Anti-inflammatory, Cyclooxygenase (COX)-2, COX-1 Inhibitory, and Free Radical Scavenging Effects of Rumex nepalensis

• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Kits, biochemicals, and chemicals
• Plant material
• Animals
• COX‐1 and COX-2 inhibition assay in vitro
• TPA-induced mouse ear edema assay
• DPPH radical scavenging assay
• ABTS radical scavenging assay
• Determination of total phenolic content
• Isolation of compounds from the ethyl acetate extract
• Statistical analysis
• Results and Discussion
• Acknowledgements
• References

Abstract

Evaluation of the topical anti-inflammatory activity of chloroform and ethyl acetate extracts of Rumex nepalensis roots in a TPA-induced acute inflammation mouse model demonstrated a significant reduction in ear edema. The extracts were further tested on purified enzymes for COX-1 and COX-2 inhibition to elucidate their mechanism of action, and a strong inhibition was observed. Six anthraquinones and two naphthalene derivatives were isolated from the ethyl acetate extract. Among the isolated compounds, emodin was found to be a potent inhibitor with slight selectivity towards COX-2, and nepodin exhibited selectivity towards COX-1. Emodin, endocrocin, and nepodin also exhibited significant topical anti-inflammatory activity in mice. Interestingly, nepodin showed better radical scavenging activity than trolox and ascorbic acid against DPPH and ABTS radicals. The strong radical scavenging activity of chloroform and ethyl acetate extracts could be explained by the presence of nepodin as well as by the high phenolic content of the ethyl acetate extract. Thus, the anti-inflammatory effect of R. nepalensis roots was assumed to be mediated through COX inhibition by anthraquinones and naphthalene derivatives and through the radical scavenging activities of naphthalene derivatives.

Abbreviations

ABTS: 2,2′-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid

AP-1: activator protein-1

COX: cyclooxygenase

DPPH: 1,1-diphenyl-2-picrylhydrazyl

EIA: enzyme immunoassay

NF-κB: nuclear factor-κB

PG: prostaglandin

TPA: 12-O-tetradecanoylphorbol-13-acetate

Trolox: 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid

Introduction

Uncontrolled inflammation often leads to the development of inflammatory diseases such as chronic asthma, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and multiple sclerosis. An inflammatory response implicates macrophages and neutrophils, which secrete a number of mediators (eicosanoids, oxidants, cytokines, and enzymes) responsible for the initiation, progression, and persistence of acute and chronic inflammation [1]. Within the inflammatory cascade, cytokines induce a wide variety of phlogistic responses, including upregulation of the enzyme cyclooxygenase (COX) [2]. COX is the rate-limiting enzyme involved in the conversion of arachidonic acid into prostaglandins. COX exists in two isoforms, COX-1 and COX-2. COX-1 is expressed constitutively in all tissues and is thus always present and active. COX-2 is predominantly present at the sites of inflammation, and it produces prostaglandins that cause swelling and pain [3]. Therefore, inhibitors of the COX-2 enzyme have the potential to be developed as an anti-inflammatory drug. Reactive oxygen species play a key role in enhancing inflammation through the activation of NF-κB and AP-1 transcription factors in various inflammatory diseases [4]. These undesirable effects of oxidative stress have been found to be controlled by the antioxidant and/or anti-inflammatory effects of compounds and plant extracts. Therefore, much attention has been given to characterize the antioxidant properties of plant extracts and identify the phytoconstituents responsible for these activities.

Rumex nepalensis Spreng. (Polygonaceae) is a tall, robust annual or perennial plant found in the temperate Himalayas from Kashmir to Bhutan and in the Western Ghats, Nilgiri, and Palni hills at altitudes between 1200 and 2700 m. The leaves are rubbed over the affected areas after injury from stinging nettles and are used to treat colic and syphilic ulcers. The roots are used to cure dysentery and as a purgative [5]. The fresh, young leaves of this plant are boiled or fried in cooking oil with spices as a vegetable [6]. R. nepalensis roots are reported to exhibit antipyretic activity in rats [7] and psychopharmacological activities in rats and mice [8]. The extracts of R. nepalensis leaves are also reported to relieve itching and pain due to stinging nettles, and they exert significant effects on wheal formation induced by histamine, acetylcholine, bradykinin, and prostaglandins [9]. Considering this ethnomedicinal information as well as the results of our preliminary screening of the ethyl acetate extract [10], we aimed to investigate the anti-inflammatory activity, COX-1 and COX-2 inhibitory activity, and free radical scavenging effects of R. nepalensis roots.

