Phytochemical studies and anti-ulcerative colitis effect of Moringa oleifera seeds and Egyptian propolis methanol extracts in a rat model

Attia H. Atta, Samar M. Mouneir, Soad M. Nasr, Doaa Sedky, Amany M. Mohamed, Shimaa A. Atta, Hassan Mohamed Desouky

1Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, Giza, 12211, Egypt

2Department of Parasitology & Animal Diseases, National Research Centre, 33 Bohouth St., Dokki, Giza, 12622, Egypt

3Immunology Department, Theodor Bilharz Research Institute, Giza, 12411, Egypt

4Department of Animal Reproduction and Artificial Insemination, National Research Centre, 33 Bohouth St., Dokki, 12622, Giza, Egypt

Keywords:Moringa oleifera seeds Propolis Ulcerative colitis Phytochemical

ABSTRACT Objective: To analyze the phytochemical constituents, and to explore potential protective effect of the methanol extract of Moringa oleifera (M. oleifera) seeds and Egyptian propolis,each alone or concurrently administered on acetic acid-induced ulcerative colitis in rats.Methods: Eight groups of 5 rats each were used: normal control group with distilled water,model group, two groups with M. oleifera seeds (100 and 200 mg/kg), two groups with propolis(50 and 100 mg/kg), one group with concurrent administration of both, and one group with prednisolone (reference drug). Macro-and microscopic picture, ulcer index and lesion scores,oxidative markers, inflammatory mediators, in vitro activity of the inflammatory enzymes and 1, 1-diphenyl-2-picrylhydrazyl free radicals scavenging activity were evaluated. The phytochemical constituents of both extracts were explored by GC-MS analysis. Results:Both treatments modulated the macro-and microscopic picture, decreased the ulcerative index, lesion score, oxidative markers and inflammatory mediators, and inhibited the COX-1 and COX-2 enzymes. Propolis appeared to be powerful free radicals scavenger. A powerful synergistic effect of both treatments in modulating the course of the disease was reported. GCMS analysis of methanol extract of M. oleifera seeds and propolis revealed the presence of 50 and 34 compounds, respectively. Conclusions: M. oleifera seeds and propolis methanol extracts have modulated the course of acetic acid-induced ulcerative colitis. Moreover, both treatments induce a good synergistic effect against the disease. Isolation of the active constituents is recommended.

1. Introduction

Ulcerative colitis (UC) is a common chronic inflammatory disease of the large intestine with unknown etiology[1]. It is characterized by erosions of the colon and rectal mucosa with the development of tiny open sores or ulcers, with no segments of normal tissue[2].Increases in certain inflammatory mediators and appearance of signs of oxidative stress are among the pathological findings associated with UC[3]. The aim of treatment of UC is to maintain remission,reduce complications and improve quality of life. Important therapeutic achievement was obtained ＞15 years ago by the introduction of biological therapies such as the anti-tumor necrosis factor alpha (anti-TNF-α) and monoclonal antibodies[4]. However,the high cost of biological drugs and their patent expiration limit their use[5]. In addition, the use of immunosuppressive treatment especially steroids is risky particularly in patients with viral hepatitis and/or bacterial infections[6]. Therefore, there is a growing need for effective and safe therapeutic agents for the treatment of UC.Alternative therapy for many diseases depends on natural products.Moringa oleifera Lam. (M. oleifera) grown in India, Pakistan,Bangladesh and Afghanistan[7] and introduced to Mediterranean region, is one of the promising plants because it contains several compounds that have antioxidant (ascorbic acid, carotenoids,flavonoids, and phenols), anti-inflammatory (isothiocianate and phenolic derivatives)[8] and immunomodulatory[9] effects. M. oleifera has been used traditionally for the treatment of many diseases[10].The root-bark extract[11] and leaves[12] of M. oleifera have been shown to induce the antiulcerogenic effect. Data on the effect of methanol extract of M. oleifera seeds on UC is lacking. On the other hand, propolis (beeswax) is a mixture containing different compounds such as flavonoids, phenolic, amino acids and trace elements[13]. Propolis has a number of pharmacological effects such as antimicrobial, anti-inflammatory, antiparasitic, antitumor, and antioxidant[14]. Brazilian red propolis extract exerts protective effects on 2,4,6-trinitrobenzenesulfonic acid- and acetic acid-induced colitis in mice and rats[15]. Data on tissue-regenerating actions of propolis are incomplete and often contradictory[16]. This variation or contradiction in its biological activity could be explained on the bases of variation between cultivars from different geographical locations as it has been reported for propolis[17], for M. oleifera[18],and for other medicinal plants[19,20]. This study aimed to analyze the phytochemical constituents, investigate the protective effect of methanol extract of M. oleifera seeds and Egyptian propolis on acetic acid-induced colitis in rats and to explore the potential synergistic effect of both extracts in minimizing the symptoms of colitis.

