The effect of Urtica Dioica leaf extract intake on serum TNF-α,stool calprotectin and erythrocyte sedimentation rate in patients with inflammatory bowel disease: A double-blind,placebo-controlled,randomized,clinical trial

Abstract

BACKGROUND:

Inflammatory bowel disease (IBD) includes Crohn’s disease and ulcerative colitis in which inflammatory factors are thought to play an important role.

OBJECTIVE:

This study examined the effects of Urtica dioica leaf extract on tumor necrosis factor alpha (TNF-α) and fecal calprotectin as inflammation markers and erythrocyte sedimentation rate (ESR) in patients with inflammatory bowel disease.

METHODS:

This study was a double-blind, placebo-controlled, randomized, clinical trial, conducted on 64 patients with IBD. The participants were divided into a Urtica Dioica group (n = 32) and a placebo group (n = 32) by permuted block randomization. Subjects received three tablets daily for 12 weeks of either hydroalcoholic nettle extract (400 mg) or a placebo. Levels of TNF-α in serum, fecal calprotectin and ESR were assessed at baseline and at the end of study.

RESULTS:

59 participants completed the trial. After 12 weeks, levels of TNF-α and fecal calprotectin showed a significant reduction in the Urtica Dioica group compared to the placebo group (p < 0.05). No significant difference was found in the level of ESR between the groups at the end of the study.

CONCLUSION:

Our results indicate that supplementation with hydroalcoholic nettle extract can improve levels of TNF-α and fecal calprotectin as inflammatory factors in patients with IBD.

Keywords

ESR fecal calprotectin inflammatory bowel disease Urtica Dioica

1 Introduction

Inflammatory bowel disease (IBD) includes Crohn’s disease (CD) and ulcerative colitis (UC) [1]. The etiology of IBD remains largely unknown, but growing evidence indicates that environmental (smoking, diet, drugs, geography, social stress, and psychological element) [2] and genetic factors contribute to promote an immunopathologic process leading to chronic inflammation [3]. The highest reported prevalence values for IBD are in Europe (UC, 505 per 100,000 persons; CD, 322 per 100,000 persons) and North America (UC, 249 per 100,000 persons; CD, 319 per 100,000 persons) [4]. Recent studies show a gradually increasing rate of IBD in many developing countries in Africa, South America, and Asia [5]. The incidence and prevalence of IBD are increasing with time and in different regions around the world, indicating its emergence as a global disease [4]. The incidence and prevalence of IBD in Iran is still unclear. However, according to recent studies, it appears that neither UC nor CD is rare in Iran; indeed their incidence appears to have increased in recent years [5]. IBDs have higher morbidity and mortality rates than most other diseases, and incur an important risk of complications and diminished quality of life in affected people [6, 7]. The goals of treatment in IBD are to induce and maintain remission and to improve nutritional status. Persons with IBD are at increased risk of nutrition problems for a host of reasons related to the disease and its treatment. Thus, the primary goal is to restore and maintain the nutritional status of the individual patient. Foods, dietary and micronutrient supplements, enteral and parenteral nutrition may all be used to accomplish that mission. Oral diet and the other means of nutritional support may change during remissions and exacerbations of the disease. Therapy in IBD has frequently focused on the short-term control of clinical disease activity [7, 8]. Nettle scientific name Urtica Dioica (UD), is a perennial plant, which belongs to the Urticaceae family, characterized by sharp hairs that line its stem and leaves and grows on non-arable and waste lands [9]. Nettle leaves are a source of fatty acids including alpha-linolenic acid, carotenoids like lutein isomer and also isomers of beta-carotene, lycopene, phenols such as ferulic acid, gallic acid, syringic acid, flavonoids such as catechins and epi-catechins, and other substances include violaxanthin, neoxanthin, caffeic acid, cytotoxic sterols and minerals [9, 10]. Nettles are a very nutritious food that is easily digested and nettle leaves are a good source of essential amino acids, ascorbic acid, available and unavailable carbohydrates, and several mineral elements [11]. The suggested doses for internal use include hydroalcoholic extracts corresponding to 8–12 g nettle leaf daily or up to 5 g as an infusion up to three times daily over an unlimited period. It has been recommended for treatment of complications associated with rheumatoid arthritis, osteoarthritis and urinary tract infection [10]. A large variety of inflammatory conditions including bacterial and viral infections, lipopolysaccharide, cytokines and various forms of stress induce NF-κB activity. Anti-inflammatory effect of Urtica extract may be ascribed to its inhibitory effect on NF-κB activation and also it has been shown that Urtica extracts inhibit the expression of several cytokines [12]. Many therapeutic effects such as diuretic, natriuretic, hypotensive, anti-rheumatic, anti-prostatic, and in-vitro antioxidant effects of the Urtica Dioica have been determined [13]. There is an increasing trend in prevalence of inflammatory bowel disease (IBD) in Iran and other countries. Access to drugs for treatment of patients with IBD and IBD-related conditions is not ideal. As reviewed above, inflammation has an important role in the pathogenesis of IBD. Based on the role of TNF-α, fecal calprotectin and ESR in inflammation, this study examined the effects of Urtica Dioica leaf extract on TNF-α and fecal calprotectin as inflammation markers, and ESR in patients with inflammatory bowel disease. To the best of the author’s knowledge, the present study is the first to evaluate the effects of Urtica Dioica leaf extract on TNF-α, fecal calprotectin and ESR in patients with inflammatory bowel disease.