Materials and Methods

Kits, biochemicals, and chemicals

The EIA kit (catalog no. 560101) for the COX (ovine) inhibitor screening assay was purchased from Cayman Chemicals. TPA, indomethacin (> 98 %), DPPH, trolox (> 97 %), and ABTS were obtained from Sigma. Celecoxib (> 99 %) was obtained as a gift sample from the Department of Pharmaceutical Technology (Formulation), NIPER. L-Ascorbic acid (> 99 %) from Spectrochem Pvt., Ltd., curcumin (99 %) from Loba Chemie, Folin−Ciocalteu reagent from S. D. Fine Chemicals, and Sephadex LH-20 from Amersham Pharmacia were purchased. The purity of the isolated compounds was determined by HPLC as follows: chrysophanol, > 99 %; physcion, > 95 %; emodin, > 96 %; chrysophanol-8-O-β-D-glucopyranoside, > 95 %; emodin-8-O-β-D-glucopyranoside, > 98 %; endocrocin, > 97 %; nepodin, > 99 %; and nepodin-8-O-β-D-glucopyranoside, > 99 %.

Plant material

The roots of Rumex nepalensis were collected from the adjoining areas of Badrinath (Uttarakhand), India, in August 2005. The roots were identified by Dr. Arvind Saklani (botanist and ex-Assistant Professor, Dept. of Natural Products, NIPER, SAS Nagar), and a voucher specimen (NIP-130) was deposited in the herbarium of the Department of Natural Products, NIPER, SAS Nagar, Punjab, India. The shade-dried, coarsely powdered roots (2 kg) were successively extracted in a Soxhlet apparatus with n-hexane, chloroform, ethyl acetate, and methanol solvents, and these extracts were concentrated to dryness in vacuo on a rotary evaporator to give n-hexane extract (yield: 34.9 g, 1.74 %), chloroform extract (yield: 21.5 g, 1.07 %), ethyl acetate extract (yield: 45.8 g, 2.29 %), and methanol extract (yield: 106.4 g, 5.32 %).

Animals

Female Swiss albino mice with a body weight of 25–30 g were procured from the central animal facility at NIPER. The animals were maintained under controlled room temperature (22 ± 2 °C) and humidity (55 ± 5 %) with a 12-h light/dark cycle. All the animals were housed in polypropylene cages in groups of six per cage and provided with standard pellet diet and water ad libitum. The protocol of this experiment was approved by the Institute Animal Ethics Committee (IAEC), and the experiments were carried out in accordance with the guidelines of CPCSEA for animal experimentation.

COX‐1 and COX-2 inhibition assay in vitro

The ability of the extracts and compounds to inhibit COX-1 and COX-2 was determined by using an EIA kit according to the manufacturer's instructions; the same kit was used in our recent study on COX-1 and COX-2 inhibitory activity [11]. COX-1 and COX-2 catalyze the biosynthesis of prostaglandin (PG) H₂ from arachidonic acid, and PGF₂ α produced from PGH₂ by reduction with stannous chloride is measured by an acetylcholinesterase-competitive EIA kit. The stock solutions of the extracts and compounds were dissolved in absolute ethanol, and their final concentrations were 50 µg/mL and 30 µM, respectively. The percentage of inhibition was calculated by comparison of compound-treated and control incubations. The concentration of the test compounds causing 50 % inhibition (IC₅₀, µM) was calculated from the concentration−inhibition response curves. Naturally occurring curcumin, a well-known anti-inflammatory agent, along with clinically used indomethacin and celecoxib were used as reference standards.

TPA-induced mouse ear edema assay

The topical anti-inflammatory activities of extracts and compounds were evaluated by the method described previously [11], [12]. For the experiment, animals were divided into groups of six per cage (n = 6). To the control group, only TPA (2.5 µg dissolved in 20 µL acetone) was applied topically to the right ear on both sides of the ear surface with a micropipette. In the treatment groups, TPA and extracts or compounds were applied simultaneously to the right ear. Indomethacin at a dose of 0.5 mg/ear was used as a standard. Ear thickness was measured before (T_o) and after (T_t) induction of inflammation using a pair of digital calipers (Mutitoyo). The maximum edema was reached 4 h after application of TPA. The edema was expressed as an increase in ear thickness due to TPA application (T_t–T_o), and edema inhibition was expressed as the percentage of thickness reduction compared with the control group.

DPPH radical scavenging assay

Free radical scavenging activity of extracts and isolated compounds was determined by the widely used DPPH assay [13]. Trolox and L-ascorbic acid were used as positive controls. The IC₅₀ parameter is defined as the concentration of the substrate that causes a 50 % reduction in DPPH activity.

