Pyrola incarnata demonstrates neuroprotective effects against β-amyloid-induced memory impairment in mice

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Pyrola incarnata demonstrates neuroprotective effects against β-amyloidinduced memory impairment in mice Shuang-Jun Lia, Qian Liua, Xiao-Bin Heb, Jin-Ping Liua, Xiao-Liu Liua, Jie Hua, Zhi-Peng Tanga, ⁎ ⁎ Qing-Yun Penga, Lian-Jie Cuia, Hua-Ni Zhangc, Xi-Liang Yanga, , Qiang Wanga, , ⁎ Zhi-Jian Zhangb, a

Department of Pharmacy, Hubei Province Key Laboratory of Occupational Hazard Identification and Control, Institute of Infection, Immunology and Tumor Microenvironments, Medical College, Wuhan University of Science and Technology, Wuhan 430081, China b Center for Brain Science, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, Wuhan Center for Magnetic Resonance, Key Laboratory of Magnetic Resonance in Biological Systems, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan 430071, China c Department of Pharmacy, Shiyan Hospital of Integrated Traditional and Western Medicine, Shiyan, Hubei 442000, China

ARTICLE INFO

ABSTRACT

Keywords: Aβ25–35 Microglia Neurodegenerative Pyrola incarnata Ursolic acid

This study aims to investigate the neuroprotective effects of Pyrola incarnata against β-amyloid-induced memory impairment in mice. Ethanol extract of Pyrola incarnata (EPI) was obtained and led to eleven phytochemicals successfully by isolation and purification, which were elucidated by spectroscopic analysis (1H NMR, 13C NMR and HR-ESI-MS). Thereinto, ursolic acid was gained as most abundant monomer. C57BL/6 mice were intracerebroventricular injected with aggregated Aβ25–35. Open-field test, Barnes maze test and Morris water maze were conducted for evaluating cognition processes of EPI and ursolic acid. EPI significantly improved learning and memory deficits, attenuated the Aβ25–35 level of deposition immunohistochemically. Further studies revealed that ursolic acid as bioactive phytochemical of P. incarnata improved spatial memory performance and ameliorated Aβ25–35 accumulation by activating microglia cells and up-regulating Iba1 level in the hippocampus. These findings suggest P. incarnata could improve the cognition of mice and be a promising natural source for the treatment of neurodegenerative disease.

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that is associated with global mental dysfunction and cognitive deterioration.1 Common pathological features of AD are extracellular accumulations of amyloid beta (Aβ) peptides and intracellular neurofibrillary tangles.2 Accumulation of Aβ leads to the deposition of insoluble neuritic or senile plaques, thereby initiating a pathological cascade, which results in synaptic dysfunction, synaptic loss, neuronal death, and cognitive impairments.3 Therefore, Aβ-induced brain injuries were widely used as a model to establish experimental animal with neuroprotective activity in medicines and foods. In traditional Chinese medicine, Pyrola incarnata (Pyrolaceae, Chinese name: Lu Xiancao) has long been used as tonifying agents to alleviate a variety of diseases contributing to strengthening muscles and bone, invigorating kidney, immunity boosting and et al.4 In China,

Pyrola incarnata was first documented in Shen Nong Ben Cao Jing, which is the first materia medica with a history of thousands of years, and listed in the top grade as a valuable medicinal and edible plant. P. incarnata is mainly distributed in the northeast of China, the main constituents of which are chimaphilin, arbutin, epicatechin, catechin, 2″-O-galloylhyperin, hyperin quercetin and etc.5 P. incarnata has been used as tonics, sedatives, analgesics against rheumatoid arthritis, and hemostatics.6 Nowadays, it was used as a famous anti-aging tea known as ‘Lushou Cha’ beverage in folks for its function of anti-aging and immunity-enhancing. However, little is known regarding the active ingredients of P. incarnata with neuroprotective activity and the underlying pharmacological mechanism involved. As a continuous study, neuroprotective effects of leaves extract and phytochemicals of P. incarnata against Aβ-induced memory impairment in mice was conducted