2 Materials and methods

2.1 Subjects

This study is a randomized double-blind, placebo-controlled trial on 64 patients with inflammatory bowel disease confirmed by a specialist, in a period of 3 months in Rasoul Akram hospital, Tehran, Iran between November, 2014 to February, 2015. Inclusion criteria were: willingness to participate in the study, inflammatory bowel disease in mild and moderate stages, no change in the type and dose of medication during the study, without any other diseases such as intestinal diseases, known autoimmune diseases, cancer, inflammatory diseases, infectious diseases, non-pregnant or breastfeeding women or non-use of contraceptive pill, not having edema caused by cardiac or renal disease, no use of dietary supplements and herbal pills, no use of anticoagulant drugs such as heparin and antibiotics, patients in the study use similar types of drugs. Exclusion criteria were: the patient’s unwillingness to cooperate, exacerbation of disease resulting in hospitalization, changes in dosage and type of medication during intervention, sensitivity to nettle, compliance rate of less than 90%.

2.2 Study design

Subjects of the present clinical trial, which involved a randomized double-blind placebo-controlled design, were randomly allocated through the permuted block randomization (size of blocks = 4) procedure into two groups. Each arm of the trial included two cases in every permuted block, and all case allocations were conducted randomly by RAS (Random Allocation Software). The sample size of our study was evaluated based on ESR factor as a dependent variable. A minimum sample size of 29 patients was determined in each group. To reach over 80% power with an estimated 10% loss of patients during follow-up, the study had to include 32 patients in each group. The sample size was evaluated based on data from the Kayahan et al. [14], study by considering α= 0.05 and β= 0.2. The patients with IBD were selected randomly according to the inclusion criteria of the study from the liver and gastrointestinal wards at Rasool Akram hospital. The aims, methods of intervention and the period of the study were described to the participants and written informed consent was obtained from all of the participants. Patients knew that they were randomly assigned to receive either supplements or placebo.

2.3 Intervention

The patients in the Urtica Dioica group received 3 tablets daily, 30 minutes before each main meal (breakfast, lunch and dinner), each tablet containing 400 mg hydroalcoholic nettle extract, whereas the patients in the placebo group received 3 tablets of wheat starch with the same appearance and weight. Similar opaque bottles were used to present both tablets of hydroalcoholic nettle extract and placebo to participants. After randomization, volunteers received supplements in compliance with allocation codes. Each volunteer received one bottle per month contained 90 tablets. In order to maintain ‘blinding’, all of the bottles were coded by a fourth subject, not involved in the study (the first three subjects being the patient, physician and researcher). In addition to participants, clinical and laboratory staff were kept ‘blind’ until the end of data analyses to ensure reliable randomization and allocation. Patients were contacted by telephone every two weeks to evaluate their compliance with the medication. Volunteers were asked to inform research staff immediately in the event of any suspicious reactions to supplements. The participants were asked to consume their medications without any change and let the researcher know if the type or dose of their medications were changed by the physician. All patients could call the study researcher if they had any question as well as any change in type and dose of their medication consumption. Patients were excluded if they had any change in the current treatment for IBD or if they could not consume the supplements regularly. Patients were asked to return any used bottles of nettle extracts tablets. Remaining tablets were counted to evaluate the adherence to the study medication and a loss of more than 10% of the tables was considered non-compliance and the patient would be excluded from the study.

