Phytomedicine 79 (2020) 153327

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Phytomedicine
journal homepage: www.elsevier.com/locate/phymed

Uncaria tomentosa reduces osteoclastic bone loss in vivo T

a,b,c d c c
Vilma Lima , Iracema Matos Melo , Thaise Mayumi Taira , Liseth Yamile Wilches Buitrago ,
Cristiane Sá Roriz Fontelesd, Luzia Kalyne Almeida Moreira Leale, Ana Sheila de Queiroz Souzae,
Talysson Silva Almeidae, Raimundo Nogueira da Costa Filhof, Manoel Odorico Moraesa,
Fernando Queiroz Cunhab, Sandra Yasuyo Fukadac,
⁎

a
School of Medicine, Department of Physiology and Pharmacology, Federal University of Ceara, Fortaleza, Brazil
b
School of Medicine of Ribeirao Preto, Department of Pharmacology, University of São Paulo, Ribeirao Preto, Brazil
c
School of Pharmaceutical Sciences of Ribeirao Preto, Department of BioMolecular Sciences, University of São Paulo, Av. Café s/n - Bloco S, 3o andar, sala 90A-S, CEP:
14040-903 Ribeirao Preto, SP, Brazil
d
School of Pharmacy, Nursing and Dentistry, Department of Dentistry, Federal University of Ceara, Fortaleza, Brazil
e
School of Pharmacy, Nursing and Dentistry, Department of Pharmacy, Federal University of Ceara, Fortaleza, Brazil
f
Department of Physics, Federal University of Ceara, Fortaleza, Brazil

ARTICLE INFO ABSTRACT

Keywords: Background: The genus Uncaria (Rubiaceae) has several biological properties significant to human health.
Cat's claw However, the mechanisms underlying the protective effect of this plant on bone diseases are uncertain.
Bone resorption Purpose: The present study investigated the role of Uncaria tomentosa extract (UTE) on alveolar bone loss in rats
Osteoclast and on osteoclastogenesis in vitro.
TRAP
Materials: UTE was characterized by an Acquity UPLC (Waters) system, coupled to an Electrospray Ionization
NFATc1
(ESI) interface and Quadrupole/Flight Time (QTOF, Waters) Mass Spectrometry system (MS). The effect of UTE
Cathepsin K
treatment for 11 days on the ligature-induced bone loss was assessed focusing on several aspects: macroscopic
and histological analysis of bone loss, neutrophil and osteoclast infiltration, and anabolic effect. The effect of
UTE on bone marrow cell differentiation to osteoclasts was assessed in vitro.
Results: The analysis of UTE by UPLC-ESI-QTOF-MS/MS identified 24 compounds, among pentacyclic or tet-
racyclic oxindole alkaloids and phenols. The administration of UTE for 11 days on ligature-induced rat atte-
nuated the periodontal attachment loss and alveolar bone resorption. It also diminished neutrophil migration to
the gingiva tissue, demonstrated by a lower level of MPO. UTE treatment also decreased the level of RANKL/OPG
ratio, the main osteoclast differentiation-related genes, followed by reduced TRAP-positive cell number lining
the alveolar bone. Additionally, the level of bone-specific alkaline phosphatase, an anabolic bone marker, was
elevated in the plasma of UTE treated rats. Next, we determined a possible direct effect of UTE on osteoclast
differentiation in vitro. The incubation of primary osteoclast with UTE decreased RANKL-induced osteoclast
differentiation without affecting cell viability. This effect was supported by downregulation of the nuclear factor
activated T-cells, cytoplasmic 1 expression, a master regulator of osteoclast differentiation, and other osteoclast-
specific activity markers, such as cathepsin K and TRAP.
Conclusion: UTE exhibited an effective anti-resorptive and anabolic effects, which highlight it as a potential
natural product for the treatment of certain osteolytic diseases, such as periodontitis.

Abbreviations: ABC, Alveolar bone crest; ALT, Alanine transaminase; AST, Aspartate transaminase; BALP, Bone-specific alkaline phosphatases; CEJ, Cement-enamel
junction; EDTA, Ethylenediaminetetraacetic acid disodium salt dehydrate; ESI, Electrospray Ionization; IL, Interleukin; MPO, Myeloperoxidase; MS, Mass
Spectrometry system; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide; NFATc1, Nuclear Factor of Activated T Cells 1; OPG, Osteoprotegerin;
PMN, Polymorphonuclear neutrophils; qPCR, quantitative real-time reverse transcriptase-polymerase chain reaction; QTOF, Interface and Quadrupole/Flight Time;
RANKL, Receptor activator of nuclear kappa ligand; SEM, Standard error of the mean; SPE, Solid-phase extraction; TNF, tumor necrosis factor; TRAP, Tartrate-
resistant acid phosphatase; UTE, Uncaria tomentosa extract; α-MEM, α-minimum essential medium
⁎
Corresponding author.
E-mail address: sfukada@usp.br (S.Y. Fukada).

