Protocatechuic Acid as a Potential Phytomedicine in a TCM Herbal Extract...

[Full title: Protocatechuic Acid as a Potential Phytomedicine in a TCM Herbal Extract Mitigates Alcohol-Induced Osteoporosis]

OPEN ACCESS International Journal of Pharmacology ISSN 1811-7775 DOI: 10.3923/ijp.2024.229.240 Research Article Protocatechuic Acid as a Potential Phytomedicine in a TCM Herbal Extract Mitigates Alcohol-Induced Osteoporosis 1,4 Dongyang Qian, 2 Hui Zhou, 2 Qiang Yang, 2 Yuancai Liu, 2 Zhe Wang, 2 Shiguang Lu, 2 Guoqiang Tong, 1 Lily Zhang, 3 Xin Wang and 1 Shuanhu Zhou 1 Department of Orthopedic Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, United States 2 Hubei Key Laboratory of Quality and Safety of Traditional Chinese Medicine Health Food, Jing Brand Co. Ltd., Wuhan, Hubei, China 3 Department of Neurosurgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, United States 4 Department of Orthopedics, The First Affiliated Hospital, Guangzhou Medical University, Guangzhou, China Abstract Background and Objective: Alcohol consumption exerts a multifaceted influence on skeletal health, with persistent gaps in understanding its contribution to bone loss. While light to moderate alcohol intake generally benefits or remains neutral for bone health in older adults, chronic excessive drinking leads to alcoholic osteoporosis and alcohol-induced osteonecrosis, hindering bone fracture recovery. Osteoporosis, a widespread bone ailment, presents a formidable global public health concern. For centuries, Traditional Chinese Medicine (TCM) has provided remedies for diverse medical conditions, including osteoporosis and alcoholism. This study aims to investigate whether protocatechuic acid (PCA) is a potential phytomedicine in a TCM herbal extract to effectively mitigate alcohol-induced osteoporosis in mice. Materials and Methods: To establish murine models of alcohol-induced bone loss, oral alcohol administration is employed via gavage in Balb/c mice and Lieber-DeCarli alcohol diet in C 57 BL/6 mice. The impacts of the herbal extract and PCA on alcohol-induced osteoporosis were investigated by bone morphological analysis using Micro-Computed Tomography (µ-CT) Results: Current study revealed that there is a preventive effect of the TCM herbal extract and PCA on alcohol-induced bone loss in Balb/c mice and the supplementation of the Lieber-DeCarli alcohol liquid diet with 50 mg kg G 1 PCA significantly attenuated alcohol-induced trabecular bone loss in C 57 BL/6 J mice. These findings highlight PCA as one of the potential phytomedicines within the herbal extract Conclusion: Natural products derived from TCM herbs have made substantial contributions to pharmacotherapies and remain an invaluable resource for drug discovery. This study demonstrates the potential of therapeutic natural products in preventing alcohol-induced osteoporosis faced by long-term drinkers Key words: Traditional Chinese Medicine, herbal extracts, protocatechuic acid, alcohol, osteoporosis Citation: Qian, D., H. Zhou, Q. Yang, Y. Liu and Z. Wang et al., 2024. Protocatechuic acid as a potential phytomedicine in a TCM herbal extract mitigates alcohol-induced osteoporosis. Int. J. Pharmacol., 20: 229-240 Corresponding Author: Shuanhu Zhou, Department of Orthopedic Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, United States Hui Zhou, Hubei Key Laboratory of Quality and Safety of Traditional Chinese Medicine Health Food, Jing Brand Co. Ltd., Wuhan, Hubei, China Copyright: © 2024 Dongyang Qian et al. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. Competing Interest: The authors have declared that no competing interest exists Data Availability: All relevant data are within the paper and its supporting information files.