Abbreviations: AD, Alzheimer’s disease; Aβ, β-amyloid; Iba1, ionized calcium binding adapter molecule 1; EPI, the ethanol extract of Pyrola incarnata; UA, ursolic acid; ICV, intracerebroventricular; OFT, open field test; MWM, Morris water maze; IOD, integral optical density; CNS, central nervous system; APP, amyloid precursor protein; BACE1, β-secretase; ROS, reactive oxygen species ⁎ Corresponding authors at: Medical College, Wuhan University of Science and Technology, No. 947, Qingshan Heping Road, Wuhan 430065, China (X.-L. Yang, Q. Wang). E-mail addresses: [email protected] (X.-L. Yang), [email protected] (Q. Wang), [email protected] (Z.-J. Zhang). https://doi.org/10.1016/j.bmcl.2019.126858 Received 19 August 2019; Received in revised form 24 October 2019; Accepted 25 November 2019 0960-894X/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Shuang-Jun Li, et al., Bioorganic & Medicinal Chemistry Letters, https://doi.org/10.1016/j.bmcl.2019.126858

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Fig. 1. The experimental schedule of the study demonstrating treatment of drugs (EPI and ursolic acid).

Fig. 2. The effects of EPI against impaired spatial memory in mice during the open-field test and Barnes maze test. Number of line crossing (A1), mean speed (A2) of mice in open-field test. Number of line crossing, mean speed were measured during five minutes session. The effects of EPI on latency time to enter the escape box during the training session (B1), latency time (B2) and mistake times (B3) during the probe trial session are presented. Data were expressed as mean ± SEM (n = 9). ns: p > 0.05 versus each group mice. **p < 0.01 versus control group mice; #p < 0.05, ##p < 0.01 versus model group mice.

and evaluated. The experimental schedule of this study was shown in Fig. 1. The OFT test was conducted to determine whether the cognitiveameliorating activity of EPI was responsible for the increased locomotor activity. There was no significant difference in the line crossing numbers or mean speeds of different groups during OFT (Fig. 2A). The results showed that treatments of EPI and ICV of Aβ25–35 had no effect on spontaneous locomotor activity of mice. To determine whether impaired memory could be reversed by the

administration of EPI, the effect of EPI on spatial memory was examined by Barnes maze test. Student's t-tests revealed that the latency for entering the escape box during the training sessions was significantly different among each group (Fig. 2B1). EPI03-treated group with the dosage of 112 mg/kg demonstrated significant decrease of its latency on day 4 (Fig. 2B1, p < 0.05), compared to the vehicle-treated Aβ25–35 group. A significant decreased time spent in the target quadrant was observed in the vehicle-treated Aβ25–35 group compared to that of the control group (Fig. 2B2 and B3, p < 0.01), and a remarkable

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Fig. 3. The attenuating effects of EPI on Aβ25–35-induced memory impairment during the Morris water maze test. EPI (28, 56 or 112 mg/kg) or the same volume of vehicle solution were administered to mice. Swimming mean speed (A) of mice in Morris water maze first day test. Escape latency apparent during the training sessions (B). Escape latency apparent during the probe sessions (C). After the platform was removed, time to platform (target circle) (D) and the number of crossing platform (E) of mice. The path map of the mice during the probe sessions (F). Data were expressed as mean ± SEM (n = 9). *p < 0.05, **p < 0.01 versus control group mice; #p < 0.05, ##p < 0.01 versus model group mice. $p < 0.05 versus EPI-treated mice.

amelioration was observed in the EPI03-treatment group (p < 0.01). The results suggested that Aβ25–35 induced a deficit in memory which was reversed by EPI03 especially. The MWM was used to evaluate whether EPI had ameliorating effects on Aβ25–35-induced long term memory deficits. As shown in Fig. 3A, no significant group effects were observed on the mean speed of swimming, suggesting that EPI had no stimulating effects on the locomotor behavior of naïve mice. During the training sessions, the administration of Aβ25–35 increased the escape latency in mice. However, the administration of EPI (28, 56, 112 mg/kg) significantly

shortened the escape latency during the training sessions on the 4th or 5th days compared with the Aβ25–35-treated mice (day 4 F2,25 = 3.472, p < 0.05, day 5 F3,33 = 4.385, p < 0.05) (Fig. 3B). These data suggested EPI ameliorated the Aβ25–35-induced cognitive deficits and improved memory during the training sessions. On the day of the final session (6th day), we conducted a probe trial. As shown in Fig. 3C and D, the escape latency and the time to the target quadrants of the EPItreated group was significantly shorter than that of Aβ25–35-treated group (F3,32 = 9.996, p < 0.01). Consistently, EPI increased the crossing numbers of target quadrants in a dose-dependent manner,