2.4 Characteristics of supplements

Tablets of hydroalcoholic nettle extract and placebo, similar in color and size, respectively, were prepared by Barij Essence Pharmaceutical Company, Kashan, Iran and Pharmaceutical Sciences Research Center of Tehran University of Medical Sciences, Iran. In the present study, the leaves of the Urtica Dioica (nettle) were washed and dried. All dried leaves were then ground into powder. The Powder of the nettle leaves (300–400 gr) was percolated with ethanol 70% as a hydroalcoholic solvent (3-4 times the weight of the U.dioica power). The extract was drawn from there by opening the valve after duration of 48 hours. As an additional step, the extracts were spray dried. Using this method, 10 kilograms of dried extract was obtained from every 60 kilograms of Urtica Dioica. Participants and researchers were not aware of treatment details, and bottles containing tablets were coded as A or B by an individual who was not involved in the trial.

2.5 Measurements

The primary outcomes of the present study were to determine the effects of Urtica Dioica leaf extract on TNF-α, fecal calprotectin and ESR in patients with inflammatory bowel disease. At baseline, and at the end of the study, anthropometric indices, physical activity, dietary intake and biochemical parameters were evaluated. Dietary intake was evaluated by means of 24-hour recall for 3 days (2 regular days and 1 weekend day). The questionnaire of general information and physical activity questionnaire were completed. We have examined some clinical symptoms by IBDQ9 in another article which is published earlier. IBDQ-9 assesses the patient’s quality of life based on gastrointestinal (e.g. flatulence), systemic (e.g. Fatigue), emotional (e.g. Satisfaction and happiness) and social disturbances (Delaying a social appointment) [15].

2.6 Anthropometric measurements

Weight and height were measured using a Seca scale (Seca725 GmbH & Co. Hamburg, Germany) while subjects were standing and wore light clothing and no shoes. The accuracy of height and weight measurements was within 0.5 cm and 100 g, respectively. Waist circumference was measured at the end of a normal expiration and in a horizontal plane, using a plastic tape measure, above the uppermost lateral border of the right ilium. Body mass index (BMI) was calculated as weight (kg) divided by height squared (m2). All measurements were conducted both before and after the intervention.

2.7 Laboratory measurements

Biochemical parameters including TNF-α, ESR drawn from 5 ml of venous blood were measured following an overnight fasting (12–14 hr) prior to and post-intervention. Accordingly, blood samples were drawn into the tubes and were kept at room temperature (18–20°C) for 18–25 minutes. Serum samples were separated by centrifugation with coagulated blood at 3000 rpm for 15 min. All serum samples were then stored at –20°C. Serum TNF-α was measured by using ELISA method (eBioscience Co. Austria) and was expressed as pg/ml. Level of calprotectin in stool was measured by using ELISA method (BÜHLMANN Co. Switzerland) and was expressed as μg/g. For the measurement of calprotectin, stool plastic containers were filled by patients at home and kept at 2 to 8°C (maximum 3 days). The erythrocyte sedimentation rate (ESR) was measured by automated ESR analyzer (Electa Lab). Approximately 1.2 ml of blood was collected in EDTA plasma tubes and then was mixed by repeated inversion of tubes on a slowly revolving rotary mixer.

2.8 Statistical Methods

Data is expressed as mean (±standard deviation) for parametric variables and as median (25th, 75th percentiles) for non-parametric variables. Categorical variables are expressed as frequency (percentage). All of the 24-recall questionnaires were analyzed using Nutritionist IV software. Normality of data was checked using the Kolmogorov-Smirnov Test. The comparison of qualitative variables as confounders between two groups was performed with chi-square test. To detect any significant differences within and between two groups of normally distributed variables, paired and independent t-test were used for parametric variables, respectively. Also, for variables that were not normally distributed Wilcoxon and Mann-Whitney nonparametric tests were applied to detect any significant differences within and between two groups respectively. As the distribution of ESR showed little-to-mild skewing toward the left, its log-transformed values were used for analysis. Analysis of covariance (ANCOVA) was used to eliminate the effects of confounders with statistically significant differences between two groups. A value of p < 0.05 was considered to be statistically significant. Statistical Package for Sciences Software version 22 (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses.