Int. J. Pharmacol., 20 (2): 229-240, 2024 INTRODUCTION The relationship between alcohol consumption and human health is intricate. While the benefits of moderate alcohol consumption remain a topic of debate, epidemiological studies indicate potential advantages of light to moderate alcohol consumption, such as supporting heart health, safeguarding against diabetes and correlating with reduced risks of mortality in older adults 1-4 . Chronic excessive drinking poses serious risks to various organs and tissues, being a significant factor in stroke, heart failure and mortality 5-7 . Osteoporosis stands as a prevalent global public health concern, being the most common bone disease and a leading cause of fractures and disability 8-12 Despite considerable advancements in comprehending postmenopausal and senile osteoporosis, the relationship between alcohol consumption and its impact on bone health remains intricate, resulting in notable gaps in our understanding of how alcoholism contributes to bone loss The complex effects of alcohol on skeleton depend on age, drinking pattern, hormonal status and the type of alcohol consumed. Light to moderate alcohol consumption is generally reported to be beneficial or have a neutral impact on bone health in older adults, while chronic excessive drinking can lead to alcoholic osteoporosis, which is a disease that compromises normal bone metabolism and increases fracture risk, diminishes bone repair and alcohol-induced osteonecrosis 13-19 Traditional Chinese Medicine (TCM) has been practiced over thousands of years for the treatment and symptom management of a wide range of medical conditions, including osteoporosis and alcoholism. Natural products, in particularly extracted from TCM, have contributed significantly to pharmacotherapy and remain unique sources for drug discovery 20,21 Preclinical and clinical studies demonstrated that an herbal extract (Jing extract) utilized a composite herbal formula consisting of nine Chinese herbal medicines, including Astragalus , Cistanche deserticola , Dioscorea polystachya , Lycium barbarum , Epimedium , Cinnamomum cassia , Syzygium aromaticum , Angelica sinensis and Curculigo orchioides , which have the anti-fatigue and immunity-enhancing properties in humans and animals 22-24 Qian et al 19 suggested that the herbal extract prevents chronic excessive alcohol consumption-induced osteopenia in male mice. This study investigates whether Jing extract holds therapeutic potential for combating alcohol-induced bone loss in female mice The HPLC, LC/MS and NMR analyses 22-24 revealed that Jing extract comprises a variety of bioactive components with antiosteoporotic properties, some of which also exhibit potential in managing alcohol use disorder, including protocatechuic acid, acteoside, calycosin, daidzin, echinacoside, epimedin, eugenol, ferulic acid, hyperoside, icariside, icariin, oleanolic acid, puerarin, sagittatoside, scopolin and ligustilide. Protocatechuic acid (3,4-dihydroxybenzoic acid, PCA) is a natural phenolic compound in various plant-based foods and traditional herbal medicines and has a wide array of health benefits, including antioxidant, anti-inflammatory, anti-hyperglycemic, antibacterial, antiviral, anticancer, antiosteoporotic, anti-aging and neuroprotective properties 25-28 . This study delves into the potential of PCA, a representative bioactive component within Jing extract, to mitigate alcohol-induced osteopenia in mice Current study not only underscores the preventive effects of TCM Jing extract against alcohol-induced bone loss in female mice, but also highlights PCA as one of the promising phytomedicines present in this herbal extract. Further investigations are warranted to explore whether other natural products identified in the extract share similar therapeutic potential in the mouse model of osteopenia MATERIALS AND METHODS Study area: This study was conducted in Brigham and