Anti-inflammatory and/or immunomodulatory activities of Uncaria tomentosa (cat’s claw) extracts: A systematic review and meta-analysis of in vivo studies

Background Uncaria tomentosa (Willd. ex Schult.) DC. (Rubiaceae) is traditionally used by Amazonian indigenous groups to treat inflammatory diseases. To date, there are no systematic reviews and meta-analyses on the use of U. tomentosa for inflammation control in animals supporting the traditional knowledge about this species. This study was conducted to evaluate the effect of U. tomentosa extracts in modulating inflammatory mediators and to determine which types of inflammatory diseases can be treated by this species. Methods We conducted a systematic review and meta-analysis of preclinical studies published before 26 July 2023, identified in PubMed, Embase, and Scopus. Four independent reviewers extracted the data and assessed the risks of bias. The effects of U. tomentosa on inflammatory diseases and the inflammatory mediators involved were extracted from the studies. Standardized mean differences (SMD) and 95% confidence intervals (95%CI) of the outcomes were estimated. The meta-analyses were conducted using RevMan 5.4 (Cochrane Collaboration). This protocol was registered in PROSPERO (CRD42023450869). Results Twenty-four of 523 studies were included. U. tomentosa extracts decreased the cytokines interleukin (IL)-6 (SMD: −0.72, 95%CI: −1.15, −0.29, p = 0.001) and transcription factor nuclear factor kappa-B (NF-κB) (SMD: −1.19, 95%CI: −1.89, −0.48, p = 0.001). However, the extracts did not significantly alter IL-1 (SMD: −0.16, 95%CI: −0.87, +0.56, p = 0.67), IL-10 (SMD: −0.05, 95%CI:–0.35, 0.45, p = 0.80), or tumor necrosis factor-alpha (TNF-α) levels (SMD: 0.18, 95%CI: −0.25, 0.62, p = 0.41). Conclusion Many extracts of stem bark, roots, and leaves of U. tomentosa, mostly aqueous and hydroethanolic, exhibited anti-inflammatory and/or immunomodulatory activities and low toxicity. The extracts decreased NF-κB and IL-6. These findings suggest that this species has the potential to treat inflammatory diseases in which these markers are increased, according to the ethnopharmacological use. These activities are not related to a specific class of compounds. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=450869, Identifier CRD42023450869.

Therefore, the present systematic review aimed to synthesize the knowledge on the preclinical anti-inflammatory and/or immunomodulatory activities of different extracts of U. tomentosa evaluated in different in vivo models and on their main mechanisms of action, including a meta-analysis of their effects on selected inflammatory mediators.

PICOS question and strategy
The review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (Page et al., 2021) and was previously registered in PROSPERO (CRD42023450869).
The research questions were as follows: against which inflammatory diseases are U. tomentosa extracts effective, as assessed in in vivo models, and which inflammatory mediators are involved?
The PICOS strategy (problem, intervention, control, outcomes, and study design) was built as follows: P: inflammatory diseases; I: treatment with U. tomentosa extracts; C: no treatment or placebo (vehicle); O: levels of inflammatory mediators; and S: in vivo preclinical studies.

Study selection and eligibility criteria
Three independent reviewers (GA, PPGA, and AMSP) analyzed the search results and selected potentially relevant studies after reading their titles and abstracts, and using the Rayyan software (Ouzzani et al., 2016).Disagreements were resolved by consensus among the reviewers, with the assistance of a fourth reviewer (JSC), when necessary.The following inclusion criteria were applied: in vivo study of inflammatory diseases in animals, administration of U. tomentosa extracts versus placebo or no treatment, assessment of effects on inflammatory mediators or cytotoxic effects, and published in English, Portuguese, or Spanish.Studies using U. tomentosa extracts mixed with other species or isolated substances, non-controlled studies, narrative or scoping review articles, abstracts, conference papers, editorials/letters, and case reports were excluded.Additionally, the reference lists of all selected studies were hand searched to identify additional primary studies for inclusion.