3 Results

59 participants completed the trial (Fig. 1). The subjects reported no serious side effects. During the 12-week follow-up, two and three patients withdrew from the intervention and placebo group respectively. The final analysis includes 30 patients in the Urtica Dioica group and 29 patients in the placebo group. Variable distribution method showed that all variables considered in this study conformed to a normal distribution except for physical activity. No significant differences were found between sex, age, height, education, smoking, drug consumption and disease duration at the beginning of study (Table-1), as well as anthropometric characteristics including weight, BMI, waist circumference and physical activity between and within the groups at the baseline and at the end of the study (Table-2). The IBDQ-9 score increased significantly in the Urtica Dioica group (p < 0.001), whereas no significant increase was observed in placebo group. These changes were significant between groups at the end of study (p < 0.001) [15]. Mean and standard deviation of energy and dietary intakes of carbohydrate, cholesterol, MUFA, zinc, dietary fiber, vitamin A and C, protein, fat, saturated fatty acid, PUFA, vitamin D and E, selenium are shown in Table-3. In the Urtica Dioica group, baseline dietary intake of vitamin E increased significantly as compared with endpoint intake. In the placebo group, baseline dietary intake of PUFA decreased significantly as compared with endpoint intake. However, these differences were not significant between the placebo and Urtica Dioica group. With regards to dietary intake at baseline, only the intake of dietary fiber was significantly different between two groups. As shown in Table-4, The ESR level showed no significant change between and within the groups at the end of 12th week (Fig. 2). The fecal calprotectin showed a significant decrease in the Urtica Dioica group at the end of 12th week (p < 0.05); however, reduction in the placebo group was not significant. Moreover, after the 12-week intervention, fecal calprotectin revealed a significant decrease between the groups (p < 0.05) (Fig. 3). The plasma level of TNF-α in the Urtica Dioica group decreased significantly during the 12-week intervention (p < 0.05), whereas no significant decrease was observed in the placebo group. These changes were significant between groups (p < 0.05) (Fig. 4). No significant changes were seen after adjustment for dietary fiber intake and age.

Fig. 1

Flow diagram of participants recruitment and randomization process.

Fig. 2

Mean values for ESR before and after treatment.

Fig. 3

Mean values for Calprotectin before and after treatment.

Fig. 4

Mean values for TNF-α before and after treatment.

Table 1

Baseline characteristics of the study participants

Baseline Characteristic	UD (n = 30)	Placebo (n = 29)	p-value
Age (year)	36.60±10.893¹	38.31±13.286	^a 0.590
Height (cm)	167.70±8.470	168.07±10.647	^a 0.883
Disease duration (year)	6.10±3.155	6.38±2.796	^a 0.721
Sex			^b 0.895
Male	15²(50.0)	15(51.7)
Female	15(50.0)	14(48.3)
Education			^c 0.640
Illiterate	0(0)	2 (6.9)
under-diploma	6(20.0)	4(13.8)
Diploma	9(30.0)	9(31.1)
Graduate	15(50.0)	14(48.3)
Smoking			^c 0.706
Yes	3(10.0)	4 (13.8)
No	27(90.0)	25(86.2)
Drug consumption			^c 0.806
5-amino salicylic acid	16(53.3)	14 (48.4)
Immunosuppressive	2(6.7)	3(10.3)
5-amino salicylic acid, immunosuppressive, corticosteroid	5(16.7)	3(10.3)
5-amino salicylic acid, corticosteroid	1(3.3)	3(10.3)
5-amino salicylic acid, immunosuppressive	6(20.0)	6(20.7)

¹Mean±standard deviation, ²Frequency (%), ^aIndependent t-test, ^bChi-square test, ^aFisher test.