2. Materials and methods

This experiment was carried out according to the guidelines of the Institutional Animal Care and Use Committee, National Research Centre, Approval Protocol No.: 18/103.

2.1. Plant material

were air dried and moderately pulverized. The powdered plant seeds(200 g) were extracted by percolation for 24 h in methanol 95% several times. The solvent was then evaporated under reduced pressure using a rotary evaporator at a low temperature not more than 50 ℃. The yield was 35.7 g. The obtained extract was then kept at -4 ℃ until used. At the start of the experiments, the extract was freshly suspended in distilled water with few drops of Tween-80. Propolis was obtained from apiaries located in the Delta region, Egypt. Propolis was extracted with methanol several times and methanol was then evaporated at low temperature. The condensed propolis methanol extract was freshly suspended in 1% carboxymethyl cellulose.

2.2. Animals and grouping

Forty Sprague Dawley male and female rats (190-200 g) of six weeks age were used. Animals were allocated randomly into 8 equal groups(GⅠ-GⅧ). Rats of GⅠ, as normal control, received an equal volume of distilled water. Rats of GⅡ, as ulcerative colitis control, received an equal volume of distilled water before acetic acid-induction of ulcerative colitis. Rats of GⅢ and GⅣ were pretreated orally with M.oleifera seeds methanol extract (MOSME) at a dose of 100 and 200 mg/kg, respectively. Rats of GⅤ and GⅥ were pretreated with propolis methanol extract orally at a dose of 50 and 100 mg/kg, respectively.Rats of GⅦ received propolis (50 mg/kg) and MOSME (100 mg/kg)orally. On the 8th day, rats of GⅡ to GⅦ were administered 2 mL of acetic acid 4% solution intrarectally. Administration of M. oleifera and propolis continued till the 11th day. Rats of GⅧ were treated with prednisolone (reference drug, 2 mg/kg, p.o., for 3 d) and acetic acid (2 mL of 4% solution, once, intrarectally) on the same day. Rats were kept in good hygienic conditions at room temperature of (25±3) ℃ with a 12 h dark/light cycle. They were fed standard laboratory feed and watered ad libitum.

2.3. Sampling

Blood samples were withdrawn by retro-orbital puncture on the 11th day under light anesthesia. Serum was obtained by centrifugation of clotted blood. Animals were euthanized by an overdose of inhalation anesthesia. Colon of each rat, about 2 cm proximal to the anus and 8 cm in length, was taken out of the body and incised longitudinally, washed with normal saline and then placed on a transparent sheet and a photo was taken using an adjusted camera. Colons were then examined macro- and microscopically. Specimens of colon were fixed in 10% formol saline solution for histopathological studies. Samples from the colon were stored immediately at -80 ℃ till biochemical analysis.

Seeds of M. oleifera were donated from the Egyptian Scientific Society of Moringa, National Research Centre, Dokki, Giza, Egypt. Seeds

2.4. Evaluation of colon macroscopic damage

2.5. Determination of ulcer index (UI)

The sum of the total length of long ulcers and petechial lesions in each group of rats was divided by its number to calculate the UI(mm)[22].

2.6. Biochemical assays

2.6.1. Determination of colonic TNF-α level

Frozen (-80 ℃) colon tissue samples were immediately weighed,minced on an ice-cold plate and maintained in a tube with 10 mmol/L sodium phosphate buffer (pH 7.4) (1:5 w/v). The tubes were shaken in a shaking water bath (37 ℃) for 20 min and centrifuged at 9 000 ×g for 10 min at 4 ℃; the supernatant was frozen at -80 ℃ until assay.TNF-α was quantified in the homogenate samples of colons by enzyme-linked immunosorbent assay (ELISA) and the results were expressed as pg/g of wet tissue.