https://doi.org/10.1016/j.phymed.2020.153327
Received 9 December 2019; Received in revised form 10 July 2020; Accepted 10 August 2020
0944-7113/ © 2020 Elsevier GmbH. All rights reserved.
V. Lima, et al.
Introduction
The Uncaria tomentosa (Willd. ex Shult) DC. is a Rubiaceae, which is
popularly known as Cat's claw due to its thorns. This plant grows in the
upper Amazon region of Peru and neighboring countries, as well as in
other tropical areas of Central and South America (Zhang et al., 2015).
Native people of these regions prepare the bark and root of this plant,
using a decoction method, and treat several inflammatory diseases in
stomach and urinary tract (Keplinger et al., 1999; Heitzman et al.,
2005; Sandoval et al., 2008; Bors et al., 2011; Nogueira Neto et al.,
2011; Ciani et al., 2018). Several studies have shown that part of the
anti-inflammatory and antioxidant activities of Uncaria tomentosa ex-
tract (UTE) is generated by polyphenols present in the extract, in-
cluding flavonols, phenol acids and proanthocyanidins (Glyn-
Jones et al., 2015; Pavei et al., 2010). More recently, U. tomentosa has
emerged as a potential alternative treatment considering its benefits in
bone diseases. A randomized, double-blind, 2 phase study for the
treatment of rheumatoid arthritis patients evidenced that the extract of
U. tomentosa reduced joint swelling and pain compared to the placebo
group (Mur et al., 2002). However, its therapeutic efficacy on bone
injury remains uncertain, and there is no consensus on the mechanisms
underlying the antiresorptive bone properties of U. tomentosa
(Sandoval-Chacón et al., 1998; Sandoval et al., 2002, 2008; Allen-
Hall et al., 2010).
Periodontitis is a disease that combines inflammation and bone re-
sorption. This type of inflammation results from an oral dysbiosis, and
the failure of local inflammation resolution leads to the alveolar bone
destruction (Van Dyke, 2017). During the inflammatory response ob-
served in periodontitis, innate immune defenses are activated against
periodontal pathogens, triggering cellular and vascular changes. Poly-
morphonuclear neutrophils (PMN), macrophages, T-lymphocytes, and
B-lymphocytes are recruited and activated, stimulating the production
of inflammatory mediators (Barbato et al., 2015). An array of these
mediators, including cytokines, such as tumor necrosis factor (TNF-α),
interleukin (IL)-1β, IL-6 and others, lead to osteoclastic activation
(Redlich et al., 2012), which in turn induce periodontal tissue break-
down.
Inflammation and bone loss are key pathophysiological events ob-
served in periodontitis (Chakravarti et al., 2009), and the previous
description of an anti-inflammatory and bone protective properties of
UTE allowed us to hypothesize that this natural product could play an
important role controlling the osteoclastogenesis and periodontitis in-
duced bone resorption. Therefore, in the present study, we aimed to
evaluate a potential pharmacological effect and the mechanisms of UTE
on osteoclastic alveolar bone loss induced in animals under thread li-
gature-induced periodontitis. Moreover, the direct effect of UTE on
osteoclast differentiation and activation was also investigated in vitro.
Material and methods
Plant extract
The Uncaria tomentosa extract (UTE) from barks (Farmafórmula,
Fortaleza, CE, Brazil) used in this study is registered in the Brazilian
Genetic Patrimony (AC3EF45).
Chemical characterization by UPLC-ESI-QToF-MS/MS
The UTE (150.8 mg) was submitted to clean-up by solid-phase ex-
traction (SPE) (Supelco® C18–500 mg/ 6 ml; Sigma-Aldrich) before the
analysis performed by an Acquity UPLC (Waters, USA) system, coupled
to an Electrospray Ionization (ESI) interface and Quadrupole/Flight
Time (QTOF, Waters) Mass Spectrometry system (MS). The analysis of
UTE compounds was based in a previous study (Wang et al., 2014). The
compounds were characterized through molecular formula provided by
MassLynx 4.1 software from their accurate masses (error < 5 ppm),
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Phytomedicine 79 (2020) 153327
isotopic patterns (i-fit), and MS fragmentation pattern as well as a
chemotaxonomic survey. Also, compounds were assigned by compar-
ison with reference standards when available and public databases,
such as ChemSpider, PubChem, SciFinder e KNApSAck (Supplementary
data).
Animals
Sixty male Wistar rats (6 weeks old) from the Federal University of
Ceara were used throughout the experiments, and three animals were
kept in each cage. The room temperature and light/dark cycles (12 h)
were controlled. The animals received food and water ad libitum. The
Institutional Ethics Committee of the Federal University of Ceara
(Fortaleza, CE, Brazil) (P. 42/08) approved all of the protocols em-
ployed in this study. Wild-type C57BL/6 mice (8 weeks old) were used
for the in vitro assays. The Committee on Ethics in Animal