Table 2

Anthropometric measurements of the UD and placebo groups before and after 12 weeks

Variable	Group	Baseline	Endpoint	p-value
Weight (kg)	UD	69.85±14.316	69.86±13.977	0.947^b
	placebo	71.51±14.352	71.08±14.324	0.158^b
	p-value ^a	0.657	0.742
BMI (kg/m²)	UD	24.76±4.400	24.77±4.260	0.979^b
	placebo	25.18±3.494	25.01±3.446	0.144^b
	p-value ^a	0.693	0.807
Waist Circumference (cm)	UD	89.53±13.477	89.33±13.171	0.363^b
	placebo	90.59±10.476	90.31±10.478	0.212^b
	p-value ^a	0.739	0.754
Physical Activity (Met.min/wk)	UD	478.50(292.250,1670.750)	565.50(330,1452.75)	0.833^d
	placebo	693(297,1321.50)	594(280.50,1222.50)	0.516^d
	p-value ^c	0.826	0.891

All values are mean±SD except physical activity in the form of median (25th, 75th percentiles). ^aBetween the groups P-values (Independent t-test), ^bWithin the group P-values (Paired t-test), ^cBetween the groups P-values (Mann Whitney test), ^dWithin the group P-values (Wilcoxon test). BMI, body mass index.

Table 3

Daily energy and dietary intake in the two groups before and after intervention

Variable	Group	Baseline	Endpoint	p-value²
Energy (kcal)	UD	1476.09±279.014	1465.13±227.602	0.583
	placebo	1502.90±218.977	1501.14±226.504	0.888
	p-value¹	0.684	0.545
Carbohydrate (g)	UD	192.46±45.714	197.40±46.578	0.256
	placebo	200.03±48.844	197.60±44.679	0.586
	p-value¹	0.541	0.987
Cholesterol (mg)	UD	308.01±175.792	257.86±79.219	0.079
	placebo	251.63±97.841	266.87±110.342	0.478
	p-value¹	0.135	0.721
Monounsaturated fatty acids (g)	UD	13.075±3.611	13.688±2.857	0.413
	placebo	14.532±4.254	14.412±3.553	0.800
	p-value¹	0.176	0.408
Zinc (mg)	UD	8.02±2.185	7.72±1.463	0.546
	placebo	8.39±1.869	8.38±2.149	0.987
	p-value¹	0.492	0.176
Dietary Fiber (g)	UD	8.14±3.163	9.45±3.403	0.102
	placebo	10.29±4.840	10.07±4.677	0.777
	p-value¹	0.048^**	0.562
Vitamin A (RE)	UD	594.366±386.770	606.171±381.478	0.833
	placebo	480.405±360.242	495.656±320.345	0.826
	p-value¹	0.247	0.234
Vitamin C (mg)	UD	61.46±43.811	64.32±42.019	0.781
	placebo	83.08±57.459	68.67±47.868	0.151
	p-value¹	0.111	0.712
Protein (g)	UD	75.81±15.114	72.73±14.019	0.296
	placebo	74.20±16.214	78.33±17.754	0.225
	p-value¹	0.695	0.183
Fat (g)	UD	45.18±13.029	43.67±7.951	0.497
	placebo	46.27±13.322	45.30±13.067	0.352
	p-value¹	0.752	0.565
Saturated Fatty Acid (g)	UD	16.20±5.920	15.48±3.647	0.499
	placebo	16.12±5.992	16.74±5.815	0.145
	p-value¹	0.957	0.326
Polyunsaturated fatty acids (g)	UD	8.75±3.672	8.91±3.089	0.787
	placebo	8.97±3.530	7.54±3.355	0.006
	p-value¹	0.819	0.109
Vitamin D (μg)	UD	0.072±0.049	0.0587±0.0479	0.274
	placebo	0.056±0.039	0.060±0.0443	0.721
	p-value¹	0.183	0.912
Vitamin E (mg)	UD	2.89±1.576	3.948±1.695	0.006
	placebo	3.272±1.874	3.109±1.654	0.673
	p-value¹	0.398	0.062
Selenium (μg)	UD	0.090±0.030	0.091±0.028	0.826
	placebo	0.092±0.039	0.090±0.033	0.858
	p-value¹	0.836	0.961
DAH (g)	UD	0.011±0/014	0/017±0/014	0/084
	placebo	0/011±0/017	0/012±0/020	0/794
	p-value¹	0.937	0.279
EPA (g)	UD	0/006±0/005	0/006±0/005	0.879
	placebo	0.004±0.006	0.003±0.005	0.819
	p-value¹	0.240	0.118

All values are mean±SD. ¹Between the groups P-values (Independent t-test), ²Within the group P-values (Paired t-test).