Data extraction
The following data were extracted from the included articles: author, part of the plant used, type of extract (solvent, method, and extraction time), concentration and/or dose, animal species, results (cytotoxicity and inflammatory mediators), and conclusions.Treatment effects for continuous outcomes were extracted as mean differences (MD) plus standard deviations (SD), which could also be estimated from standard errors or confidence intervals.In the studies where such values were not reported, they were estimated from charts using ImageJ software (National Institutes of Health, Bethesda, USA).The authors of the included studies were contacted when necessary (when some data or articles were not available).

Quality assessment
For the risk of bias, two investigators (AMSP and FC) independently reviewed the selected studies according to a modified CAMARADES checklist (Macleod et al., 2004) and reported the risks of bias in a table.After the initial analysis, the authors reassessed the articles analyzed previously by each other.Any discrepancies were resolved by a third author (GMA) after discussion with the team.The information is presented as a risk of bias summary (Table 2).

Statistical analysis
Review Manager 5.4 (Nordic Cochrane Centre, The Cochrane Collaboration, Copenhagen, Denmark) was used for statistical analysis.Heterogeneity was evaluated using Cochrane's Q test and I 2 statistics, with a p-value <0.10 and I 2 > 50% being considered significant, respectively.We built a fixed-effects model for endpoints with I 2 < 50% (low heterogeneity).In the case of pooled outcomes with high heterogeneity, a random-effects model was applied.The results were reported as standardized mean differences (SMD) and their respective 95% confidence intervals (95%CI).We also performed a subgroup analysis to assess the effects of different extracts (aqueous, hydroethanolic, and ethanolic) on the outcomes.

Study selection and characteristics
The initial search retrieved 523 studies published between 1989 and 2023; of these, 124 were found in PubMed, 279 in Embase, and 120 in Scopus.There were 217 duplicated articles, and 273 were excluded after reading the title and the abstract.Thus, 33 studies were selected for fulltext reading.Two additional articles were later excluded because they were conference abstracts, whereas seven could not be obtained in full.The final number of articles was 24 (Figure 1).The rats treated with UT took less time to reach the platform, and the STZ-treated rats that received UT spent significantly more time in the target quadrant than vehicle-treated rats.UT treatment inhibited the hyperphosphorylation of tau protein at the sites of S396 and S404.UT suppressed the elevated levels of IL-1β, IL-6, and TNF-α.UT exerted antioxidant effects by increasing the activities of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) and the expression of the HO-1 protein UT ameliorated cognitive impairments in SZT-induced Alzheimer's disease rats.This activity has been attributed in part to the protective effects of the alkaloids rhynchophylline and isorhynchophylline XU et al., 2021 (China) In this, 1,000 g of stem was macerated in 10 L of 70% ethanol:water for 24 h at room temperature and then extracted in an ultrasonic bath for 1 h.The material was freeze-dried.The yield of the extracts was 13.88%.The contents of alkaloids were as follows: rhynchophylline (0.278%), isorhynchophylline (0.531%), corynoxeine (0.010%), and isocorynoxeine (0.028%) Frontiers in Pharmacology frontiersin.org07 Research workers from eleven countries across the American, European, and Asian continents have published preclinical studies with extracts of U. tomentosa, demonstrating the scope of interest and use of this plant in the world.The countries that have contributed most to the publications of in vivo studies are Brazil (41.6%), the United States (12.5%), and Peru (12.5%).
The results of the included studies show that U. tomentosa extracts are well tolerated by the animals and have little or no toxicity at the doses evaluated (Table 1).