Experimentation at the University of Sao Paulo (Ribeirao Preto, SP,
Brazil) approved the in vitro protocols. All animals were kept in the
same conditions
Ligature-induced periodontitis model
A model for the induction of periodontitis in rats, as previously
described, was used (de Lima et al., 2000; Lima et al., 2004). Nylon
(3.0) thread ligature was placed around the second left upper molar of
the anesthetized rats with 20 mg/kg xylazine (Xilazin, Syntec do Brasil
Ltda, Santana do Parnaíba, SP, Brazil) and 90 mg/kg ketamine (Ce-
tamin, Syntec do Brasil Ltda, Santana do Parnaíba, SP, Brazil), in-
tramuscular. The ligature remained subgingival on the palatal side and
supragingival on the buccal side, due to the knot on the buccal side of
the tooth.
Experimental groups
The ligature-induced periodontitis rats were divided into four
groups, which received three different subcutaneous doses of UTE
(9 mg/kg, 27 mg/kg or 81 mg/kg) or sterile saline as a vehicle (non-
treated group). The contralateral maxilla with no ligature of the saline-
treated group was considered as the control group.
The UTE was administered for 11 days, and the first treatment was
performed 30 min before the ligature placement. All the analysis was
performed on day 11 based on a previous experiment that showed the
peak of the bone resorption at this day (data not shown).
Measurement of alveolar bone loss
The maxillae previously fixed in 4% neutral formalin and washed
with 1% sodium hypochlorite were dissected and stained with aqueous
methylene blue (1%) to highlight the cement-enamel junction (CEJ)
and alveolar bone crest (ABC). Hemimaxillae specimens with period-
ontitis were photographed and analyzed using computer software
(ImageJ 1.32j, National Institute of Health; EUA). The bone loss mea-
surements were performed considering the CEJ and ABC area of the
three molars (Kuhr et al., 2004). The area was expressed as square
millimeters (mm2).
Histomorphometry and immunohistochemical staining analysis of the
periodontium
Maxillae of rats were demineralized with 10% buffered ethylene-
diaminetetraacetic acid disodium salt dihydrate (EDTA) and processed
to paraffin for histological analysis. Then, 4 μm-thick sections were cut
along the molars on the sagittal plane for Mallory trichrome staining.
Only slices that exhibited the first, second, and third molars in the same
plane were considered.
For the attachment levels, the distance between CEJ and the base of
V. Lima, et al.
the gingival sulcus (periodontal pocket) of the mesial portion of the
second molar was measured and expressed in millimeters (Sima et al.,
2016).
For immunohistochemistry, slices were processed on slides with
poly-L-lysine for receptor activator of nuclear factor-κB ligand
(RANKL), osteoprotegerin (OPG) and tartrate-resistant acid phospha-
tase (TRAP) immunostainings (Santa Cruz Biotechnology, Dallas, TX,
USA). The deparaffinized sections were treated with an endogenous
peroxidase blockade using H2O2, and incubated with polyclonal pri-
mary antibodies anti-RANKL (1:100), anti-OPG (1:100) and anti-TRAP
k17 (1:100) except for the negative controls. After washing, incubation
with anti-goat biotinylated secondary antibodies (1:300), as well as
with streptavidin-conjugated and peroxidase diluted in PBS solution.
The immunoperoxidase reaction disclosure was done with diamino-
benzidine/hydrogen peroxide chromogen and contra-stained with
Harris hematoxylin. All the reactions were accompanied by negative
control. The quantitative analysis was performed with appropriated
software (Infinity Analyze Software, Ottawa, ON, CA). The regions of
interest involved the periodontal ligament and margin of the alveolar
bone in the furcation area of the second molar. Mononuclear cells ex-
hibiting cytoplasmic positivity for RANKL and OPG were counted in
five fields. Brownish stained cells with multiple nuclei and irregular
branches were considered as being TRAP+-multinucleated osteoclasts
(mature osteoclasts). The values were expressed as the mean of the
stained cell per mm2 and compared between different groups.
Analyses of gingival tissue
The gingival tissues of the rats were used for the myeloperoxidase
(MPO) activity assays or quantitative real-time reverse transcriptase-
polymerase chain reaction (qPCR). The gingival tissue of the con-
tralateral hemi-maxilla of the rats that received saline was used as the
control.
A modified version of Bradley et al. (1982) was used following the
kinetics of the reaction front to hydrogen peroxide and coloring reagent
composed of ortho-dianisidine hydrochloride and H2O2 in phosphate
buffer to determine the MPO activity, a marker for neutrophils in the
inflamed tissue. The absorbance was measured at a wavelength of
450 nm.
For the qPCR, total RNA was extracted (Promega SV Total Isolation
System, Madison, WI, USA) from the gingival tissue. The com-
plementary DNA (cDNA) was synthesized by a reverse transcription