Womenʼs Hospital, Harvard Medical School, United States during the period from January, 2019 to December, 2022 Materials: In this study, a composite herbal formula consisting of nine Chinese herbal medicines was utilized These herbs include Astragalus , Cistanche deserticola , Dioscorea polystachya , Lycium barbarum , Epimedium , Cinnamomum cassia , Syzygium aromaticum , Angelica sinensis and Curculigo orchioides (Fig. 1). These herbs were prepared in accordance with our description 19 . The collection of these Traditional Chinese Medicine herbs was carried out by researchers at the Jing Brand Research Institute. Voucher specimens, as outlined in a report by researchers 22-24 , have been deposited at the Herbarium of Jing Brand Research Institute, located in Daye, Hubei, China. The raw Chinese herbal medicines underwent a meticulous process, which included washing, drying and slicing into pieces, followed by superfine pulverization, in accordance with the protocols outlined in the Chinese Pharmacopoeia. The traditional Chinese medicine herbal extracts, referred to as Jing extract, were prepared using a percolation extraction method, 230

Int. J. Pharmacol., 20 (2): 229-240, 2024 231 Fig. 1: Experimental design A concentrated TCM herbal extract, referred to as Jing extract, from nine Traditional Chinese Medicine herbs and bioactive com pounds in the herbal extract were investigated in animal models of alcoholinduced bone loss As tr agal us C is tanche des er tico la C ur cul igo or chi oi des Ep imed iu m L yci um bar bar um D ios cor ea polys tachya Angel ic a s inens is C innam om um cas si a Syz ygi um ar om at ic um al A lco ho lin du ced os teopen ia O O HO OH OCH 3 HO HO HO O O OH O O OH O O O OH OH OH H O OH OH OH O OH HO O OH O O O O O OH OH OH OH HO HO H OH O O OH O HO O OH HO O O O O OH OH OH HO OH OH OH HO H H H O OH HO HO O OH HO O OH O O O O OH OH OH H O O OH OH OH O O Acteoside Calycosin Daidzin Echinacoside Epimedin A Oleanolic acid Icariin HO O OH O O O HO O OH HO Icariside II Hyperoside Ferulic acid Eugenol Protocatechuic acid Sagittatoside A Sagittatoside B Scopolin (Z)-Ligustilide HO HO HO OH O O O O OH HO HO OH OH O O O O O O O O HO HO HO OH OH OH OH OH O OH OH HO O O OH O O H C 3 CH 3 OH HO HO OH O O O O O OH HO HO O OCH 3 CH 3 OH O O OH HO O O O OH OH OH H O O OH OH

Int. J. Pharmacol., 20 (2): 229-240, 2024 as detailed by Qian et al 19 . The extracts of these Chinese herbal medicines (Jing extract) have been subjected to extensive characterization through chemical constituent analysis with LC/MS and NMR 22,23 and high-performance liquid chromatogram-gas chromatography fingerprint analysis 24 Protocatechuic acid (PCA) (3,4-dihydroxybenzoic acid) and vitamin D 3 were procured from MilliporeSigma (Burlington, Massachusetts). Xian-Ling-Gu-Bao (XLGB) capsule was obtained as an over-the-counter Traditional Chinese Medicine (TCM) product from Sinopharm Group Tongjitang Pharmaceuticals Co. Ltd., based in Guizhou, China Animals: The Balb/c and C 57 BL 6 mice from Jackson Laboratory (Bar Harbor, Maine, USA) were procured, two mating pairs per mouse strain, to establish breeding colonies within the animal facilities of Brigham and Womenʼs Hospital (BWH). This study used 70 Balb/c mice and 20 C 57 BL 6 mice (2 months old). The animal facilities at BWH, fully accredited by Association for Assessment and Accreditation of Laboratory Animal Care, were employed for housing the mice. The mice were maintained under controlled conditions, including a temperature of 22±2/-2.5 E C with alarm setpoints at+4/-3 E C and a humidity level of 40% with alarm setpoints at +15/-10%. Lighting was provided on a 12 hrs light/12 hrs dark cycle. Mice were given unrestricted access to standard chow and water Ethical consideration: All aspects of animal maintenance and experimental procedures were conducted in strict adherence to ethical guidelines for animal research, which were established