Interleukin-1 (IL-1)
Three studies that used four animal models and aqueous and hydroethanolic extracts of U. tomentosa were analyzed.The results showed no effect on IL-1 levels (SMD: −0.16, 95%CI: −0.87, +0.56, p = 0.67).The overall assessment of the data revealed high heterogeneity (p < 0.001, I 2 = 90%) (Figure 2).Forest plot of the efficacy of Uncaria tomentosa extracts on levels of interleukin (IL)-6.UT has anti-asthmatic activity comparable to methylprednisolone in mice 100 g of ground leaves were extracted in 1 L of ethanol for 24 h at a temperature of 37 °C, and after evaporation, the dry extract was resuspended in ethanol (1 L) Forest plot of the efficacy of Uncaria tomentosa extracts on levels of interleukin (IL)-10.

Quality assessment
There was a high risk of bias regarding sample size calculation, missing outcome data (excluded animals), and possible conflicts of interest, and a moderate risk of bias for temperature control, the number of animals appropriate to the model, and compliance with animal welfare regulations.The risk of bias was low for peer reviewing and the disease model (Table 2).Publication bias was high for IL-6 as the funnel plot is shifted toward the left (data not shown).

Discussion
In this systematic review and meta-analysis, U. tomentosa decreased the levels of IL-6 and NF-κB but not of IL-1, IL-10, or TNF-α, in animal models of inflammatory diseases.These models included asthma, diabetes, arthritis, obesity, gastric ulcers, and intestinal diseases (Azevedo et al., 2018;Mendes et al., 2014;Castilhos et al., 2015;Araujo et al., 2018;Sandoval et al., 200;Sandoval-Chacón et al., 1998).Although some ethnic groups use this species for religious purposes only, such as the Asháninkas (Keplinger et al., 1999), our findings confirm the anti-inflammatory and/or immunomodulatory activities of the species, as advocated by other indigenous groups of the Amazon.
Inflammatory diseases are usually accompanied by a significant production of reactive oxygen and nitrogen species, as well as the expression of pro-inflammatory cytokines, most notably IL-1, IL-6, and TNF-α; the anti-inflammatory cytokine IL-10; and the  activation of NF-κB (Hata et al., 2004;Kany et al., 2019).The studies using extracts of U. tomentosa included in this review show decreases in levels of IL-6.These findings explain, at least in part, the anti-inflammatory activity of this species.Most of the in vivo studies have demonstrated, directly or indirectly, that the inhibition of the activity of the transcription factor NF-κB by the extracts was accompanied by decreased levels of IL-1 or IL-6 (Aguilar et al., 2002;Sandoval et al., 2002;Sandoval et al., 2002;Lozada-Requena et al., 2015;Araujo et al., 2018;Azevedo et al., 2018;Elgawish et al., 2019;XU et al., 2021).Targeting IL-6 is an important strategy to treat inflammatory diseases.Therapeutic monoclonal antibodies against cytokines or their receptors, such as tocilizumab (Ohsugi, 2020), are among the most effective, yet very expensive, therapies.
Another important aspect to be considered is that U. tomentosa extracts preserve CD4 + and CD8 + T cells and possibly stimulate cytokines that favor the polarization of CD4 + Th2 cells.These cells play an important role in autoimmune diseases, such as rheumatoid arthritis, by modulating the excessive activity of Th1 cells (Domingues et al., 2011a;Domingues et al., 2011b;Lozada-Requena et al., 2015;Azevedo et al., 2018).In fact, an extract of U. tomentosa was superior to placebo in 50 patients with rheumatoid arthritis, decreasing the number of painful joints, morning stiffness, pain intensity, and joint edema (Mur et al., 2002).Therefore, U. tomentosa should be more broadly investigated for the treatment of autoimmune diseases.
Interestingly, U. tomentosa extracts can either stimulate or inhibit the release of different cytokines, depending on the animal's health status or the modeled disease.This was observed for IL-1: in the study by Eberlin et al. (2005), a sepsis model, U. tomentosa increased IL-1 levels throughout the infection; in the study by Araujo et al. (2018), a nonalcoholic fatty liver disease model, extracts of U. tomentosa decreased IL-1 levels.Some authors refer to this regulatory effect as immunomodulation (Domingues et al., 2011a;Domingues et al., 2011b;Lozana-Requena et al., 2015;Elgawish et al., 2019;Aldayel et al., 2021;Xu et al., 2021).In our study, U. tomentosa did not alter IL-10 levels in a sepsis model, suggesting immunomodulatory activity in infectious diseases.
The chemical composition of U. tomentosa extracts is often diverse.In addition to alkaloids, the presence of other compounds such as quinovic acid and polyphenols, which contribute to the pharmacological activity of the species, has been reported (Aquino et al., 1991;Yépez et al., 1991;Dietrich et al., 2014).Furthermore, synergism between different compounds usually contributes to the pharmacological effect of medicinal plants (Carmona and Pereira, 2013).
Interestingly, 54% of the studies included in this review used extracts directly related to traditional formulations (aqueous and hydroethanolic extracts).So important is the traditional use that only these studies could be included in the meta-analysis.On the other hand, although 46% of the studies have used extracts not directly related to traditional formulations, they are important because they can help elucidate mechanisms of action (Table 1).
Overall, the studies were at high risk of bias because most of them did not report sample size calculations, the number of excluded animals, or possible conflicts of interest.The main limitation of this study is the small number of studies included in the meta-analysis.Therefore, as more studies are conducted, other pharmacological effects of this species might be demonstrated.Another limitation is the considerable variation in the plant parts used and in the chemical profiles of the extracts, which is a fact that makes interpretation of these results challenging.Nevertheless, as preclinical studies confirmed the anti-inflammatory and/or immunomodulatory effects and the low toxicity of U. tomentosa extracts, clinical studies should be encouraged.