reaction (High capacity cDNA Reverse Transcription kit 4,368,813,
Applied Biosystems, Carlsbad, CA, USA) using 1 μg of total RNA. qPCR
was carried out on the sequence detection system (StepOnePlus,
Applied Biosystems, Carlsbad, CA, USA) using TaqMan primers and
probes. The tumor necrosis factor (ligand) superfamily, member 11
(Rn00589289_m1; Tnfsf11; Rankl) and tumor necrosis factor receptor
superfamily member 11b (Rn00563499_m1; Tnfrsf11b; Opg) genes were
evaluated, and glyceraldehyde-3-phosphate dehydrogenase
(Rn01775763_m1; Gapdh) was used as an endogenous control (re-
ference gene).
Systemic parameters analyses
Dosages of bone-specific alkaline phosphatases (BALP) (Moss and
Whitby, 1975), hepatic transaminases, urea, and creatinine were mea-
sured on blood samples of animals collected before and after 11 days of
ligature placement. All the reactions were performed following the
manufacturer's recommendations (Labtest, Lagoa Santa, MG, Brazil).
The ratios between the wet weights of the liver or kidney and total body
masses were expressed as an index of the respective organ to determine
UTE toxicity.
3
Phytomedicine 79 (2020) 153327
Culture of the osteoclasts
Bone marrow cells were aseptically flushed from the femur and tibia
of 5 C57BL/6 mice with α-minimum essential medium (α-MEM) sup-
plemented with 10% fetal bovine serum (v/v), 100-units/ml of peni-
cillin and 100 mg/ml of streptomycin (Invitrogen Corporation,
Carlsbad, CA, USA) over 3 days. The adhered cells (pre-osteoclasts)
were plated in 96-well microtiter plates at 2 × 104 cells/well with M-
CSF (30 ng/ml) and RANKL (10 ng/ml; R&D Systems, Minneapolis, MN,
USA), and UTE (3 μg/ml, 9 μg/ml and 27 μg/ml). The formation of
mature multinucleated osteoclasts (more than three nuclei) was con-
firmed by TRAP staining. To test the effect of UTE on cell viability, the
3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-
based cytotoxicity assay was used with absorbance values set at 570 nm
(Sigma Aldrich, St. Louis, MO, USA) (Martins et al., 2009). For qPCR
and western blotting, osteoclast precursors were plated in 24-well mi-
crotiter plates at 2 × 105 cells/well with the same M-CSF and RANKL
concentrations and 27 μg/ml UTE.
Statistical analysis
The data were expressed as mean ± standard error of the mean
(SEM) of 6 animals per group. Comparisons between 2 groups were
performed using the unpaired 2-tailed Student's t-test. When appro-
priate, univariate analysis of variance (ANOVA) followed by
Bonferroni's test was used for multiple comparisons of means. A p-value
of < 0.05 was considered to be significant. The statistical analysis was
performed using GraphPad Software (Prism 6 for Mac OS X, San Diego,
CA, USA).
Results
Chemical characterization of Uncaria tomentosa extract
Uncaria tomentosa extract (UTE) was analyzed by UPLC-ESI-QTOF-
MS/MS, and as shown in the chromatograms (Fig. 1A and B), twenty-
four compounds were identified, mainly alkaloids and phenols. The
chromatography run show the positive ionization mode (A) and nega-
tive ionization mode (B). The majority of the compounds were penta-
cyclic or tetracyclic oxindole alkaloids such as mitraphyline (10),
rhynchophylline (12) and isorhynchophylline (15), besides indole al-
kaloids such as 5-carboxystrictosidine (7), corynantheine (18), hirsu-
teine (19) and hirsutine (20). The phenols detected included chloro-
genic acid (3) and vincosamide 11,6-di-glucopyranosidec Rutin (2),
supplementary Table 1.
Administration of Uncaria tomentosa extract reduce inflammatory-induced
bone loss in periodontitis animal model
The present study investigated the effect of UTE on an inflammatory
induced bone loss model. Initially, we show that the ligature-induced
periodontitis groups (NT group) exhibited great alveolar bone de-
struction evidenced by the root exposure (Fig. 2A). The CEJ/ABC area
(indicated by the dotted line) in the NT group (Fig. 2A) was 5.7-fold
higher than the respective area in the control group (Fig. 2B). Macro-
scopically, it was demonstrated that the treatment with UTE at con-
centrations of 9 mg/kg, 27 mg/kg and 81 mg/kg reduced the alveolar
bone resorption area by 19.7%, 36.7%, and 45.9%, respectively, when
compared to NT group (Fig. 2A and B).
Measurements of the distance between CEJ and the attached peri-
odontal ligament fibers were used to evaluate the level of periodontal
attachment further. Histometric analysis (dotted arrows in Fig. 2C)
indicates that a severe attachment loss observed in the non-treated
group was inhibited by UTE treatment (Fig. 2C and D). Moreover, the
non-treated periodontitis group exhibited a higher level of neutrophil
infiltration in the gingival tissue compared to control, evidenced by
V. Lima, et al.
increased MPO activity (Table 1). Interestingly, UTE treatment (81 mg/
kg) significantly reduced MPO activity by 46% (Table 1).
Subsequently, we assessed the level of RANKL and OPG expressed in