and approved by the Brigham and Womenʼs Hospital Institutional Animal Care and Use Committee (#2016 N 000361) Oral alcohol administration models of alcohol-induced bone loss: To establish murine models of alcohol-induced osteoporosis and investigate the impacts of the herbal extract and PCA, oral alcohol administration via gavage in female Balb/c mice was employed. This approach follows the procedures outlined in previous studies 19,29 In this study, vitamin D 3 , a recognized preventive agent for alcohol-induced osteopenia 30 , was employed as a control for the potential small molecule phytomedicine, PCA. Xian-Ling-Gu-Bao (XLGB) capsule is a potent traditional Chinese medicine prescription widely utilized for the prevention and treatment of osteoporosis in China, as supported by studies 31-33 The XLGB is composed of several key ingredients, including Epimedium brevicornum , Dipsacus fullonum , Salvia miltiorrhiza , Anemarrhena asphodeloides , Psoralea corylifolia and Rehmannia glutinosa 32-33 . This study employed XLGB as the control for Traditional Chinese Medicine (TCM) anti-osteoporotic treatment. The 0.4 g kg G 1 dosage administered to the mice corresponds to the recommended human dosage (taken 3 capsules twice per day, each capsule containing 0.3 g of XLGB) based on mouse-human dose conversion 34,35 Chronic alcohol-induced bone loss model using the Lieber-DeCarli liquid diet: The C 57 BL/6 is a strain of mice known for its preference for alcohol and is frequently utilized in alcohol-related experiments 29 . For current study, C 57 BL/6 J male mice were randomly divided into age-matched groups at 8 weeks old. They were then subjected to dietary conditions, including the Lieber-DeCarli liquid control diet (Bio-Serv) or the Lieber-DeCarli alcohol diet (Bio-Serv), which contained 5% (v/v) alcohol and maltose dextrin. These dietary conditions were administered according to previously established protocols 29,30,36,37 . A third experimental group received the Lieber-DeCarli alcohol diet containing 5% (v/v) alcohol along with the phytomedicine candidate PCA (at a dose of 50 mg kg G 1 ). The C 57 BL/6 J male mice receiving the Lieber-DeCarli alcohol liquid diet were provided with food ad libitum , while calorically matched control and Lieber-DeCarli alcohol plus PCA (50 mg kg G 1 ) diets were adjusted based on the previous dayʼs consumption by the alcohol-exposed groups. To ensure consistent experimental dosages over the 6-week study period, the quantity of PCA added to the Lieber-DeCarli alcohol liquid diet was adjusted daily in accordance with body weight and the previous dayʼs consumption Bone morphological analysis Micro-Computed Tomography (µ-CT): Bone morphological analysis of the proximal tibia was conducted using Micro-Computed Tomography System (µ-CT 35, Scanco Medical, Switzerland), following the procedures as described by Qian et al 19 . For trabecular bone analysis, selected the scanned region proximal to the growth plate, extending 1.4 mm, as indicated by the outlined box in Fig. 2 a. The 3-dimensional microstructural characteristics of the bone, encompassing Trabecular Bone Mineral Density (Tb. BMD), tibia Trabecular relative Bone Volume (Tb. BV/TV), trabecular number (Tb. N) and cortical bone mineral density (C. BMD), were evaluated using software provided by the manufacturer, Scanco Medical, Switzerland. Furthermore, a second segment, measuring 0.6 mm in length and positioned at the midpoint of the tibia as indicated by the enclosed box in Fig. 2 c, was employed to compute diaphyseal parameters 232