2.6.2. Determination of colonic malondialdehyde (MDA)contents

Colonic MDA level was determined in tissue homogenates by the reaction with thiobarbituric acid as described by Ohkawa et al.[23]using commercial kits from Biodiagnostic Co., Egypt. The values were expressed as nmol MDA/g protein.

2.6.3. Determination of colonic myeloperoxidase (MPO)contents

The activity of MPO is defined as the quantity of enzyme degrading 1 μmol of peroxide per min at 25 ℃ and is expressed in U/g of wet scrapings. The colonic MPO activity was determined according to Krawisz et al.[24].

2.6.4. Determination of colonic nitrite/nitrate level

As soon as the sun rose the King came, and when he perceived the gold he was astonished and delighted, but his heart only lusted more than ever after the precious metal

Nitric oxide (NO) level in the colon samples was determined by the acidic Griess reaction after reduction of nitrate to nitrite by vanadium trichloride as described by Miranda et al.[25]. A pink azo-product is produced as a result of the reaction between nitrite, sulfonamide,and N-(1-naphthyl) ethylenediamine. The intensity of the color was then measured at a maximum absorbance of 543 nm. A standard curve of sodium nitrate was established and the concentrations were expressed as μg/mg of wet tissue.

2.6.5. In vitro effect on the inflammatory enzymes (COX-1 and COX-2)

The ability of M. oleifera and propolis to inhibit COX-1 and COX-2 was evaluated using an ovine COX-1/COX-2 inhibitor screening assay kit (catalog no. 760111, Cayman Chemicals, MI, USA) that utilizes the peroxidase component of COX. The peroxidase activity is assayed colorimetrically by monitoring the appearance of oxidized N,N,N',N'-tetramethyl-1,4-phenylenediamine according to Kulmacz and Lands[26]. In addition, the selectivity index (SI values) for the tested compounds was calculated as IC50(COX-1)/IC50(COX-2).Celecoxib was used as a reference COX-2 inhibitor.

2.7. Determination of in vitro antioxidant effect

The antioxidant activities of MOSME and propolis methanol extract were tested in vitro by free radical scavenging assay method described by Sreelatha and Padma[27] using 1, 1-diphenyl-2-picrylhydrazyl (DPPH).

2.8. Histopathological evaluation

Colon samples were washed off formol saline with tap water and dehydrated with serial dilutions of ethanol. Specimens were cleared in xylene and embedded in paraffin at 56 ℃ in a hot air oven for 24 h. Paraffin tissue blocks were sectioned at 5 microns thickness by slide microtome. The obtained tissue sections were mounted on glass slides, deparaffinized and stained by hematoxylin & eosin (H&E)and examined under light microscope[28]. Inflammatory changes,necrotic foci, and damage to tissue structures or to the nucleus in the stained sections were recorded.

2.9. GC-MS analysis

Gas chromatograph (GC, Agilent Technologies 7890A) interfaced with a mass-selective detector (Agilent 7000 Triple Quad) was used. Agilent HP-5ms capillary column (30 m×0.25 mm ID and 0.25 μm film thicknesses) was used. The flow rate was 1 mL/min.The injector and detector temperatures were 200 ℃ and 250 ℃,respectively. The acquisition mass range was 50-600. The chemical structure of the compounds was identified and recorded after comparing their mass spectra and retention time (RT) with those of NIST and WILEY library.

2.10. Statistical analysis

Data were presented as mean±SD. Differences between means were tested for significance by ANOVA test using SPSS version 16 computer program. Probability level of P＜0.05 was taken as indication of significance.

3. Results

3.1. Effect on the macroscopic picture

Acetic acid produced severe colonic damage characterized by inflammation, swelling, edema, hemorrhages, and necrosis. Dosedependent mild to moderate inflammatory changes appeared in the colon of rats treated with either MOSME or propolis methanol extract. Concurrent administration of both MOSME and propolis methanol extract completely protected colonic mucosa against acetic acid-induced changes (Figure 1a-1h).

Figure 1. Macroscopic pictures of colon.