the periodontal tissue and further associated with the osteoclastogen-
esis and alveolar bone loss. The expression of mRNA for Rankl (Tnfsf11)
was higher in the periodontitis group, while mRNA expression of Opg
(Tnfrsf11b) was not altered (Fig. 3A) by ligature-induced periodontitis.
The UTE treatment slightly reduced the expression of RANKL and in-
creased OPG expression. Together, the treatment with UTE resulted in a
significant reduction of the ratio Rankl/Opg (75%) compared with the
NT group, which was associated with reduced osteoclastogenesis
(Fig. 3A).
In order to confirm mRNA results, protein expression of RANKL and
OPG was evaluated by immunohistochemistry. The ligature-induced
periodontitis groups (NT) showed an increased number of RANKL+
cells in the periodontal ligament close to the dentin, coupled with a
reduced OPG+ cell count compared to control (Fig. 3B–D). Interest-
ingly, the treatment with UTE (81 mg/kg) significantly reduced the
number of RANKL+ cells, while increased OPG+ cells in the period-
ontal ligament (Fig. 3B–D). Similarly to the PCR assay, the RANKL/OPG
ratio in the UTE group was 82% lower than the observed ratio in the NT
group (p < 0.01). To further investigate whether UTE would impact on
the osteoclast number, we identified the osteoclasts lining the inter
radicular alveolar bone, by TRAP immunostaining (Fig. 4A). A high
number of TRAP+ cells were observed in the remaining alveolar bone
of the periodontitis NT group (P< 0.01), which was reduced in the UTE
treated group (Fig. 4B and C). Altogether, these experiments indicate
that UTE reduced alveolar bone loss by attenuating osteoclast forma-
tion.
Effect of Uncaria tomentosa extract on systemic parameters
Bone remodeling is mainly controlled by the balanced action of
osteoblasts and osteoclasts, which are responsible for bone formation
and resorption, respectively. To have an insight into whether UTE
4
Phytomedicine 79 (2020) 153327
Fig. 1. UPLC-ESI-QTOF-MS/MS chromatograms for
extract of Uncaria tomentosa extract (UTE) in po-
sitive ionization mode (A) and negative ionization
mode (B). The numbers in the chromatograms are
referenced to those on Table 1 (supplementary ma-
terial): 1. p-Coumaric acid hexa-hexoside derivative,
2. Loganic acid, 3. Chlorogenic acid, 4. Vincosamide
11,6-di-glucopyranoside, 5. Rutin, 6. Cadambine, 7.
5-Carboxystrictosidine, 8. 3β-Dihydrocadambine, 9.
Isorhynchophyllic acid, 10. Mitraphyline, 11. Glab-
ratine, 12. Rhynchophylline, 13. Corynoxeine/ Iso-
corynoxeine, 14. Unknown, 15. Isorhynchophylline,
16. Rhynchophylline N-oxide, 17. Strictosamide, 18.
Corynantheine, 19. Hirsuteine, 20. Hirsutine, 21.
Vincoside lactam, 22. Trihidroxy-octadecenoic acid,
23 and 24 unknowns.
would affect osteoblast, we also evaluated the plasma level of bone-
specific alkaline phosphatase (BALP). The BALP level on the non-
treated periodontitis group was significantly reduced compared with
the respective control. Interestingly, the UTE treated group exhibited a
higher level of plasma BALP compared to non-treated (Table 1). UTE
treatment did not modify the kinetic curve of the body mass variation
(data not shown).
We further checked the effect of UTE treatment for 11 days, on liver
or kidney function, assessing the level of transaminases (AST and ALT)
and renal markers (creatinine and urea). The data shows that the level
of AST, ALT, creatinine, urea, as well as liver and kidney indexes were
not altered compared with the non-treated group, evidencing that UTE
does not affect liver or kidney function (Table 1).
Uncaria tomentosa extract inhibits osteoclast differentiation in vitro
Given that UTE inhibited bone loss and osteoclast formation in-
duced by the periodontitis model, we next performed an in vitro ex-
periment to elucidate if UTE would have a direct effect on osteoclas-
togenesis. Thus, bone marrow macrophages (BMMs) were cultured with
M-CSF and RANKL, under three concentrations of UTE (3, 9 and 27 μg/
ml). After 96 h, RANKL-stimulated osteoclasts evidenced as TRAP+
multinucleated cells were assessed. UTE (9 μg/ml and 27 μg/ml) in-
duced a significant reduction in osteoclast formation, while the lower
concentration of UTE (3 μg/ml) did not show an effect on osteoclas-
togenesis (Fig. 5A and B). To rule out a possible cytotoxic effect of UTE
on RANKL-induced osteoclasts formation, it was performed a cell via-
bility assay using thiazolyl blue tetrazolium bromide (MTT). UTE
(3–27 μg/ml) did not exhibit cytotoxic effects at any of the tested
concentrations, showing that the inhibitory effect of UTE was not due to
a cytotoxic effect (Fig. 5C).
The expression of different osteoclastogenic and function markers
on cell-cultured with UTE demonstrated that UTE treatment down-
regulated the mRNA level of TRAP (Acp5), nuclear factor activated T-
cells, cytoplasmic 1 (Nfatc1), and cathepsin K (CtsK), compared with
V. Lima, et al. Phytomedicine 79 (2020) 153327