ABTS radical scavenging assay

ABTS radical scavenging activity of the extracts and compounds was determined using an ABTS radical cation decolorization assay employing Trolox and ascorbic acid as positive controls [14].

Determination of total phenolic content

The Folin-Ciocalteu reagent assay was used to determine the total phenolic content of the extracts [15]. An aliquot (0.2 mL) of the extract in methanol (1.0 mg/mL) was mixed with Folin-Ciocalteu reagent (0.5 mL) and the mixture was allowed to stand for 3 min at room temperature. Then the saturated sodium carbonate solution (0.2 mL) was added to the mixture and allowed to stand for another 2 h before the absorbance at 725 nm was measured. Gallic acid was used as a standard for the calibration curve (20–100 µg/mL). The total phenolic content was expressed as micrograms of gallic acid equivalents (GAE) per milligram of extract (µg/mg).

Isolation of compounds from the ethyl acetate extract

The ethyl acetate extract (30 g) was subjected to CC (12 × 7.5 cm) on silica gel (60–120 mesh, 700 g). The extract was eluted from the column using a stepwise gradient of n-hexane containing increasing amounts of ethyl acetate (from 10 % up to 100 %) and finally washed with 100 % methanol (each 500 mL). A total of 24 fractions (each of 250 mL) were collected and evaluated on TLC using a solvent system of n-hexane−ethyl acetate in different ratios. The chromatograms were derivatized with Borntrager reagent. The fractions showing the common TLC profile were combined to obtain seven pooled fractions: Fr 1 (500 mL, 1.3 g), Fr 2 (1000 mL, 1.6 g), Fr 3 (750 mL, 1.2 g), Fr 4 (750 mL, 1.4 g), Fr 5 (1000 mL, 6.6 g), Fr 6 (1500 mL, 13.2 g), and Fr 7 (500 mL, 2.4 g). Fraction 1 (1.3 g) was subjected to CC (18 × 3 cm) over silica gel (60–120 mesh, 60 g) eluting with n-hexane and ethyl acetate (9.5 : 0.5, 9 : 1, and 8 : 2; 400 mL each) to afford 1 (140 mg), 2 (8 mg), and 7 (62 mg). Fraction 3 (1.2 g) was further subjected to CC (15 × 3 cm) on silica gel (60–120 mesh, 50 g) eluting with n-hexane and ethyl acetate (7 : 3, 6 : 4 and 5 : 5; 500 mL each) to afford 3 (105 mg) and 6 (7 mg). A portion of Fr 6 (2 g) was chromatographed onto a Sephadex LH-20 column (70 × 2.5 cm) eluting with methanol (100 %), and after repeated chromatography on a Sephadex LH-20 (50 × 2 cm) column with methanol (100 %) as an eluent, compounds 4 (60 mg), 5 (35 mg), and 8 (75 mg) were obtained.

Compounds 1–8 ([Fig. 1]) were identified using mass spectrometry and ¹H- and ¹³C‐NMR experiments, and by comparison of their spectral data with that reported in the literature as chrysophanol (1) [16], physcion (2) [16], emodin (3) [16], emodin-8-O-β-D-glucopyranoside (5) [17], endocrocin (6) [18], and nepodin (7) [19]. Nepodin was reported earlier as musizin; however, the compounds are structurally identical [20], [21].

The earlier phytochemical investigation of R. nepalensis roots reported chrysophanol-8-O-β-D-galactopyranoside and musizin-1-O-β-D-glucopyranoside [21]. However, in our investigation we have identified the structures of these compounds as chrysophanol-8-O-β-D-glucopyranoside (4) and nepodin-8-O-β-D-glucopyranoside (8). Compound 4 was obtained as a yellow amorphous powder and gave a red color with Borntrager reagent. ¹H- and ¹³C‐NMR spectral data were found to be in agreement with that reported in the literature [22], and the glycosidic linkage of the sugar on the 8 position of the aglycone (chrysophanol) was confirmed by an HMBC experiment. Furthermore, the sugar moiety was confirmed by preparing the acetate derivative of 4, which showed the characteristic splitting pattern of glucose protons: ¹H‐NMR (400 MHz, CDCl₃, δ ppm): 5.51 (t, 1H, J = 7.92 Hz, H-2′), 5.33 (t, 1H, J = 9.54 Hz, H-3′), 5.21 (t, 1H, J = 9.56 Hz, H-4′), 5.15 (d, 1H, J = 7.92 Hz, H-1′), 4.31–4.19 (m, 2H, H-6′), and 3.93–3.89 (m, 1H, H-5′). The ¹H- and ¹³C‐NMR spectral data of 8 were found to be in agreement with that reported in the literature [19], and the glycosidic linkage of the sugar on the aglycone (nepodin) was found to be at the 8 position, as confirmed by an HMBC experiment. Recently, some new seco-anthraquinone glycosides have been reported from the roots of R. nepalensis [23].