Table 4

Biochemical factors in the two groups before and after intervention

Variable	Group	UD (n = 30)	Placebo (n = 29)	p-value²	Adjusted P-value³
ESR (mm/hr)⁴					0.991
	Baseline	2.473±0.973	1.898±1.151	0.043
	Endpoint	2.368±1.027	1.891±1.122	0.094
	p-value¹	0.479	0.940
TNF-α (pg/ml)					<0.001
	Baseline	3.19±0.979	2.82±0.754	0.112
	Endpoint	2.17±0.691	3.14±1.177	<0.001
	p-value¹	<0.001	0.135
Calprotectin (μg/g)					<0.001
	Baseline	538.133±202.196	438.414±204.346	0.065
	Endpoint	345.933±180.847	450.197±197.123	0.039
	p-value¹	<0.001	0.355

All values are mean±SD. ¹Within the group P-values (Paired t-test), ²Between the groups P-values (Independent t-test), ³Between the groups P-value based on ANCOVA with variables Age, Dietary fiber and baseline value of each variable as covariates, ⁴Log-transformed data were used for analysis.

4 Discussion

In the present study, 400 mg pure stinging nettle (Urtica dioica) extract taken 3 times a day, led to decreased TNF-α as well as calprotectin levels compared to the placebo group in people suffering from inflammatory bowel disease (IBD) over a period of 12 months. No side effects from taking nettle extract and/or placebo was reported during the intervention. IBDQ-9 score increased significantly in the Urtica Dioica group compared to placebo group so Urtica Dioica leaf extract is effective and useful for decreasing the clinical symptoms (gastrointestinal, systemic, emotional and social disturbances) in patients who consumed Urtica Dioica leaf extract [15]. Nettle leaf extract’s in improving IBD can be attributed to its anti-inflammatory, which lead to reduced levels of pro-inflammatory cytokines. In recent years, various potential effects of the stinging nettle extract have been observed in vivo in animal models of inflammatory bowel disease. So far, different studies have revealed various potential mechanisms on how nettle extract affects the inflammatory factors. Oberteris et al showed that caffeoylmalic acid, the main phenolic component of stinging nettle preparations, inhibits cyclooxygenase-derived reactions in a dose-dependent manner as well as having a partial inhibitory effect on 5-lipoxygenase-catalyzed reactions. In this manner, caffeoylmalic acid constrains the production of pro-inflammatory cytokines [16]. Stinging nettle may restrain cytokine production by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB). Among the numerous target genes of NF-KB are those encoding inflammatory and chemotactic cytokines such as interleukin-1 (IL-1), IL-2, IL-6, IL-8 and TNF and proinflammatory enzymes such as inducible nitric oxide synthase and cyclooxygenase-2. In many inflammatory diseases NF-KB activation is increased leading to the overexpression of proinflammatory gene products. NF-KB exists in the cytoplasm in the form of an inactive complex and has a binding pattern with inhibitory subunit IkB-α. The phosphorylation of this subunit leads to changes in its spatial transformation, complex separation, and NF-KB activation. Stinging nettle inhibits the phosphorylation and transformation of IKB- protein structure and results in the constancy of IkB-α inhibitory subunit and accordingly, prevents NF-KB activation. In this way, stinging nettle restrains a significant proinflammatory signaling pathway [12]. Konrad et al investigated the effect of IDS 30, a stinging nettle leaf extract, on disease activity of murine colitis in different models. They found that the long-term use of IDS 30 is effective in the prevention of chronic murine colitis. The mechanism which is used in this study includes the suppression of cytokine production by IDS 30 via an inhibition of NF-kB activation. the inhibition of NF-kB or the pro-inflammatory cytokine TNF-a by an antisense oligonucleotide strategy, led to an improvement in the disease [17]. Flavonoid, sesquiterpene, lactone, glytoksin, and other unknown components in stinging nettle extract may intermediate the inhibitory effects on NF-KB [12]. Carotenoids are also other constituents found in the leaves of stinging nettle which can induce anti-inflammatory effect. In the current study, the inhibitory impact of stinging nettle in the production of TNF-α could be a potential mechanism to explain its anti-inflammatory properties. Also, Konrad et al examined the healing effects of nettle leaf extract on chronic colitis. They observed reduced interleukin-1 beta (IL-1beta) in the stool and mucus cytokines in patients with chronic colitis. On the same basis, the authors concluded that long-term intake of nettle leaf preparations is effective in preventing chronic colitis in mice [17]. The results of our study about the anti-inflammatory effects of nettle are consistent