Fig. 2. Effect of Uncaria tomentosa extract (UTE) on ligature-induced periodontitis in rats. Macroscopic (A and B) and histological (C and D) aspects of the
control maxilla of rats, and maxilla of rats with ligature-induced periodontitis non-treated or treated with UTE. The alveolar bone loss area was measured along the
region between the cement-enamel junction (CEJ) and alveolar bone crest (ABC) for 6 rats/group (dotted area). The histometric analysis considered the attachment
levels from the CEJ to the base of the gingival sulcus or periodontal pocket (dotted arrow) in the distal portion of the 2nd molar for 6 rats/group. Lowercase letters
indicate: ab = alveolar bone; pl = periodontal ligament. # p < 0.05 compared with the control values; * p < 0.05 compared with the non-treated group [The data
were analyzed using analysis of variance (ANOVA) and Bonferroni's test].

Table 1 Discussion
Effects of Uncaria tomentosa extract (UTE) on gingival inflammation and sys-
temic parameters of rats submitted to ligature-induced periodontitis. The Uncaria tomentosa, recently registered and incorporated in the
Groups Analysis Control Non-treated UTE 81 mg/kg list of Brazilian Genetic Patrimony, represents a great medicinal and
social significance for Brazilian Health System. It is popularly used by
MPO (U/mg) 1.1 ± 0.1 2.5 ± 0.2 # 1.4 ± 0.4 * natives to treat stomach and urinary tract inflammation, dysmenorrhea,
BALP (U/l) 136.5 ± 11.9 63.5 ± 10.7 # 246 ± 16.2 #*
rheumatoid arthritis symptoms relief, among other diseases. In the
AST (U/l) 101.0 ± 3.7 99.7 ± 6.1 94.6 ± 8.1
ALT (U/l) 44.6 ± 2.3 47.0 ± 2.9 37.1 ± 2.0 present study, we investigated the effect of UTE on a periodontitis ex-
Urea (mg/dl) 46.3 ± 4.1 48.8 ± 3.1 37.1 ± 1.9 perimental model. Our data show that UTE effectively attenuated the
Creatinine (mg/dl) 1.7 ± 0.2 2.6 ± 0.1 # 2.8 ± 0.2 # development of periodontitis, reducing the periodontal ligament at-
Liver index 0.035 ± 0.002 0.037 ± 0.002 0.042 ± 0.002 tachment loss and alveolar bone destruction. Moreover, we show a
Renal index 0.003 ± 0.000 0.004 ± 0.0003 0.004 ± 0.0001
direct effect of this natural product inhibiting osteoclastogenesis and
Ligature-periodontitis was induced in rats (Non-treated) or UTE during 11 days, osteoclasts demineralization activity.
and gingival tissues were used to myeloperoxidase activity (MPO). Blood The experimental periodontitis is characterized by an intense neu-
samples were used for biochemical analysis of bone alkaline specific phos- trophil infiltration followed by lymphocytes and macrophages at the
phatase (BALP), aspartate aminotransferase (AST), alanine aminotransferase chronic stage. The production of inflammatory mediators such as TNF,
(ALT). The data represent the mean value ± standard error of the mean (SEM) interleukins and chemokines contributes to the inflammatory response
#
p < 0.05 compared to control; * p < 0.05 compared to respective non-treated in animals and humans (Guimarães et al., 2016; Castro-Alcaraz et al.,
groups. [Data were analyzed by using analysis of variance (ANOVA) and 2002; Hayden et al., 2011). Therefore, the positive correlation between
Bonferroni's test].
inflammation and the severity of periodontitis, shown in this study,
resemble the periodontal disease in humans (Hajishengallis et al.,
their respective non-treated groups (Fig. 5D). Confirming these results, 2015). Using a periodontitis experimental model, this study shows an
UTE downregulated the protein levels of NFATc1 and CtsK during os- anti-inflammatory effect of UTE, as the neutrophil infiltration, evi-
teoclast differentiation (Fig. 5E). denced by the MPO level, was significantly reduced in the gingival
tissue. Moreover, the presence and the depth of periodontal pockets are
parameters clinically used to define the severity of periodontitis