TLC profiling of the chloroform extract revealed nepodin, chrysophanol, and emodin as major compounds, whereas the glycosides were absent.

TLC profiling of the ethyl extract showed the eight isolated compounds as described above. Chrysophanol-8-O-β-D-glucopyranoside, chrysophanol, nepodin, and emodin were found to be the major compounds in the ethyl acetate extract.

Statistical analysis

Numerical data are expressed as mean (%) ± SEM. Statistical differences between the treatments and the control groups were evaluated by ANOVA and Dunnett's t-tests. A value of p < 0.05 was considered significant.

Results and Discussion

The chloroform and ethyl acetate extracts of R. nepalensis roots were evaluated in an acute inflammation mouse model based on the topical application of TPA in a single-dose regimen. The extracts showed significant (p < 0.05) topical anti-inflammatory activity at 0.5 and 1.0 mg/ear when ear edema was measured 4 h after application. The anti-inflammatory effect of the ethyl acetate extract was greater than that of the chloroform extract and, at 1 mg/ear, was comparable to that of the clinically used anti-inflammatory drug indomethacin ([Table 1]). TPA, an inflammatory agent, exerts its effect through protein kinase C activation, which in turn is thought to evoke primarily three events: release of hydrolytic enzymes and vasoactive amines (serotonin, histamine), an increase in the level of lipid mediators (eicosanoids), and stimulation of peptide-releasing nerves (neurogenic inflammation) [24], [25]. Prostaglandins and related compounds produced from arachidonic acid by the action of COX-1 and/or COX-2 enzymes are collectively known as eicosanoids. The inhibition of COX-1 results in some undesirable side effects, whereas COX-2 inhibition provides therapeutic effects on pain, inflammation, arthritis, cancer, and Alzheimer's disease [2]. Therefore, we intended to examine the COX-1 and COX-2 inhibitory activities of ethyl acetate and chloroform extracts on purified enzymes as a mechanism of their topical anti-inflammatory action. The COX-1 and COX-2 inhibitory effects of the ethyl acetate extract were better than that of the chloroform extract at 50 µg/mL. The effects of these extracts were compared with the standardized dichloromethane (DCM) extract of Curcuma longa ([Fig. 2]). The composition of the DCM extract was determined by HPLC; it contained curcumin (66.3 %), demethoxycurcumin (22.8 %), and bisdemethoxycurcumin (9.8 %).

From the ethyl acetate extract, six anthraquinones and two naphthalene derivatives were isolated. In the preliminary screening, all the isolated compounds were tested for COX-1 and COX-2 inhibitory activity at a concentration of 30 µM. Three compounds, namely, emodin (3), endocrocin (6), and nepodin (7), exhibited moderate to strong inhibitory effects on COX-1 and COX-2 activity, whereas all other compounds showed mild to moderate inhibition of enzymes ([Table 2]). Compounds 3, 6, and 7 showed 76.04 %, 73.46 %, and 59.42 % inhibition of COX-2, respectively, and 54.94 %, 56.79 %, and 68.24 % inhibition of COX-1 activity, respectively, at 30 µM.

The study was further elaborated to examine the inhibitory effect of compounds 3, 6, and 7 on the COX-1 and COX-2 enzymes, and their IC₅₀ values were calculated from the concentration-versus-inhibition curve. Emodin was found to exhibit potent inhibitory effects on COX-2, followed by endocrocin, whereas nepodin exhibited more pronounced inhibitory effects towards the COX-1 enzyme ([Table 2]). The COX-2 inhibitory effects of these three compounds were three times greater than that of curcumin, whereas their COX-1 inhibitory effects were comparable to that of curcumin. The selective COX-1 inhibitor indomethacin and the selective COX-2 inhibitor celecoxib were used as positive controls and exhibited IC₅₀ values of 0.18 and 0.15 µM, respectively. Our results are in concurrence with previous reports of a few anthraquinones and naphthalene derivatives that possess anti-inflammatory activity. Emodin exhibited an anti-inflammatory effect by inhibiting the TPA-induced degradation of IkBα, the nuclear translocation of p65, and the NF-κB DNA-binding activity [26], and knipholone, a binary compound composed of anthraquinone chrysophanol and an acetylphloroglucinol, showed an anti-inflammatory effect by inhibiting leukotriene metabolism through selective inhibition of 5-LOX [27]. Naproxen [(S)-(+)-6-methoxy-α-methyl-2-naphthaleneacetic acid] and its 7-methoxy isomer isolated from a natural source are reported to possess COX-1 and COX-2 inhibitory activity [28].

