor cell proliferation and a key mediator of inflammation [37,38]. Interleukin1 (IL-1) is a prototypical pro-inflammatory cytokine that stimulates both local and systemic responses, also known as endogenous pyrogen, leukocyte endogenous mediator, mononuclear cell factor and lymphocyte activating factor [39]. Treatment with methanol extract produced a significant reduction in the concentrations of TNF-α and IL-1 in the blood samples of the rats used in the cotton pellet assay as compared to the control. The inhibition of TNF-α and IL-1 levels in macrophages suggests that methanol extract exerts its anti-inflammatory effects by suppressing the ability of macrophages to produce pro-inflammatory mediators. Both TNF-α and IL-1 synergistically are involved in chronic inflammatory reactions through induction of COX-2 expression in activated immune cells [38]. PGE2 is produced by COX-2 at sites of inflammation, whereas the constitutive isoform, COX-1, is relevant in the production of prostaglandins that regulate normal cellular processes [34]. Nonsteroidal anti-inflammatory drugs (NSAIDs) act by inhibiting COX-1 and COX-2 enzymes, thereby reducing the production of prostaglandins, resulting in their anti-inflammatory effects. The major adverse effect of the non-selective NSAIDs resulting from inhibition of COX-1 is injury to the upper gastrointestinal tract (ulcers and bleeding). COX-2 selective inhibitors are associated with fewer upper gastrointestinal tract disturbances [41]. Selective inhibition of COX-2 has been suggested to be an approach for treating inflammatory disorders [42]. Consequently, the main purpose of developing COX-2 selective inhibitors is not to increase potency, but to increase therapeutic safety. The present study found that the methanol extract was comparatively more effective against COX-2 (86.8% inhibition) than COX-1 (55.0%) and comparatively more effective than celecoxib (selective COX-2 inhibitor) as an inhibitor of COX-2. These results suggest that the reduction of COX-2 activity is another potential mechanism responsible for the anti-inflammatory activity of methanol extract of G. segetum.
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result suggests that the methanol extract contained phytochemical constituents that are capable of donating hydrogen to a free radical to remove the extra electron [24,25]. Peroxidation inhibition by hydrogen atom donation is the underlying basis of beta-carotene–linoleic acid assay [14]. In this test, betacarotene underwent the rapid discoloration in the absence of an antioxidant. This is because of the coupled oxidation of beta and linoleic acid, which generates free radicals [16]. The oxidation of linoleic acid generates peroxyl free radicals due to the abstraction of a hydrogen atom from diallylic methylene groups of linoleic acid [26]. The free radical will then attack the highly unsaturated beta-carotene molecules. This process leads to the loss of conjugation in the beta-carotene molecule and this is accompanied by the loss of the characteristic orange color of carotenoids; this process was monitored spectrophometrically [27]. The presence of antioxidants in the plant extracts will minimize the oxidation of beta-carotene by neutralizing the linoleate free radical and other free radicals formed in system, and the orange color was retained for a long time [15]. As reported by Safaei-Ghomi et al. [14], the compound containing hydrogen atom in the allylic and or benzylic positions will show better activity because of relatively easy abstraction of atomic hydrogen from the functional groups by peroxy radicals formed in the test circumstance. In this study, methanol extract showed pronounced ability to prevent the bleaching of beta-carotene (N80% inhibition). Therefore, the high antioxidant activity of the methanol extract may be a consequence of the presence of allylic/benzylic groups.
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Acknowledgment
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The authors acknowledge the financial support given by the Universiti Sains Malaysia (USM). The authors also thank the School of Pharmaceutical Sciences, USM for providing the required facilities and excellent technical assistance. The first author was supported by MyBrain 15 (MyPhD) scholarship sponsored by the Ministry of Higher Education (MOHE) Malaysia.
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