with the previous studies. Rezazarandi et al also studied the anti-inflammatory effects of the stinging nettle alcoholic extract on male rats. The effect of nettle alcoholic extract was assessed in both experimentally-induced acute and chronic inflammation of the soft tissues (including foot and ear) and peritoneum. The researchers found that the extract can diminish the acute inflammation to a significant extent, and is associated with low but significantly reduced chronic inflammation in high doses [18]. These findings are in line with what was found in our study. Fecal calprotectin is a protein in neutrophil granulocytes and macrophages. It is stable and well-distributed in feces which make it reflect the entire state of the feces when we detect a part of it. Calprotectin is considered as one type of damage associated with molecular pattern protein (DAMP). It is released by activated innate immunity cells when cell stresses and damages, which also reflect the process of inflammation. More and more studies focus on fecal calprotectin in IBD and confirm its value in diagnosis, disease activity evaluation, effect evaluation, and relapse monitor [1]. Since no study has previously assessed the impact of stinging nettle on the fecal calprotectin, we have no basis with which to compare our findings, but the anti-inflammatory effect of stinging nettle was showed in more studies. Erythrocye Sedimentation Rate (ESR), as an acute phase reactant, was measured in this study. But its baseline and post-treatment values were not significantly different in either group, though the values were normal at baseline limiting the usefulness of this measure. ESR’s slow response to acute inflammation leads to normal values during the primary inflammatory process. It also has less sensitivity in an acute phase reaction compared to CRP [19]. Since no study has previously assessed the impact of stinging nettle on the ESR, we have no basis with which to compare our findings. Of the most valuable strong points of the present study is that it is the first double-blind randomized controlled trial, which has examined the dried hydroalcoholic extract of nettle leaves on the factors of fecal calprotectin, TNF-α and erythrocyte sedimentation rate in IBD patients. Also, since the randomized block design of classification was used in our study to assign participants into two groups, the residual destructive effects between 2 groups are similar. Among the limitations of this study, include the inability to control for stressors during the study. This can, in turn, be one of the contributing factors to the recurrence of symptoms of inflammatory bowel disease. Given the fact that the effects of nettle on these parameters has not previously been assessed in patients with inflammatory bowel disease, and also due to the variation in the preparation of nettle extract, terms of intervention, as well as the dose of preparations in animal studies, the mechanism of nettle’s impact in IBD cannot be expressed definitively. Therefore, there exists a need to do more research in order to identify the active ingredients in nettle and its anti-inflammatory mechanisms. It is also suggested that more studies with a larger sample size and longer term of intervention be conducted to explore definitive results confirming the remedial effects of nettle. This could open a new gate of using the herb as an adjunct to other methods in the treatment of patients with IBD.

5 Conclusion

In our series of patients, it seems that the dried hydroalcoholic extract of nettle leaves leads to decreased levels of TNF-α and stool calprotectin in patients with IBD demonstrating its anti-inflammatory effects. It seems that the dried hydroalcoholic extract of nettle may be recommended as an adjuvant therapy in people suffering from IBD.

Statement of Ethics

The study protocol was approved by the medical ethics committee of Iran University of Medical Sciences, Tehran, Iran with certificate No 25212. The research has also been registered in the Iranian Center for Clinical Trials (No. IRCT201410062709N30) and is accessible at the following website: www.irct.ir.

Disclosure Statement

The authors have no conflicts of interest to declare.

Funding sources

This study was funded by Iran University of Medical Sciences.

Author contributions

F.Sh., and Sh.NG designed the research; Sh.NG contributed to the data collection; L.J., Sh.NG performed the statistical analysis; F.Sh., and Sh.NG wrote the paper and has primary responsibility for the final content; and F.Sh., Sh.NG., Sh.A., A.F., Sh.h. interpreted the data, reviewed the paper, and revised it critically. All authors read and approved the final manuscript.