Emodin (3), endocrocin (6), and nepodin (7) were also evaluated for acute topical anti-inflammatory activity in TPA-induced inflammation in the mouse ear; they exhibited a 65.3 %, 57.7 %, and 43.2 % reduction in ear edema, respectively ([Table 1]). Thus, the anti-inflammatory and COX-1 and COX-2 inhibitory activities of the ethyl acetate and chloroform extracts of R. nepalensis roots could be related to the presence of these anthraquinones and naphthalene derivatives.

The role of oxygen free radicals is well understood in the inflammation process. The free radicals liberated from phagocytes are important in the inflammation process, as they are implicated in the activation of transcription factor NF-κB, which induces the formation of inflammatory cytokines and COX-2 [29], [30]. Therefore, during the inflammatory response, the rise of free radical generation is one of the tissue-damaging factors. This encouraged us to evaluate the free radical scavenging effects of the chloroform and ethyl acetate extracts and the compounds isolated from these extracts by employing two widely used radicals, i.e., DPPH and ABTS. In both assays, the chloroform and ethyl acetate extracts exhibited radical scavenging activity of about half that shown by the positive controls trolox and ascorbic acid ([Table 3]). Among the isolated compounds, none of the anthraquinones showed activity against the two radicals, and these results were found to be in agreement with the fact that anthraquinones are devoid of antioxidant activity [31].

Nepodin (7) and its glucoside (8) were found to scavenge both radicals strongly; the effects of 7 were better than that of 8. When both the compounds were evaluated at various concentrations to establish concentration-dependent inhibition, nepodin (7) exhibited more potent radical scavenging activity than Trolox and ascorbic acid. The IC₅₀ value of nepodin was found to be better than that of trolox but was about one-half that of ascorbic acid ([Table 3]). In the case of 8, the introduction of a glucose moiety to nepodin is a likely reason for its reduced radical scavenging activity. The radical scavenging activity of the ethyl acetate extract of R. nepalensis roots could be due in part to the presence of nepodin and its glycoside, which was found to be in agreement with the earlier finding [32].

The high radical scavenging activity of the ethyl acetate extract led us to determine its phenolic content using Folin−Ciocalteu reagent employing gallic acid as a standard. The ethyl acetate extract was found to have a high phenolic content ([Table 4]); thus, the radical scavenging activity of the extract may be correlated to its phenolic content. The phenolic content of the chloroform extract was found to be one-third that of the ethyl acetate extract; therefore, its radical scavenging activity was lower than that of the ethyl acetate extract. The higher radical scavenging activity of the ethyl acetate extract could be due to the higher phenolic content as well as the presence of nepodin. In the case of the chloroform extract, its radical scavenging activity may be attributed to nepodin alone, because nepodin was found to be the major compound among the four (nepodin, emodin, chrysophanol, and physcion) present in this extract and because the anthraquinones are devoid of radical scavenging activity.

In summary, the anti-inflammatory effects of R. nepalensis root extract could be at least in part due to COX-1 and COX-2 enzyme inhibition and free radical scavenging activity. The presence of anthraquinones, mainly emodin and endocrocin, and the naphthalene derivative nepodin in the chloroform and ethyl acetate extracts could account for their anti-inflammatory and radical scavenging activities.

Acknowledgements

The authors are thankful to the Director of NIPER for providing financial assistance and necessary facilities to carry out this work.

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Dr. Sanjay M. Jachak Associate Professor

Department of Natural Products
National Institute of Pharmaceutical Education and Research (NIPER)

Sector 67, SAS Nagar

Mohali 160062 (Punjab)

India

Phone: + 91 17 22 21 46 83

Fax: + 91 17 22 21 46 92

Email: sanjayjachak@niper.ac.in

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Dr. Sanjay M. Jachak Associate Professor

Department of Natural Products
National Institute of Pharmaceutical Education and Research (NIPER)

Sector 67, SAS Nagar

Mohali 160062 (Punjab)

India

Phone: + 91 17 22 21 46 83

Fax: + 91 17 22 21 46 92

Email: sanjayjachak@niper.ac.in