Cyclic Hexapeptide from Bouvardia ternifolia (Cav.) Schltdl. and Neuroprotective Effects of Root Extracts

Bouvardia ternifolia (Cav.) Schltdl. is a shrub that belongs to the Rubiaceae family and is distributed throughout México; it has been used for its antioxidant, neuroprotective, and anti-inflammatory properties. This work aimed to evaluate the protective effects of B. ternifolia root extracts on the blood-brain barrier and the positive regulation of cytokines IL-1β, IL-6, and TNF-α, and the characterization of compounds present in the dichloromethane (BtD) and hexane (BtH) extracts. Male ICR mice were orally administered with B. ternifolia extracts for 5 days before a single injection of LPS. Administration of BtH and BtD significantly decreased Evans blue leakage into brain tissue by 70% and 68%, respectively. Meloxicam (MX) decreased the concentration of IL-1β by 39.6%; BtM by 53.9%; BtAq by 48.4%; BtD by 31.9%, and BtH by 37.7%. BtH was the only treatment that significantly decreased the concentration of IL-6 by 32.2%. The concentration of TNF-α declined with each of the treatments. The chemical composition of BtD and BtH was characterized by GC–MS, and the cyclic hexapeptide was identified by 13C, 1H NMR, and two-dimension techniques. In the BtD extract, seven compounds were found and in BtH 13 compounds were found. The methanolic (BtM) and aqueous (BtAq) extracts were not subjected to chemical analysis, because they did not show a significant difference in the BBB protection activity. Therefore, the results suggested that the extracts BtD and BtH protect the blood-brain barrier, maintaining stable its selective permeability, thereby preventing LPS from entering the brain tissue. Simultaneously, they modulate the production of IL-1β, IL-6, and TNF-α. It is important to note that this research only evaluated the complete extracts.


Introduction
Neurodegenerative diseases are predicted to be the most significant health concern in this century and the second leading cause of death by 2050. Related to the increase in life expectancy, there has been a surge in the incidence of these disorders. Neurodegenerative diseases encompass a range of conditions in which neuronal structure and function are altered, affecting the brain and spinal cord central nervous system (CNS) and worsening over time [1]. The main cellular and molecular events that cause neurodegeneration are oxidative stress, deposition of protein aggregates, neuroinflammation, impaired mitochondrial function, induction of apoptosis, and alteration of autophagy [2].
Lipopolysaccharides (LPS) are endotoxins formed of an O-antigen and are found in the outer membrane of Gram-negative bacteria and play a role as stimulants for microglial activation. Toll-like receptor 4 (TLR4) is expressed on microglial cells and is responsible for the inflammatory cascade in microglia by binding to LPS [3].
Active microglia initiate a process of brain inflammation and play a crucial role in regulating neuroinflammatory reactions [4]. Hyperactive microglia are known to release a variety of neurotoxic mediators, such as nitric oxide (NO), inducible nitric oxide in the brain. Regarding the experimental treatments, it was observe decreased by 70% the concentration of Evan's blue per mg of brain by 68%. The BtM was reduced by 13%, and the aqueous BtAq by 2 statistically significant difference with respect to the MX group, ternifolia treatments have a higher percentage of reduction in the blue than the treatment with meloxicam. Figure 1. Effect of B. ternifolia extracts on Evans blue extravasation to br VEH (animals administered with LPS 5 mg/kg). Values represent the m were evaluated by one-way analysis of variance (one-way-ANOVA) and (****) p < 0.0001 vs. VEH group. No significant difference (ns). Figure 2A,B shows that the administration of LPS (5 mg/kg fo icant increment of IL-1β and IL-6 on the brain in comparison with (* p < 0.05). However, these IL-1β values significantly decreased w 53.9%, BtAq 48.4%, BtD 31.9%, and BtH 37.7%. IL-6 was only sign the BtH extract by 32.2%.

Effect of B. ternifolia Root Extracts on Cytokine Levels in the Brain o LPS-Administered Mice
An increment of TNF-α ( Figure 2C) in the brain tissue with L pared to the basal control group. At the same time, meloxicam sh concentration of TNF-α by 18%, methanol extract by 34%, aqueou chloromethane extract by 31%, and hexane extracts by 24.3% with control; these values were statistically different with respect to the ternifolia extracts on Evans blue extravasation to brain tissue with respect to VEH (animals administered with LPS 5 mg/kg). Values represent the mean ± SEM. The variables were evaluated by one-way analysis of variance (one-way-ANOVA) and the Dunnett post hoc test (****) p < 0.0001 vs. VEH group. No significant difference (ns). Figure 2A,B shows that the administration of LPS (5 mg/kg for 4 h) induced a significant increment of IL-1β and IL-6 on the brain in comparison with the basal control group (* p < 0.05). However, these IL-1β values significantly decreased with MX by 39.6%, BtM 53.9%, BtAq 48.4%, BtD 31.9%, and BtH 37.7%. IL-6 was only significantly decreased by the BtH extract by 32.2%.