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Fig. 4. Effects of EPI treatment on Aβ25–35 deposition in the hippocampus of mouse brains slices. Hippocampus were stained with specific antibodies against Aβ25–35 (red). Nuclei were stained with DAPI (blue). (A) Representative Aβ25–35 photographs were shown for the mice hippocampal in different groups. (B) Integral optical density (IOD) of the Aβ25–35. Data were expressed as mean ± SEM. ***p < 0.001 versus control group mice; #p < 0.05, ##p < 0.01, ###p < 0.001 versus model group mice. $p < 0.05, $$p < 0.01 versus EPI-treated group.

especially in the EPI03 group. The hippocampal sections were immuno-histochemically stained for Aβ25–35 to further confirm the anti-Aβ effects of EPI on cellular level. From the results of immunohistochemistry, we observed intuitively that the level of Aβ25–35 deposition was extremely higher compared with the model group, however significantly decreased after long-term EPI administration (Fig. 4). This result indicated that EPI treatment could effectively reduce Aβ25–35 deposition in vivo in dose-dependent manner. Compounds 1–11 isolated from P. incarnata were identified and elucidated by comprehensive analysis of their 1H NMR, 13C-NMR and HR-ESI-MS spectra compared with literature data (details in Supplementary Figs. 1–11), including stearic acid (1) (11 mg), pinellic acid (2) (19 mg), 7,4′-dihydroxyflavone (3) (36 mg), ursolic acid (4) (2460 mg), oleanolic acid (5) (25 mg), maslinic acid (6) (20 mg), asiatic acid (7) (18 mg), 2-hydroxyursolic acid (8) (26 mg), myricadiol acid (9) (11 mg), monotropein (10) (24 mg) and ilekudinoside A (11) (37 mg). Four pentacyclic triterpenoids named asiatic acid (7), ilekudinoside A (11), myricadiol (9), and 2-hydroxyursolic acid (8) were reported from P. incarnata for the first time. The scheme of extraction and isolation of P. incarnata was showed in Fig. 5. The chemical structures of

compounds were drawn by chemdraw 7.0 and showed in Fig. 6. Taken together, UA was gained as the most abundant phytochemical from P. incarnata. Whether it is owing to noticeable neuroprotective effects of EPI on the performance of AD mice in behavioral tests? It is worthy further investigation concerning to its underlying pharmacological mechanisms of neuroprotective effects against β-amyloid-induced memory impairment in vivo. There was no significant difference in the line crossing numbers or mean speeds of different groups during OFT (Fig. 7A). The results showed that treatments of UA and ICV of Aβ25–35 had no effect on the locomotor activity of mice. Student's t-tests revealed that there were significant differences in group effects in the latency for entering the target box during training session (Fig. 7B). We found that ICV of Aβ25–35 significantly increased latency behavior of C57BL/6 mice compared with normal group on the 4th day (p < 0.01). Moreover, UA03-treated group with the dosage of 56 mg/kg demonstrated significant decrease of its latency on day 4 (p < 0.05), compared to the vehicle-treated Aβ25–35 group. During probe session, UA treatment significantly decreased the escape latency, mistake times in a dose-dependent manner. It is noted that remarkable

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Fig. 5. Scheme of extraction and isolation of P. incarnata.

behavior alterations were observed including escape latency (p < 0.05), mistake times (p < 0.01) in the UA03-treatment group. The results suggested that Aβ25–35 induced a deficit in memory in Barnes maze test of AD mice which was reversed by UA treatment especially UA 03 group. There was no significant difference observed on the mean speed of swimming, suggesting that UA has no stimulating effects on the locomotor behavior of naïve mice (Fig. 8A). During the training sessions, the model group took longer time in training days on escape latency compared with the normal group, especially on the fifth day (p < 0.01). However, the administration of UA (14, 28, 56 mg/kg) significantly shortened the escape latency during the training sessions on the 5th day compared with the Aβ25–35-treated mice (day 5 F2,21 = 4.575, p < 0.05) (Fig. 8B). These data suggested that UA ameliorated the Aβ25–35-induced cognitive deficits and improved memory during the training sessions. On the day of the final session (6th day), a probe trial was conducted. As shown in Fig. 8C and D, the escape latency (F3,28 = 6.189, p < 0.01) and the time to the target quadrants (F3,28 = 3.095, p < 0.05) of the UA-treated group was significantly shorter compared with Aβ25–35-treated group. Additionally, UA increased the crossing numbers of target quadrants in a dose-dependent manner, there into UA03 group possessed outstanding