Footnotes

Acknowledgments

The authors would like to thanks the Barij Essence Pharmaceutical Company and the Pharmaceutical Sciences Research Center of Tehran University of Medical Sciences for preparing the hydroalcoholic extract of nettle and placebo. This research was financially supported by Vice chancellor for research of Iran University of Medical Sciences. This study is a part of results of Sh.Gorgani MSD dissertation.

References

Beaven

, Abreu

. Biomarkers in inflammatory bowel disease. Current Opinion in Gastroenterology. 2004;20(4):318–27.

Zhang

Y-Z

, Li

Y-Y

. Inflammatory bowel disease: Pathogenesis. World J Gastroenterol. 2014;20(1):91–9.

Monteleone

, Pallone

, Monteleone

. Interleukin-23 and Th17 cells in the control of gut inflammation. Mediators of Inflammation. 2009; 2009.

Molodecky

, et al. Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review. Gastroenterology. 2012;142(1):46–54. e42.

Vahedi

, et al.

Epidemiologic characteristics of 500 patients with inflammatory bowel disease in Iran studied from 2004 through 2007

Arch Iran Med. 2009;12(5):454–60.

Basso

, Zambon

C-F

, Plebani

. Inflammatory bowel diseases: From pathogenesis to laboratory testing. Clinical Chemistry and Laboratory Medicine. 2014;52(4):471–81.

Rutgeerts

. A critical assessment of new therapies in inflammatory bowel disease. Journal of Gastroenterology and Hepatology. 2002;17(s1):S176–S185.

Mahan

, Escott-Stump

, Raymond

. Medical Nutrition Therapy for Lower Gastrointestinal Tract Disorders, in Krause’s Food & The Nutrition Care Process, A. Yvonne, Editor. 2012, Elsevier Health Sciences. pp. 610-644.

Ahmadi

, et al. Therapeutic effects of Urtica dioica methanolic extract on gentamicin induced nephrotoxicity in rats. Koomesh. 2014;15(2):Pe220–Pe231, En31.

10.

Chrubasik

, et al. A comprehensive review on nettle effect and efficacy profiles, Part I: herba urticae. Phytomedicine. 2007;14(6):423–35.

11.

Guil-Guerrero

, Rebolloso-Fuentes

, Isasa

. Fatty acids and carotenoids from Stinging Nettle (Urticadioica L.). Journal of Food Composition and Analysis. 2003;16(2):111–9.

12.

Riehemann

, Behnke

, Schulze-Osthoff

. Plant extracts from stinging nettle (Urtica dioica), an antirheumatic remedy, inhibit the proinflammatory transcription factor NF-κB. FEBS Letters. 1999;442(1):89–94.

13.

Cetinus

, et al. The role of urtica dioica (urticaceae) in the prevention of oxidative stress caused by tourniquet application in rats. The Tohoku Journal of Experimental Medicine. 2005;205(3):215–21.

14.

Kayahan

, et al. Evaluation of early atherosclerosis in patients with inflammatory bowel disease. Digestive Diseases and Sciences. 2012;57(8):2137–43.

15.

Nematgorgani

, et al. Effects of Urtica dioica leaf extract on inflammation, oxidative stress, ESR, blood cell count and quality of life in patients with inflammatory bowel disease. Journal of Herbal Medicine. 2017;9:32–41.

16.

Obertreis

, et al. Anti-inflammatory effect of Urtica dioica folia extract in comparison to caffeic malic acid. Arzneimittel-Forschung. 1996;46(1):52–6.

17.

Konrad

, et al. Ameliorative effect of IDS 30, a stinging nettle leaf extract, on chronic colitis. International Journal of Colorectal Disease. 2005;20(1):9–17.

18.

Rezazarandi

, Khalili

, Vahidi

. Anti-inflammatory effect of alcoholic Urtica dioica extract in male NMRI rats. Journal of Basic and Clinical Pathophysiology. 2012;1(1):24–8.

19.

Harrison

. Abnormal laboratory results: Erythrocyte sedimentation rate and C-reactive protein. Australian Prescriber. 2015;38(3):93.