5
V. Lima, et al. Phytomedicine 79 (2020) 153327

Fig. 3. Effect of Uncaria tomentosa extract (UTE) on bone markers. The data represent the mean ± SEM of Rankl and Opg mRNA expressions, and Rankl/Opg
ratio (A). Photomicrographs of the RANKL and OPG staining of the region of interest corresponding in the furcation area of 2nd molar (100 x magnification) (B). The
square in the schematic illustration of the tooth indicates the region of interest corresponding to the furcation area of the first molar (C). The data represent the
mean ± SEM of positive cells number of RANKL and OPG per mm2 in 5 fields for 3 animals/group (D). Lowercase letters indicate: ab = alveolar bone; d = dentin;
pl = periodontal ligament. The arrows indicate stained cells. # p < 0.05 compared with the control; * p < 0.05 compared with the non-treated group [The data were
analyzed using analysis of variance (ANOVA) and Bonferroni's test].

(Papapanou et al., 2018). Together, our data show that this natural
product attenuated the periodontal disease parameters.
Previous report has shown that intense inflammatory process may
stimulate the osteoclast activity, which results in bone resorption
(Adamopoulos, 2018). Reportedly, the osteoclastogenesis is controlled
by the RANK–RANKL–Osteoprotegerin (OPG) system, which is a tri-
molecular complex acting as receptors and ligand that belongs to the
tumor necrosis factor (TNF) superfamily (Kobayashi et al., 2015). We
observed that the UTE administration altered the gene and protein le-
vels of RANKL-OPG molecules, which resulted in a reduction of the
RANKL/OPG ratio in the inflamed gingival tissue. Although we can not
identify exactly which cell type (osteoblasts, fibroblast, B cell) are ex-
pressing them, the effects of UTE on RANKL and OPG expression are
consistent with an inhibition of osteoclastogenesis and consequently
bone resorption. Corroborating this data, UTE treated group exhibited a
reduced number of TRAP+ cells lining the alveolar bone, suggesting

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V. Lima, et al. Phytomedicine 79 (2020) 153327

Fig. 4. Effect of Uncaria tomentosa extract (UTE) on TRAP immunostainings. The square in the schematic illustration of the tooth indicates the region of interest
corresponding to the furcation area of the first molar (A). TRAP immunostaining aspects of the contralateral (control), non-treated (NT), and 81 mg/kg UTE groups
(B). The mean of the number of positive cells expressed per mm2 in 5 fields/mm2 for 3 animals/group (C). Lowercase letters indicate: ab = alveolar bone;
pl = periodontal ligament. # p < 0.05 compared with the control; The arrows indicate stained cells. * p < 0.05 compared with the non-treated group [The data were
analyzed using analysis of variance (ANOVA) and Bonferroni's test].