activity of ameliorating memory. In the hippocampus, the level of Aβ25–35 deposition was markedly increased compared with the control group. However, the results of immunohistochemistry demonstrated that the level of Aβ25–35 deposition could be significantly attenuated after long-term UA administration compared with the model group (Fig. 9A and C). The data indicated that UA treatment could effectively reduce Aβ25–35 deposition in vivo in dose-dependent manner. Thereinto, UA03 (56 mg/kg) possessed outstanding ability of scavenging Aβ25–35. Microglia have been reported to play key roles in mediating the clearance of Aβ. The activation of microglia were detected by immunofluorescence, and Iba-1 was tested as a marker.7,8 Our data showed that the number of microglia was significantly increased after ICV of Aβ25–35 (F3,28 = 16.75, p < 0.01) (Fig. 9B and 9D). What’s more, Iba1 expressing level of UA-treated group was dramatically increased in a dose-dependent manner. These results suggested that UA treatment could effectively mediate the clearance of Aβ25–35 in mice model via activating microglia cells. Pyrola incarnata has been known not only as functional food material, but also as an important component involved in traditional Chinese prescriptions for over 2000 years. P. incarnata is one of the most important plants because of its anti-aging, renal function enhancement

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Fig. 6. Constituents isolated from P. incarnata.

and immunity boosting properties.6,9 To the best of our knowledge, there is still no evidence that P. incarnata could possess neuroprotective ability against Aβ-induced memory impairment in vivo. Hence, the present study demonstrated that P. incarnata improved the ability of cognition, behavior, exploration of C75BL/6 mice by ameliorating Aβ25–35 accumulation. Additionally, UA might be one of the principal monomers contributed to the memory protective effects of EPI. The most frequent neurodegenerative disorders, such as AD and senile dementia, are characterized by the impairments in memory and cognition.10,11 An increasing number of studies indicate that the Aβ peptide is the major component of senile plaques and is regarded to play a critical role in the pathogenesis of AD.12–14 Accumulations of Aβ are associated with hippocampal network dysfunction and result in cognitive deficits.15,16 Microglia are the resident macrophage – like cells of the CNS, tasked with surveilling and clearing harmful substances including Aβ to maintain brain integrity under physiological and pathological conditions.17,18 Additionally, microglia are involved

in modulating higher cognitive functions such as learning and memory. Recent works showed that microglia could be mobilized to promote Aβ clearance and reduce amyloid deposition through multiple mechanisms. Apart from phagocytosis of Aβ, microglia could also degrade Aβ by secreting proteolytic enzymes, such as insulin-degrading enzyme, neprilysin, matrix metalloproteinase 9, and plasminogen.19,20 Iba1 is a calcium binding protein and a marker specifically expressed in microglia in brain cells of rat or mice.21 Our results demonstrated that expressed Iba1 protein was remarkably increased in microglia in immunocytochemical and immunohistochemical assays, indicating that UA attenuated neurotoxicity via activating microglia pathway and clearance of Aβ directly. In brief, UA was revealed to improve learning and memory deficits remarkably, and ameliorate Aβ25–35 levels by activating microglia and upregulating Iba1 level in the mouse hippocampus of AD mice. UA (3β-hydroxy-urs-12-en-28-oic acid) is an ursane pentacyclic triterpenoid widely distributed in many plants, such as apples,

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Fig. 7. The effects of ursolic acid against impaired spatial memory in mice during the open-field test and Barnes maze test. Number of line crossing (A1), mean speed (A2) of mice in open-field test. Number of line crossing, mean speed were measured during five minutes session. The effects of ursolic acid on latency time to enter the escape box during the training session (B1), latency time (B2) and mistake times (B3) during the probe trial session are presented. Data were expressed as mean ± SEM. ns: p > 0.05 versus each group mice. **p < 0.01 versus control group mice; #p < 0.05, ##p < 0.01 versus model group mice; $p < 0.05 versus UA-treated mice.

hawthorn, plantain, gardenia, cranberries, peppermint and etc. Previous research demonstrated UA possessed various pleiotropic biological activities, including anti-oxidant, -inflammatory, -hyperlipidemic, -ulcer, -microbial, and -tumoral activities.22–24 However, the neuroprotective effects and the underlying mechanisms of UA involved the neurodegenerative related targets remain limited available. Our previous study have proved that UA showed neuroprotective effects against the Aβ25–35 induced apoptosis25 and anti-oxidant activity against H2O2-incduced cytotoxicity in PC12 cells.25 Aβ is generated from the amyloid precursor protein (APP) by proteolytic processing of β-secretase (BACE1) and γ-secretase. The former is a key enzyme in the production of Aβ and becomes a prime target for the therapeutic intervention in AD. UA also demonstrated significant attenuation of BACE1 rather than TACE (α-secretase), suggesting that it is relatively selective and specific inhibitor of BACE1.26 Microglia of alternative (M2) activation rather than classical (M1) activation could inhibit inflammation and tissue repair, and play an important role in reducing neuroinflammation.27 Activated microglia cells in chronic inflammation could generate reactive oxygen species (ROS) and cause the degradation of brain tissue. Another study reported that UA possessed neuroprotective activity by inhibiting the expression of iNOS and COX-2 in Aβ25–35-injured PC12 cells, blocking nuclear translocation of the p65/NF-κB and phosphorylation of IκB-α, reducing ERK1/2, p-38, and JNK phosphorylation.28