Effect of B. ternifolia Root Extracts on Cytokine Levels in the Brain of LPS-Administered Mice
An increment of TNF-α ( Figure 2C) in the brain tissue with LPS was observed compared to the basal control group. At the same time, meloxicam showed a decrease in the concentration of TNF-α by 18%, methanol extract by 34%, aqueous extract by 23.4%, dichloromethane extract by 31%, and hexane extracts by 24.3% with respect to the damage control; these values were statistically different with respect to the damage group. Figure 2. Effect of B. ternifolia extracts on the concentration of (A) IL-1β, (B) IL-6, and (C) TNF-α in the brain with respect to the VEH (animals administered with LPS (5 mg/kg). Values represent the mean ± SEM. The variables were evaluated by one-way analysis of variance (one-way-ANOVA) and the Dunnett post hoc test. (*) p < 0.05 (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001 vs. VEH group. No significant difference (ns).

Chemical Analysis
Chemical analysis of BtD and BtH extracts led to the identification of twenty compounds from the root of B. ternifolia: one cyclic hexapeptide, seven fatty acids, one alkane, two triterpenes, two phenanthrene-carboxylic acids, one isoprenoid lipid, one phytosterol, one tocopherol, one monoterpenoid, one tetramethyl, and one indanylidene.

Chemical Analysis
Chemical analysis of BtD and BtH extracts led to the identification of twenty compounds from the root of B. ternifolia: one cyclic hexapeptide, seven fatty acids, one alkane, two triterpenes, two phenanthrene-carboxylic acids, one isoprenoid lipid, one phytosterol, one tocopherol, one monoterpenoid, one tetramethyl, and one indanylidene.