Int. J. Pharmacol., 20 (2): 229-240, 2024 Fig. 2(a-c): Oral alcohol administration models of alcohol-induced bone loss in Balb/c female mice. The Balb/c female mice (2 months old) were administrated with different doses of alcohol, 0.7, 1.4 and 2.8 g kg G 1 (alcohol/body weight) for 40 days, (a) Representative µ-CT 3-D microstructures of trabecular bone were obtained from female mice with alcohol gavage (alcohol) or water gavage (control) for 40 days, (b) Quantitative analysis of Trabecular Bone Mineral Density (Tb. BMD) and (c) Representative µ-CT 3-D microstructures of cortical bone were obtained from female mice with 2.8 g kg G 1 alcohol gavage (alcohol) or water gavage (control) for 40 days (a) Bars represent 100 µm. The 3-D microstructural properties of the tibia were calculated using software supplied by the manufacturer, (b) (Alcohol of 0.7 g kg G 1 , n = 3, 1.4 g kg G 1 , n = 7, 2.8 g kg G 1 , n = 14, vs control, n = 18; *p<0.05, ***p<0.001, t-test, NS: Not significant and (c) Bars represent 100 mm. The quantitative analysis of cortical bone mineral density (C. BMD), 2.8 g kg G 1 alcohol did not induce cortical bone loss (alcohol, n = 9, vs control, n = 14, NS: Not significant and t-test) Statistical analysis: All experiments were conducted with a minimum of three replicates. Quantitative data were treated as continuous variables and analyzed using standard statistical tests. The Kolmogorov-Smirnov test was employed to assess the normal distribution of data sets. Group data is presented as Mean±SEM (Standard Error of the Mean). Unless specified otherwise, quantitative data were subjected to non-parametric Mann-Whitney tests for group comparisons. If the data met the assumptions of parametric analysis, t-tests were employed for two-group comparisons, while one-way ANOVA was utilized for multiple group comparisons, all conducted using GraphPad Instat (GraphPad Software, La Jolla, California). Statistical significance was defined as a p-value less than 0.05 RESULTS Optimal dosages for alcohol-induced bone loss in Balb/c female mice: To determine the optimal dosages for inducing alcohol-induced osteopenia in female Balb/c mice, a study was conducted using 2 months old mice and administered oral alcohol at doses of 0.7, 1.4 and 2.8 g kg G 1 of body weight. This administration took place once per day between 3-4 PM, five days a week, over a 40-day period Current study showed that the dosage of 0.7 g kg G 1 significantly increased Bone Mineral Density (BMD) in Balb/c female mice that received alcohol via oral gavage, while the 1.4 g kg G 1 alcohol dosage did not induce significant bone loss after the 40 days administration (Fig. 2 a-b). However, following 40 days of oral alcohol administration, the 2.8 g kg G 1 dosage of alcohol did lead to significant bone loss in Balb/c female mice (Fig. 2 a-b). Consequently, the 2.8 g kg G 1 alcohol dosage was selected and maintained a 40 days duration for further investigations into the effects of the herbal extract and PCA on alcohol-induced osteopenia. It is noteworthy that, under these conditions, using the same 2.8 g kg G 1 dosage and 40 days duration of alcohol administration did not result in bone loss in cortical bone (Fig. 2 c). Therefore, current report primarily focuses on the impact of alcohol on trabecular bone 233 Control Alcohol (0.7 g kg G 1 ) Alcohol (1.4 g kg G 1 ) Alcohol (2.8 g kg G 1 ) (a) 1 mm 100 µm - 0.7 1.4 2.8 (b) * NS *** 300 200 100 0 Tb . BMD (m g c m G 3 ) Alcohol (g kg G 1 ) - 2.8 Alcohol (g kg G 1 ) 1500 1200 900 600 300 0 C. BMD ( m g c m G 3 ) NS (c) DDW Alcohol (2.8 g kg G 1 ) 100 mm 100 mm

Int. J. Pharmacol., 20 (2): 229-240, 2024 Fig. 3(a-b): An herbal extract (Jing extract) prevents chronic alcohol consumption-induced osteopenia in young adult female Balb/c mice, (a) Representative µ-CT 3-D microstructures of trabecular bone were obtained from female mice with alcohol gavage (alcohol)±Jing extract or without alcohol (DDW control) and (b) Quantitative analysis of Trabecular Bone Mineral Density (Tb. BMD) (a) Bars represent 100 µm. The 3-D microstructural properties of the tibia were calculated using software supplied by the manufacturer and (b) 2.8 g kg G 1 of alcohol, n = 14, vs control, n = 18, ***p<0.001, t-test; alcohol+0.125 g kg G 1 of Jing extract, n = 6, vs alcohol, *p<0.05; alcohol+0.25 g kg G 1 of Jing extract, n = 16 vs alcohol, **p<0.01; alcohol+0.5 g kg G 1 of Jing extracts, n = 8, vs alcohol, ***p<0.001, t-test); the quantitative analysis of tibia Trabecular Relative Bone Volume (Tb. BV/TV) (*p<0.05, **p<0.01, ***p<0.001; control, n = 18; alcohol, n = 9; alcohol+0.125 g kg G 1 , n = 6, +0.25 g kg G 1 , n = 12, +0.5 g kg G 1 , n = 8, of Jing extract; t-test); the quantitative analysis of tibia trabecular number per mm (Tb. N) (***p<0.001, **p<0.01 and NS: Not significant; n is same as BV/TV; t-test) Herbal extract prevents chronic alcohol-induced bone loss in Balb/c mice: In present investigation, the study aimed to determine the efficacy of an herbal extract in mitigating the bone loss induced by chronic alcohol consumption in Balb/c female mice. To establish the model for alcohol-induced osteopenia, mice were administered a dose of 2.8 g kg G 1 of alcohol via oral gavage for a duration of 40 days. As depicted in Fig. 2 a, this regimen led to significant bone loss in Balb/c female mice. Subsequently, mice were assessed the impact of the herbal extract, specifically Jing extract from traditional Chinese medicine, on alcohol-induced osteopenia. In this experiment, 2 months old Balb/c female mice were orally administered 2.8 g kg G 1 of alcohol via gavage, with or without varying doses of the herbal extract (Jing extract) at 0.125, 0.25 or 0.5 g kg G 1 , as outlined in Fig. 3 a. After 40 days of oral administration of alcohol, with or without the herbal extract, the mice were sacrificed for bone morphological analysis through µ-CT. Figure 3 revealed that, the three-dimensional microstructures of trabecular bone (Fig. 3 a) and the quantitative analysis of tibial microstructural properties (Fig. 3 b), encompassing Trabecular Bone Mineral Density (Tb. BMD), Trabecular Relative Bone Volume (Tb. BV/TV) and trabecular number per mm (Tb. N), indicated a dose-dependent preventive effect of the herbal extract on alcohol-induced bone loss in Balb/c female mice. Current data demonstrated that the administration of 2.8 g kg G 1 of alcohol via oral gavage induced substantial bone loss in Balb/c mice following 40 days of treatment (Fig. 3). However, all three doses of the herbal extract-low, middle and high-ameliorated the trabecular bone damage caused by chronic alcohol consumption. Additionally, all three doses mitigated the alcohol-induced reductions in Bone Mineral Density (Tb. BMD) and relative Bone Volume or Bone Volume fraction (BV/TV) (Fig. 3 b). Furthermore, the high dose of the herbal extract also alleviated the decrease in trabecular number (Tb. N) induced by alcohol (Fig. 3 b) 234 200 100 0 Tb B M D (mg cm ) G 3 Alcohol - + + - - 0.125 Jing (g kg ) G 1 *** * ** *** + 0.25 + 0.5 0.20 0.15 0.10 0.05 0.00 Tb B V /T V Alcohol - + + - - 0.125 Jing (g kg ) G 1 *** * ** *** + 0.25 + 0.5 6 4 2 0 Tb. N/mm Alcohol - + + - - 0.125 Jing (g kg ) G 1 *** NS NS ** + 0.25 + 0.5 (b) (a) Control Alcohol (2.8 g kg G 1 ) Alcohol+Jing (0.125 g kg G 1 ) Alcohol+Jing (0.25 g kg G 1 ) Alcohol+Jing (0.5 g kg G 1 ) 1 mm 100 µm

Int. J. Pharmacol., 20 (2): 229-240, 2024 Fig. 4(a-b): Protocatechuic acid (PCA) prevents chronic alcohol consumption-induced osteopenia in C 57 BL 6 mice, (a) Overview of the chronic alcohol Lieber-DeCarli liquid diet feeding procedure and (b) µ-CT analysis of proximal tibia Scanned regions proximal to the growth plate were indicated with the box. Representative 3-D microstructures of trabecular bone were obtained from proximal tibia of C 57 BL 6 male mice with Lieber-Decarli 5% alcohol diet (alcohol)±PCA (50 mg kg G 1 ) or Lieber-Decarli control diet (Control) for 6 weeks; bar in bottom right-hand represents 100 µm. The quantitative µ-CT data of proximal tibia Tb. BMD, Tb. BV/TV and Tb. N (alcohol, n = 7; Control, n = 9; alcohol+PCA, n = 4) demonstrated that supplementation of the Lieber-DeCarli alcohol liquid diet with 