3.2. Effect on the UI

Administration of acetic acid dramatically increased the UI(438.17 ± 59.26) as compared to that of normal rats (3.53 ±0.60). Administration of MOSME induced a dose-dependent significant (P＜0.05) decrease in the UI. The lesion scores were also dramatically increased by acetic acid and decreased by pretreatment with MOSME or propolis. Concurrent administration of both MOSME and propolis methanol extract significantly decreased the UI (P＜0.05) and lesion scores to values nearly similar to the normal control or to prednisolone. The percent inhibition of colonic damage in rats concurrently pretreated with MOSME and propolis was nearly similar to that pretreated with prednisolone (Table 1).

3.3. Effect on the inflammatory mediators and oxidative markers

Rectal administration of acetic acid significantly increased the inflammatory mediators (TNF-α and NO) and the oxidative markers(MPO and MDA) levels in colonic tissue of treated rats as compared to normal one. Administration of MOSME or propolis methanol extract induced a dose-dependently significant (P＜0.05) decrease in both the inflammatory mediators and the oxidative markers.Concurrent administration of both MOSME and propolis methanol extract significantly decreased the levels of the inflammatory mediators and the oxidative markers which were comparable to normal control and prednisolone (Table 2).

Table 1 Effect of methanol extract of M. oleifera seeds and Egyptian propolis on UI and lesion scores in rats with UC (Mean ± SD, n=5).

Table 2 Effect of methanol extract of M. oleifera seeds and Egyptian propolis on the oxidative markers and the inflammatory mediators in rats with UC (Mean ±SD, n=5).

3.4. In vitro effect on COX-1 and COX-2

The present results showed that both MOSME and propolis methanol extract are potential inhibitors to the inflammatory enzymes; COX-1 and COX-2. The high selectivity for COX-1 inhibition was recorded for the MOSME followed by propolis and then celecoxib (reference COX-2 inhibitor). In contrast, the high selectivity for COX-2 inhibition was recorded for the celecoxib (SI:302) followed by propolis and then MOSME (Table 3).

Table 3 In vitro COX-1, COX-2 anti-inflammatory activities of M. oleifera seeds and Egyptian propolis methanol extracts.

3.5. In vitro antioxidant effect

Scavenging activity of MOSME against DPPH was weak with only 6.2% scavenging level at 100 μg/mL of the extract. However,propolis methanol extract had high activity against DPPH with an IC50value of 10.34 μg/mL and 92.5% scavenging rate of free radicals at the same concentration.

3.6. Effect on the histopathological picture

Microscopic examination of colons sections of normal rats showed a normal histological structure of the mucosa, glandular structure with activation of goblet cells, submucosa and muscularis and serosa (Figure 2a). Colon of rats with experimental UC showed focal ulceration, hemorrhages and necrosis with inflammatory cells infiltration in the mucosa, submucosa, muscularis and serosa of the colon wall (Figure 2b). Colon of rats with UC treated with MOSME at a dose of 100 mg/kg showed less focal ulceration and necrosis with hemorrhage and inflammatory cells infiltration in the mucosa, submucosa and muscularis (Figure 2c). Colon of rats with UC treated with MOSME at a dose of 200 mg/kg showed moderate focal ulceration and necrosis associated with inflammatory cells infiltration and mild hemorrhages in the submucosa, muscularis and serosa (Figure 2d). Colon of rats with UC treated with propolis methanol extract at a dose of 50 mg/kg showed focal ulceration and necrosis in the mucosa and inflammatory cells infiltration and hemorrhages in the submucosa (Figure 2e). Colon of rats with UC treated with propolis methanol extract at a dose of 100 mg/kg showed mild focal ulceration and necrosis in the mucosa associated with inflammatory cells infiltration (Figure 2f). Colon of rats with UC and treated with both MOSME (100 mg/kg) and propolis extract 50 mg/kg (Figure 2g) as well as those treated with prednisolone in a dose of 2 mg/kg (Figure 2h) showed intact mucosa with inflammatory cells infiltration in the underlying submucosa.

Figure 2. Histopathological pictures of colon sections.

3.7. GC-MS analysis of the methanol extract

GC-MS analysis revealed the presence of 50 and 34 compounds in MOSME and propolis extract respectively (Tables 4 & 5 and Figure 3). There were 34 and 17 compounds of more than 1% in MOSME and propolis extract, respectively. The major components are 2,5-Di-tert-butyl-1,4-benzoquinone (15.43%) and 4',6-dimethoxyisoflavone-7-O-β-D-glucopyranoside (9.25%) in MOSME and fraxidin (30.15%), methyl α-D-rhamnopyranoside(17.28%) and 6-ethyl-3-formylchromone (5.24%) in propolis extract.