that UTE inhibits osteoclastogenesis in vivo. Given that bone formation
and resorption are essential events that occur in the bone remodeling
process, this study suggests that the UTE acted not only as an anti-
resorptive agent reducing the number of osteoclasts, but also indicates
that the anabolic phase might be stimulated as the BALP levels are
increased by the UTE in this model.
The bone loss observed in this study is mainly caused by the activity
of osteoclasts, which are unique cells with the ability to solubilize the
bone matrix. Here we show, for the first time, that UTE has a direct
effect controlling osteoclast formation and activity in vitro. We found
that the expression of osteoclast markers' genes was down-regulated by
UTE and that this natural product also inhibited osteoclastogenesis.
RANKL-RANK is a critical pathway activated during osteoclast differ-
entiation, and it results in NF-κB activation, which in turn activates
further transcription factors, such as the NFATc1 (Kim and Kim, 2014).
Mechanistic investigation showed that UTE inhibits RANKL-mediated
osteoclastogenesis by decreasing the expression of Nfatc1 and Acp5.
NFATc1 is known to play an essential role as a master transcription
regulator of osteoclast differentiation. Furthermore, NFATc1 also in-
duces the transcription of cathepsin K, an enzyme involved with os-
teoclast resorption function (Wilson et al., 2009). Therefore, our find-
ings identified that UTE not only acts as an antiresorptive agent by
inhibiting osteoclasts formation but also osteoclasts function by
blocking the expression of cathepsin K. Although our data show a direct
effect of UTE inhibiting osteoclasts formation in vitro, it is well known
that several cell types expressing RANKL and OPG can control osteo-
clastogenesis. Therefore, we cannot rule out the effect of UTE on other
cells involved indirectly in bone resorption.
The present report highlights the natural compound UTE as a safe
anti-inflammatory, antiresorptive, and potential bone anabolic product
to be considered as a therapeutic alternative to control bone diseases,
opening new perspectives for a further clinical study.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Author contributions
V.L. designed the study, data collection, drafted the manuscript.
I.M.M. designed and carried out the in vivo experiments. T.M.T. and
L.Y.W.B. designed and carried out the in vitro experiments. C.S.R.F.,
R.N.F.C. and M.O.M. performed data analysis. L.K.A.M.L., A.S.Q.S. and
T.S.A. contributed for chemical characterization of U. tomentosa ex-
tract. F.Q.C. and S.Y.F. conceived the idea, supervised all research and
revised the manuscript. All authors approved the final version of the
manuscript.
All data were generated in-house, and no paper mill was used. All
authors agree to be accountable for all aspects of work ensuring in-
tegrity and accuracy.
Acknowledgments
The authors acknowledge Mayara dos Santos Gomes (chemical en-
gineer in the Department of Physics and Chemistry of the School of
Pharmaceutical Sciences of Ribeirao Preto at University of São Paulo)
for her technical assistance. This work was supported by grants from
Brazilian government agencies: National Council for Scientific and
Technological Development (CNPq: 110935/2016-0), Center for
Research in Inflammatory Diseases (CRID)/Sao Paulo Research
Foundation (FAPESP: 2013/08216-2), Sao Paulo Research Foundation
(FAPESP: Regular Project 2015/09034-0), Coordination for the

7
V. Lima, et al. Phytomedicine 79 (2020) 153327

Fig. 5. Effect of Uncaria tomentosa extract (UTE) on osteoclast formation in vitro. Bone marrow cells were incubated with M-CSF, and the adhered cells were
incubated by M-CSF and RANKL in the presence of UTE (3 μg/ml, 9 μg/ml and 27 μg/ml). Photomicrographs of the Tartrate-resistant acid phosphatase (TRAP) +
staining (100 x magnification) (A). TRAP multinucleated cells characterized by more than three nuclei were counted (B). UTE had no effect on the viability of cells for
a 48 h culture period, which was evaluated by MTT assay (C). The data represent Acp5, Nfatc1 and CtsK mRNA fold change (D). UTE also inhibited the Rankl-induced
expressions of Nfatc1 and CtsK proteins (E). The data were expressed as mean ± standard error mean (n = 5) for each group. Data represent at least 3 independent
experiments. * p < 0.05 compared with the non-treated group [The data were analyzed using analysis of variance (ANOVA) and Bonferroni's test].

Improvement of Higher Education Personnel (CAPES-REUNI) and Ceara
Foundation of Scientific and Technological Development (FUNCAP)/
CNPq/Support Program for Centers of Excellence (PRONEX: PR2-0101-
00054.01.00/15). Part of the work was carried out with the infra-
structure support of Embrapa Agroindústria Tropical, Fortaleza-CE,
Brazil.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.phymed.2020.153327.
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