50 mg kg G 1 PCA was able to attenuate trabecular bone loss in C 57 BL/6 J mice (*p<0.05, **p<0.01, ***p<0.001 and t-test). There are no differences in cortical BMD among the experimental groups (data not shown) Protocatechuic acid alleviates chronic alcohol-induced bone loss in C 57 BL 6 mice: Present study aimed to investigate whether protocatechuic acid (PCA), a component found in the Jing herbal extract, mitigates alcohol-induced osteopenia in C 57 BL/6 J male mice (8 weeks old) fed with the Lieber-DeCarli alcohol diet. Following six weeks of dietary intervention, during which C 57 BL/6 J mice were fed either the Lieber-DeCarli liquid control diet or the Lieber-DeCarli alcohol diet containing 5% v/v alcohol, with or without the inclusion of the phytomedicine candidate PCA at a dosage of 50 mg kg G 1 (Fig. 4 a), the study analyzed bone microstructures in the tibia using µ-CT (Fig. 4 b). Current data demonstrated that the alcohol liquid diet-induced trabecular bone loss in the tibias of male C 57 BL/6 J mice (Fig. 4 b). Intriguingly, the supplementation of the Lieber-DeCarli alcohol liquid diet with 50 mg kg G 1 PCA significantly attenuated this trabecular bone loss in male C 57 BL/6 J mice (Fig. 4 b). These findings highlight PCA as one of the potential phytomedicines within the herbal extract Protocatechuic acid mitigates alcohol-induced osteopenia in Balb/c mice: This study aimed to evaluate the preventive effects of protocatechuic acid (PCA) on alcohol-induced bone loss in Balb/c female mice, following a similar experimental approach as detailed in Fig. 3 for the herbal extract. Current study involved a comparative analysis of PCA, administered at a dosage of 50 mg kg G 1 and vitamin D 3 , delivered at a dosage of 2000 IU kg G 1 . The objective was to assess the individual impacts of PCA and vitamin D 3 on alcohol-induced osteopenia in 2 months old female Balb/c mice. Over a period of 40 days, 235 6 weeks Lieber-DeCarli control diet 6 weeks Lieber-DeCarli alcohol (5% v/v) diet ( ) ad libitum 6 weeks Lieber-DeCarli alcohol (5% v/v) diet+phytomedicine 5 days Lieber-DeCarli control diet ( ) ad libitum (a) 0.25 0.20 0.15 0.10 0.05 0.00 Tb B V /T V Alcohol - + + - - + PCA *** * 250 200 150 100 50 0 Tb B M D (mg cm ) G 3 Alcohol - + + - - + PCA *** * 8 6 4 2 0 Tb. N/mm Alcohol - + + - - + PCA ** * (b) Control Alcohol Alcohol+PCA 1 mm

Int. J. Pharmacol., 20 (2): 229-240, 2024 Fig. 5(a-b): Effects of PCA and herbal extract on alcohol-induced osteopenia in Balb/c mice, (a) Balb/c female mice (2 months old) were orally administrated 2.8 g kg G 1 body weight of alcohol with or without 50 mg kg G 1 PCA or 2000 IU kg G 1 of vitamin D 3 (Vit. D); after 40 days treatment, both PCA and vitamin D 3 significantly prevented alcohol-induced osteopenia and (b) Balb/c female mice (2 months old) were orally administrated 2.8 g kg G 1 body weight of alcohol with or without 0.5 g kg G 1 Jing extract or 0.4 g kg G 1 of Xian-Ling-Gu-Bao (XLGB) (a) **p<0.01, alcohol+PCA, n = 5, vs alcohol, n = 9; *p<0.05, vitamin D+alcohol, n = 5, vs alcohol; PCA vs vitamin D 3, NS: Not significant and t-test and (b) After 40 days, both Jing extract and XLGB significantly prevented alcohol-induced osteopenia in Balb/c female mice (***p<0.001, alcohol, n = 9, vs control, n = 18; **p<0.01, alcohol+Jing, n = 8, vs alcohol, t-test; alcohol+XLGB, n = 4 vs alcohol, **p<0.01; Jing vs XLGB, NS: Not significant and Mann-Whitney test) mice were administered either 2.8 g kg G 1 of alcohol per body weight alone or in combination with either 50 mg kg G 1 of PCA or 2000 IU kg G 1 of vitamin D 3 (Vit. D) via gavage in Balb/c female mice. Results demonstrated significant protective effects of both PCA and vitamin D 3 against alcohol-induced osteopenia in these mice (Fig. 5 a). Remarkably, the bone health of mice treated with alcohol in conjunction with either PCA or vitamin D 3 exhibited substantial improvements compared to