Conclusion
Extracts of the stems, stem barks, roots, and leaves of U. tomentosa, mostly aqueous and hydroethanolic extracts, exhibited antiinflammatory and/or immunomodulatory activities and low toxicity.These extracts decreased NF-κB and the cytokine IL-6 without altering IL-1, IL-10, or TNF-α.These findings suggest that this species has the potential to treat inflammatory diseases associated with increased IL-6 and/or NF-κB, according to the ethnopharmacological use.These activities are not related to a specific class of compounds.

FIGURE 1 PRISMA
FIGURE 1 PRISMA flow diagram of study screening and selection.

FIGURE
FIGUREForest plot of the efficacy of Uncaria tomentosa extracts on levels of interleukin (IL)-1.

FIGURE 6
FIGURE 6plot of the efficacy of Uncaria tomentosa extracts on levels of tumor necrosis factor kappa B (NF-κB).

TABLE 1
Characteristics of the included studies regarding plant part, type of extract, disease model, treatment, and main results.
of aqueous extract per gram of dry bark.Chemical composition: not reported Route: oral.Frequency and duration: ad libitum for 8 days Stem bark Male BALB/c mouse model of listeriosis.Doses: 10, 50, 100, 150, and 200 mg/kg.Route: oral.Frequency and duration: 7 days prior to infection Treatment with 100 mg/kg of UT decreased levels of IL-1 at 72 h and maintained elevated levels of IL-6 throughout the experiment.The dose of UT indirectly modulates immune activity by inducing a greater reserve of myeloid progenitors in the bone marrow as a result of the release of Eberlin et al., 2005 (Brazil) (Continued on following page)

TABLE 1 (
Continued) Characteristics of the included studies regarding plant part, type of extract, disease model, treatment, and main results.

TABLE 1 (
Continued) Characteristics of the included studies regarding plant part, type of extract, disease model, treatment, and main results.

TABLE 1 (
Continued) Characteristics of the included studies regarding plant part, type of extract, disease model, treatment, and main results.

TABLE 1 (
Continued) Characteristics of the included studies regarding plant part, type of extract, disease model, treatment, and main results.

TABLE 2 (
Continued) Risk of bias among the included studies.