Discussion
B. ternifolia has been used in traditional medicine to treat inflammation-related conditions. Its anti-arthritic, anti-inflammatory, and inhibitory effect on the NF-kB signaling pathway was recently demonstrated [12]. Cytokines and interleukins contribute to the amplification of inflammatory pathways involved in neurodegenerative disorders, including Alzheimer's disease, multiple sclerosis, Parkinson's disease, and amyotrophic lateral sclerosis. LPS, a neurotoxin, is commonly used as an experimental model to induce systemic inflammatory responses. The present study examined the effect of B. ternifolia root extracts on LPS-induced damage to the permeability and stability of the blood-brain barrier and pro-inflammatory cytokine alterations in experimental mice. In addition, the extracts that demonstrated greater BBB neuroprotection activity were chemically characterized. Our results showed a decrease in the extravasation of Evans blue into brain tissue, indicating that the BtD and BtH extracts helped to protect the permeability of the blood-brain barrier, as well as the decrease in proinflammatory cytokines in the brain of mice after LPS-induced damage.
Previous studies reported in the literature have shown that LPS induces inflammatory responses both at the systemic level and at the brain level that include the activation of resident immune cells, such as macrophages and microglial cells. These immune cells release a variety of pro-inflammatory molecules, such as cytokines and interleukins, which promote inflammation in the central nervous system [16]. In the present study, the induction of neuroinflammation in the mice brain was shown by the increase in IL-1β, TNF-α, and IL-6 production.
LPS can activate the NF-κB pathway through interaction with its receptor, TLR-4. Once in the nucleus, NF-κB binds to its binding sequence to activate the relevant promoters and cause the expression of inflammatory cytokines, such as IL-1β, IL-6, COX-2, and TNF-α. These facts indicate that NF-κB plays a critical role in the regulation of inflammation, and that NF-κB inhibition may protect against neuroinflammation and neurodegeneration [17]. In this study, the extracts of the root of B. ternifolia at a dose of 25 mg/kg attenuated the production of the interleukins IL-1β, IL-6, and TNF-α; the mechanism of action by which the compounds present in the extracts would be acting is by inhibiting the NF-κB pathway. Numerous natural products have demonstrated their potential in exerting antineuroinflammatory effects by employing various mechanisms. These mechanisms include inhibiting the activation of microglia, reducing the release of pro-inflammatory cytokines from activated microglia, as well as inhibiting the activation of NF-κB and p38 MAPK [18].
It has been reported that some natural products can interfere with the intracellular signaling pathways that regulate the production and activity of cytokines. For example, they can modulate the signaling pathway of protein kinases (MAPK) or the signaling pathway of Janus kinases (JAK)/signal transducers and activators of transcription (STAT), which are important in the regulation of the expression of cytokines [19].
To assess the effects of B ternifolia extracts on the permeability of the blood-brain barrier after intraperitoneal injection with LPS, we evaluated Evans blue extravasation to the brain; dichloromethane and hexane extracts were the most effective treatments compared to the VEH and meloxicam groups (70% and 68%, respectively). Evans blue is a dye that specifically binds to albumin. Under physiological conditions, the endothelium acts as a barrier, preventing the passage of albumin and thus restricting the movement of Evans blue within the blood vessels. However, under pathological conditions characterized by increased vascular permeability, endothelial cells undergo changes that result in the loss of the tight interconnection between them. Consequently, the endothelium becomes permeable to small proteins such as albumin. This altered state allows extravasation of Evans blue from blood vessels into surrounding tissues, including the brain. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue [20].
When the permeability of the BBB is compromised, it allows the passage of high concentrations of LPS from the bloodstream into the brain, along with inflammatory cells and mediators. This condition results in a significant worsening of neuroinflammation [21]. Several studies have shown that plant extracts have protective effects on the BBB. These extracts contain bioactive compounds, such as polyphenols, flavonoids, and terpenoids, which have antioxidant and anti-inflammatory properties [22].
The protection of the BBB by plant extracts has been attributed to several beneficial actions. First, these extracts can strengthen the tight junctions between the endothelial cells that make up the BBB. Tight junctions play a crucial role in regulating the flow of molecules and cells from the bloodstream to the brain. By strengthening these junctions, plant extracts can help prevent the infiltration of harmful substances or inflammatory cells into the brain [23,24].
Furthermore, plant extracts can modulate the inflammatory response in the BBB. Chronic inflammation can weaken the barrier and compromise its protective function. Compounds present in plant extracts can inhibit the activation of immune cells, such as macrophages and microglial cells, thus reducing the production of pro-inflammatory cytokines and preventing damage to the BBB [24].
Another mechanism by which plant extracts can protect the BBB is through their antioxidant capacity. These compounds can counteract oxidative stress, which is a major cause of barrier damage. By reducing the production of free radicals and promoting a proper redox balance [25]. In this study, the characterization of the compounds present in the BtD and BtH extracts was also carried out by means of gas chromatography analysis coupled to masses. The dichloromethane extract presented a great chemical diversity in its composition, highlighting the presence of seven compounds, including, for example, the α-tocopherol, which has been reported as a neuroprotector as it is a powerful antioxidant that neutralizes reactive oxygen and nitrogen species [26]; squalene, which has been used for its antioxidant properties [27] and immunomodulatory activity; 2-Nonadecanone has antioxidant properties, which may help to protect cells from oxidative damage caused by free radicals, and anti-inflammatory properties [28]; 3-Carene has anti-inflammatory properties and antioxidant activity [29]; lupeol has neuroprotective effects [30].
In the BtH extract, 13 compounds were found. Among them, for example, was ursolic acid, which has been reported to decrease the level of proinflammatory markers such as COX-2, iNOS, TNF-α, IL-1β, IL-2, and IL-6 in the brain of mice [31]; β-sitosterol has been reported to have an anti-inflammatory effect and act as a modulator of the expression of proinflammatory markers, such as IL-6, iNOS, TNF-α, and COX-2. [17].
In the BtD4.1 fraction of the dichloromethane extract, a glycosylated hexapeptide cyclic-type compound was identified; it is the first time that it has been reported for the root of B. ternifolia. However, it has been isolated before under the name of Rubiyunnanins H from the roots of Rubia yunnanensis (Franch.) Diels, a plant belonging to the Rubiaceae family, such as Bouvardia ternifolia. Previous research has identified bicyclic hexapeptides from Rubiaceae family plants, also known as RAs. The first reported RAs were bouvardin and deoxybouvardin, isolated from the stems, leaves, and flowers of B. ternifolia. Since then, an additional twenty-eight RAs have been identified in three Rubia plants: Rubia cordifolia, Rubia akane, and Rubia yunnanensis. Rubiyunnanins H has been reported for its cytotoxic activity in in vitro cultures of cancer cell lines, as well as its ability to inhibit nitric oxide production in the LPS model and IFN-,-induced RAW 264.7 murine macrophages, and to inhibit NF-κB and TNF-α activation [32].
In this study, meloxicam was employed as a positive control, representing a nonsteroidal anti-inflammatory drug (NSAID) commonly used for the management of pain, inflammation, and fever. Meloxicam exerts its effects through selective inhibition of the enzyme cyclooxygenase-2 (COX-2), which plays a pivotal role in prostaglandin synthesis. Prostaglandins serve as critical mediators in the inflammatory cascade, contributing to the sensitization of nerve endings to pain and the amplification of the inflammatory response [33]. By targeting COX-2, meloxicam effectively diminishes the production of proinflammatory prostaglandins, leading to a reduction in the overall inflammatory response. Consequently, this includes a decrease in the generation of pro-inflammatory interleukins, such as interleukin IL-1β, IL-6, and IL-8, which are recognized for their involvement in mediating inflammation and facilitating the transmission of pain [34]. The plant material was air-dried at room temperature for six days. Once dry, it was ground to achieve a particle size of 1-5 mm using a mill (Pulvex S.A de C.V, Mexico City, Mexico). Plant extracts were obtained through a maceration process, starting with hexane (BtH), followed by dichloromethane (BtD), methanol (BtM), and finally, water (BtAq). Each extract underwent filtration, a process repeated three times for each extract. The solvents from the filtrate were recovered through reduced-pressure distillation using a Büchi 490 rotary evaporator (Büchi, Postfach, Flawil, Switzerland). To achieve complete drying, the extracts were lyophilized and stored at −4 • C. Subsequently, thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) were employed to monitor the extracts.