Table 4 GC-MS analysis of MOSME.

Table 5 GC-MS analysis of Egyptian propolis methanol extract.

4. Discussion

Figure 3. GC-MS peaks of (a) MOSME and (b) Egyptian propolis methanol extract.

In this work, an acetic acid model of experimental colitis in rats was used as it has been reported that acetic acid produces rapid and reproducible colitis suitable for screening of drugs for anti-colitis effects[29]. In this study, acetic acid induced severe colonic damage and the macroscopic lesions characterized by inflammation, swelling, edema, hemorrhages, and necrosis. It also increased colonic major inflammatory mediators in similar patterns to inflammatory bowel disease (IBD). TNF-α is one of the cytokines that are involved in systemic inflammation and make up the acute phase reaction. It is produced by activated lymphocytes,macrophages, neutrophils, mast cells, eosinophils, and neurons[30]and can be released as a result of an inflammatory stimulus (e.g.lipopolysaccharide)[31]. MDA is a product of degradation by reactive oxygen species of polyunsaturated lipids[32]. It is a reactive aldehyde that causes toxic effects in the cells[33]. Its level is used as an indicator of the degree of oxidative stress in an organism[34]. MPO is an important enzyme released by activated polymorphonuclear neutrophils during phagocytic lysis of engulfed foreign particles. It activates the synthesis of hypochlorous acid from hydrogen peroxide and chloride anion[35,36]. Hypochlorous acid radicals are cytotoxic to bacteria and other pathogens[37]. However, it may also cause oxidative damage in host tissue. Its increased level is considered as a biomarker of inflammation[38]. NO is a free radical associated with fatty liver disease and is involved in hepatic lipid metabolism under starvation[39]. Pro-inflammatory mediators, such as cytokines and NO are released by inflammatory cells massively infiltrating the inflamed intestine of patients with IBD[40]. In this study, acetic acid produced a dramatic increase in TNF-α, NO, MDA, and MPO which are evident in IBD disease[8]. The decreased pro-inflammatory mediators and improvement of macro- and microscopic picture reported in this study could confirm the strong antioxidant and antiinflammatory effect of both M. oleifera and propolis[8,14]. The acetic acid-induced tissue damage was suggested to be ameliorated by antioxidants that inhibit the enzymes and/or chelating trace elements involved in the free-radical production. The decrease in the MDA and NO level in the MOSME and propolis extract indicates the role of the extracts as antioxidants. It has been previously reported that the protective effect against acetic acid-induced damage includes scavenging reactive species, protecting antioxidant defenses, and activating antioxidant enzymes[41]. The protective effects of MOSME are also probably due to its anti-inflammatory. Other mechanisms for the antioxidant effect of M. oleifera have been reported such as superoxide anion and NO radical scavenging activity that prevents oxidative damage of major biomolecules[26,42]. In this study,propolis has been proved to induce a strong in vitro DPPH free radicle scavenging activity, providing its antioxidant activity. The antioxidant activity is usually attributed to the total phenolics and flavonoids[43]. M. oleifera seeds extract was shown to have significant antioxidant effect which was attributed to its phenolic content[44].Other bioactive constituents from M. oleifera such as glucosinolates,isothiocyanates, and thiocarbamates were suggested to be responsible for antioxidant effect[45]. Isothiocyanates have been proved to have anti-inflammatory as well as immunomodulatory activities[8] to which the anti-ulcerative effect was attributed[9,46]. Moreover, the antioxidant properties of M. oleifera were suggested to be mediated through direct trapping of the free radicals and also through metal chelation which was attributed to the presence of phenolic acids and flavonoids[47]. It has been reported that flavonoids possess potent antioxidant effect[48]. In this study, the GC-MS analysis of the methanol extract of M. oleifera seeds revealed the presence of at least 10 flavonoid flavones namely, 5,7,3',4',5'-pentamethoxyflavone(1), 3,7,3',4',5'-pentahydroxyflavone (2), 3',4'-dihydroxy-β-naphthoflavone (3), 5,7,3',4'-tetramethoxyflavone (4), 5,7,3',4',5'-pentahydroxyflavone (5), 4',6-dimethoxyisoflavone-7-O-β-D-glucopyranoside (6), 7,4'-dimethoxy-3-hydroxyflavone (7), 6,4'-dimethoxy-7-hydroxyisoflavone (8), 3,6,2',3'-tetrahydroxyflavone(9), 5,3'-dihydroxy-6,7,4'-trimethoxyflavone (10). The flavonoid flavones may be contributed to the antioxidant effect of MOSME.Moreover, the phytochemical analysis in the present study revealed that MOSME contains the essential oil alloaromadendrene which has been previously reported to have an antioxidant effect[49]. More recently, phytochemical analysis of MOSME revealed the presence of isovitexin which has been reported to be responsible for inhibition of TNF-α level, MPO activity and MDA content as it occurs in animals with liver intoxication[50]. In this study, the antioxidant effect is also confirmed in vitro by the high scavenging activity against DPPH free radicals. This antioxidant effect is nearly similar to that previously reported for Moringa peregrina[51,52] and M. oleifera[53]extracts. On the other hand, phytochemical analysis of propolis revealed the presence of at least 9 flavonoid flavones namely;3,3',7,8-tetramethoxyflavone (1), 5,7,3',4',5'-pentahydroxyflavone(2), 4',6-dimethoxyisoflavone-7-O-β-D-glucopyranoside (3),7-hydroxy-6-methoxyisoflavone (4), 7,8,3',4'-tetrahydroxyflavone(5), 3-hydroxy-6,2',4'-trimethoxyflavone (6), 4'-benzyloxy-5,7-dimethoxyflavone (7), 4'-hydroxyflavanone (8) and 3,2',4',5'-tetramethoxyflavone (9). These flavones may also be contributed to the antioxidant effect of propolis. It should be pointed out that MOSME and propolis methanol extract have common compounds with an identical structural formula such as 5,7,3',4',5'-pentahydroxyflavone and 4',6-dimethoxyisoflavone-7-O-β-D-glucopyranoside which could explain the synergistic antioxidant effect between them. Moreover, the methanol extract of both M.oleifera and propolis in this work showed a potent inhibitory effect on the inflammatory enzymes (COX-1 and COX-2). However, a high selectivity index was recorded for the celecoxib followed by propolis and then MOSME proving a selective inhibition of COX-2, which plays a key role in prostaglandin biosynthesis and hence protects colonic mucosa against acetic acid-induced damage. Moreover,M. oleifera flower hydroethanolic extract was reported to suppress the secretion of NO, prostaglandin E2, TNF-α, inducible NO synthase, and COX-2[54]. M. oleifera has also been reported to have a suppressive effect on COX-1[55].