Resent data also reported that UA suppressed the generation of ROS, attenuated DNA fragmentation and eventually attenuated Aβ-induced apoptosis in a dose-dependent manner.29 Therefore, it could be deduced that the beneficial effect of UA is closely connected with clearance of Aβ, anti-neuroinflammation and antioxidant activities, indicating multifactorial mechanisms involved in activated microglia pathway. Natural products have long been the major molecular resources for the discovery of AD drugs based on features of structural diversity. The biological activities of the P. incarnata can be attributed to various secondary metabolites. In this study, eleven compounds were successfully purified and identified from EPI including seven triterpenoids, an iridoid glycoside and a flavone. Four pentacyclic triterpenoids named asiatic acid (7), ilekudinoside A (11), myricadiol (9), and 2-hydroxyursolic acid (8) were reported from P. incarnata for the first time. UA obtained from both PE and AE partitions simultaneously, was the most abundant phytochemical and characteristic pentacyclic triterpenoid from P. incarnata. Triterpenoids has not been paid much more attention than flavone and quinones in the genus of Pyrola plants in the previous study. Hyperin, 2-O’-galloylhyperin and chimaphilin are considered as the primary bioactive constituents of P. incarnata for decades, and the pharmacological research focused on their anti-inflammation, antioxidant, and anti-bacterial activities. This is the first report regarding the characteristic triterpenoid of P. incarnata and its pharmacological

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Fig. 8. The attenuating effects of ursolic acid on Aβ25–35-induced memory impairment during the Morris water maze test. Ursolic acid (14, 28 or 56 mg/kg) or the same volume of vehicle solution were administered to mice. Swimming mean speed (A) of mice in Morris water maze first day test. Escape latency apparent during the training sessions (A). Escape latency apparent during the probe sessions (B). After the platform was removed, time to platform (target circle) (C) and the number of crossing platform (D) of mice. The path map of the mice during the probe sessions (E). Data were expressed as mean ± SEM (n = 9). *p < 0.05, **p < 0.01 versus control group mice; #p < 0.05, ##p < 0.01 versus model group mice. $p < 0.05 versus UA-treated mice.

screening in vivo. In conclusion, we demonstrate that EPI and its active phytochemical could improve the learning and memory deficits in Aβ-induced mice model and ameliorate Aβ25–35 level by activating microglia and upregulating Iba1 level. These novel findings suggest that P. incarnata contains biologically active component that could attenuate the progression of Aβ-related neurodegenerative diseases. Collectively, it suggests Pyrola incarnata could be a valuable natural plant both as neuroprotection therapeutic agents and functional foods through the improvement of cognitive function. Furthermore, it is worth testing for further pharmacological investigations in the treatment of neurological disease.

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 31900286 to X.Y), the Scientific Research Projects of Hubei education department for Young Scholars (Grant No. Q20171109 to X.Y), China Postdoctoral Science Foundation funded project (Grant No. 2018M632946 to Z.Z), and the National Natural Science Foundation of China (Grant No. 31800885 to Z.Z). 8

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Fig. 9. Effects of ursolic acid on the expression of immunoreactivity of Aβ25–35 in the mouse hippocampus. Hippocampus were stained with specific antibodies against Aβ25–35 (red) and Iba-1 (Green). Nuclei were stained with DAPI (blue). (A) Representative Aβ25–35 photographs were shown for the mice hippocampal in different groups. (B) Representative Iba-1 photographs were shown for the mice hippocampal in different groups. (C) Integral optical density of the Aβ25–35. (D) Integral optical density of the Iba-1. Data were expressed as mean ± SEM. *p < 0.05, ***p < 0.001 versus control group mice; #p < 0.05, ##p < 0.01, ### p < 0.001 versus model group mice. $p < 0.05, $$p < 0.01 versus UA-treated group.

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Appendix A. Supplementary data

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