LPS-Induced Acute Brain Inflammation Model in Mice
For this assay, female mice of the ICR strain weighing between 30 and 42 g were used and were divided into 7 groups, with 6 mice in each group, for a total of 84 mice. Animals were provided by the animal center of the Health Research Coordination of the Siglo XXI Medical Center (Mexico City) and were strictly handled according to Mexican regulations (NOM-062-ZOO-1999). The protocol was approved by the local Ethics Committee (R-2020-1702-033). The mice were administered with the extracts at a dose of 25 mg/kg vo, twice a day for three days. (Table 4). After the pretreatment, LPS was intraperitoneally administered at 5 mg/kg and left to act for 4 h.

Quantification of Evans Blue Extravasation
A sterile 0.5% Evans blue solution in PBS was prepared and 200 µL of Evans blue solution was intravenously placed in the lateral tail vein of the mouse. The mouse was then placed in the cage and observed for 30 min. The mice had previously been administered the crude extracts for 5 days, and 4 h before the injection with Evans blue they had been injected with LPS at 5 mg/kg i.p. (Table 4).
Mice were euthanized by a lethal dose of pentobarbital at 100 mg/kg i.p., the ribcage was opened and transcardially perfused through the left ventricle with 100-150 mL of saline. A concomitant small cut was made in the right atrium to remove intravascular blood and tracer, and perfusion continued for 15 min until the atrial fluid was clear. Subsequently, the brain was collected in 1.5 mL tubes and weighed, and then 500 µL of formamide was added to each tube and transferred to a 55 • C water bath for 24-48 h to extract the dye from brain tissue. The supernatants were cooled and centrifuged at 14,000× g for 15 min. Albumin-Evans blue concentration was spectrophotometrically quantified at 610 nm and using a standard curve [35]. Protein concentration in the samples was quantified using a modified Bradford method [36].

Quantification of Cytokines
Mice were euthanized with a lethal dose of pentobarbital at 100 mg/kg i.p. The ribcage was opened and transcardial perfusion was conducted through the left ventricle using 100-150 mL of saline solution. Simultaneously, a minor incision was performed in the right atrium to extract intravascular blood and Evans blue, while perfusion was maintained for 15 min. Following this, the brain tissue was homogenized in 1× PBS solution containing 0.1% protease inhibitor (phenyl-methyl-sulfonyl fluoride, PMFS from Merck KGaA, Darmstadt, Germany). The homogenate was then subjected to centrifugation at 3000× g for 5 min, and the resulting supernatant was preserved at −80 • C until further analysis. Cytokine concentrations (IL-1β, IL-6, and TNF-α) were quantified using ELISA according to the manufacturer's instructions (Becton, Dickinson and Co., Franklin Lakes, NJ, USA).