On the other hand, the extract of M. oleifera and propolis suppresses leukotriene biosynthesis (TNF-α); a mechanism which is similar to glucocorticoids anti-inflammatory effect in relieving human IBD[56].Additional mechanisms such as improvement of cellular and humoral immunity by M. oleifera[57] and by propolis[58] can also be involved in the protective effect against acetic acid-induced colitis. Furthermore,M. oleifera contains β-caryophyllene which has been shown to have anti-inflammatory and anti-edematogenic action[59]. Moreover, propolis contains abundant polyphenolic compounds[60] which were claimed to exhibit significant anti-inflammatory effects, probably by blocking the activation of inflammatory mediator NF-κB[61,62]. In this study, propolis has been also proved to inhibit the inflammatory mediators; TNF-α and NO. The synergistic effect between MOSME and propolis was marked in inhibition of these mediators. The synergistic effect of MOSME and propolis was also marked in an improvement of the histopathological picture of livers of treated rats as compared to those treated by each one alone. The outcome of the combined treatment with M. oleifera seeds and propolis extracts was better than the use of each one alone or even the use of prednisolone regarding the inflammatory mediators (TNF-α,NO), and the oxidative markers (MDA, MPO) as well as the macro and microscopic picture confirming a synergistic effect of both M. oleifera and propolis.

Our findings suggest a useful therapeutic activity for the methanol extract of M. oleifera seeds and of propolis as an anti-ulcerative remedy. A synergistic effect between MOSME and propolis was pronounced suggesting their possible use as an alternative therapy for the colonic inflammatory condition.

Conflict of interest statement

The authors declare that there is no conflict of interest.