GC-Mass Spectrometry (CG-MS)
BtD and BtH extracts (5 mg) were analyzed by GC-MS. Analysis was performed using an Agilent/HP 6890 gas chromatograph coupled to a quadrupole mass spectrometer (5973 MSD) and fitted with a capillary column (5MS-l 30 m × 0.25 mm, i.d.; 0.25 µm film thickness). The oven temperature was programmed at 40 • C for 1 min and was then increased at 10 • C/min to 280 • C. The inlet temperature was set at 250 • C. The mass spectrometer was operated in positive electron impact mode (EI, 70 eV). Samples were injected in 1 µL volume using helium as a carrier gas (1 mL/min). Detection was performed in selective ion monitoring (SIM) mode, and peaks were identified and quantified using target ions. Compound characterization was based on comparing their mass spectra with the National Institute of Standards and Technology (NIST) library version 1.7a. Relative percentages were determined by integrating the peaks using GC Chem Station software (v C.00.01). The composition was reported as a percentage of the total peak area [37].

Isolation and Identification of Cyclic Hexapeptide
A fractionation of BtD3 (600 mg) was carried out, obtaining 75 fractions; in TLC, it was observed that most of the compounds presented a blue coloration, and it was possible to isolate the BtD4 subfraction. This fraction was analyzed by HPLC and 4 peaks with similar retention times and UV spectra were observed. Therefore, it was necessary to perform acetylation of 50 mg of the fraction and then carry out another chromatographic separation to isolate the four compounds in BtD4. Subsequently, BtD4.1 was analyzed by NMR ( 1 H and 13 C NMR) and two-dimensional techniques (HMBC, HSQC, and COSY).

Nuclear Magnetic Resonance
BtD4.1 was subjected to structural chemical characterization using mass spectrometry techniques. In addition, proton and carbon nuclear magnetic resonance spectra ( 1 H and 13 C NMR), as well as two-dimensional (2-D) correlated spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond coherence (HMBC); experiments were performed using a Varian INOVA-400 instrument at 400 MHz. BtD4.1 was appropriately diluted in deuterated methanol (CD 3 OD). The software ACD/Labs Predictors, ACD/Processor 12.0, and MestreNova LITE were used for subsequent analysis and interpretation, serving as theoretical simulators to elucidate the structure of the compound.

Conclusions
In the present study, we report the anti-inflammatory and neuroprotective properties of B. ternifolia root extracts and the compounds contained in the active extracts. This study demonstrates that B. ternifolia root extracts inhibit the production of the proinflammatory interleukins IL-1β, IL-6, and TNF-α in an LPS-induced mice model. The inhibitory effect of interleukins was attributed to the suppression of the transcriptional activation of NF-κB through its membrane receptor TLR-4 since NF-κB is one of the key transcription factors responsible for regulating inflammation-related genes ( Figure 5). In addition, the B. ternifolia root extracts that best protected the blood-brain barrier were BtD and BtH. The main compounds found in the BtD extract were Rubiyunnanins H, terpene-type compounds, α-tocopherol, and squalene; the main compounds found in the BtH extract were fatty acidtype compounds, α-tocopherol, β-sitosterol, and terpene-type compounds. In summary, the inhibition of proinflammatory molecules IL-1β, IL-6, and TNF-α through NF-κB by BtD and BtH extracts from the B. ternifolia root, as well as the protection of the blood-brain barrier, would be a possible therapeutic approach for the treatment of neuroinflammation.
factors responsible for regulating inflammation-related genes ( Figure 5). In addition, the B. ternifolia root extracts that best protected the blood-brain barrier were BtD and BtH. The main compounds found in the BtD extract were Rubiyunnanins H, terpene-type compounds, α-tocopherol, and squalene; the main compounds found in the BtH extract were fatty acid-type compounds, α-tocopherol, β-sitosterol, and terpene-type compounds. In summary, the inhibition of proinflammatory molecules IL-1β, IL-6, and TNF-α through NF-κB by BtD and BtH extracts from the B. ternifolia root, as well as the protection of the blood-brain barrier, would be a possible therapeutic approach for the treatment of neuroinflammation. Figure 5. The schematic representation illustrates the anti-inflammatory and neuroprotective properties of B. ternifolia root extracts on the blood-brain barrier. Created with Biorender.com.

Supplementary Materials:
The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Figure S1: GC-MS Analysis of the BtD extract ; Figure S2: GC-MS Analysis of the BtH extract; Figure S3: Spectrum UV and HPLC of cyclic hexapeptide; Figure S4: Mass spectrum (MS) of cyclic hexapeptide; Figure S5

Institutional Review Board Statement:
The animal study protocol was approved by the Ethics Committee of Centro de Investigación Biomédica del Sur, Instituto Mexicano del Seguro Social (R-2020-1702-033) in strict accordance with Mexican regulations for the use of experimental animals (NOM-062-ZOO-1999).

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.