those treated solely with alcohol. It is worth mentioning that no significant difference was observed between the effects of 50 mg kg G 1 of PCA and 2000 IU kg G 1 of vitamin D 3 (Fig. 5 a) Herbal extracts (Jing) and Xian-Ling-Gu-Bao (XLGB) in preventing alcohol-induced osteopenia: In our experiments (Fig. 5 b), we employed XLGB as the control for Traditional Chinese Medicine (TCM) anti-osteoporotic treatment. After a 40 days period of oral administration involving 2.8 g kg G 1 of alcohol, either with or without 0.5 g kg G 1 of Jing extract or 0.4 g kg G 1 of XLGB, we observed that both Jing extract and XLGB significantly prevented alcohol-induced osteopenia in 2 months old female Balb/c mice. Importantly, their preventive effects did not exhibit significant differences (Fig. 5 b). These findings underscore the potential of both PCA and Jing extract as promising interventions for mitigating the detrimental consequences of chronic alcohol consumption on bone health DISCUSSION Traditional Chinese Medicine (TCM) has been practiced over thousands of years for the treatment and symptom management of a wide range of medical conditions, including osteoporosis and alcoholism. For thousands of years, guided by TCM principles, alcohol has been combined with TCM herbal ingredients to create Chinese herbal Liqueur, which serve both preventive and therapeutic purposes 38,39 . The herbal extract (Jing extract) utilized in this study is derived from a Traditional Chinese Medicine (TCM) herbal formula found in the renowned Chinese Herbal Liqueur, Chinese Jing Liqueur 19,22-24 . Europe also has a longstanding tradition of consuming medicinal wines, including Absinthe, Chartreuse, Bénédictine, Dubonnet, Strega, Galliano, Gin, Kräuterlikör, Becherovka and Unicum, etc. Given that alcoholic osteoporosis primarily stems from diminished bone formation rather than heightened bone resorption 17,19 , anabolic anti-osteoporotic agents that stimulate bone formation, such as vitamin D and estrogen, are likely to prevent alcohol-induced bone loss as reported by researchers 30,37 The TCM formulas have a longstanding history of use in preventing and treating osteoporosis and the phytochemicals derived from TCM formulas hold significant potential for the development of novel anti-osteoporotic drugs 39-41 . A key molecular mechanism of TCM anti-osteoporotic drugs lies in their ability to promote osteoblast-mediated bone formation 42 , rendering TCM a promising candidate for therapy targeting alcoholic osteoporosis 236 200 100 0 Tb B M D (mg cm ) G 3 Alcohol - + + - - + PCA *** NS ** * - - - + - + Vitamin D (a) 200 100 0 Tb B M D (mg cm ) G 3 Alcohol - + + - - + Jing *** NS ** ** - - - + - + XLGB (b)

Int. J. Pharmacol., 20 (2): 229-240, 2024 To investigate the potential of the herbal extract (Jing extract) and protocatechuic acid (PCA), a component of the herbal extract, in ameliorating alcohol-induced osteopenia in murine models (Fig. 1), the study initially focused on determining the optimal dosage and duration for oral alcohol administration in Balb/c female mice. Among the various oral alcohol dosages tested, namely 0.7, 1.4 and 2.8 g kg G 1 of body weight administered over a 40 days period, it was observed that the 0.7 g kg G 1 dosage significantly increased Bone Mineral Density (BMD), the 1.4 g kg G 1 alcohol dosage did not induce significant bone loss, while the 2.8 g kg G 1 alcohol dosage resulted in substantial bone loss in Balb/c female mice (Fig. 2) Epidemiological studies suggested that light to moderate alcohol consumption have a beneficial or neutral impact on bone health and chronic excessive drinking leads to a loss of bone mass and increases the risk of osteoporosis 13-18 . Current findings from a female murine alcohol model in this study align with these epidemiological observations and our prior analysis involving male mice 19 . Doses exceeding 2.8 g kg G 1 or longer durations (50 days) led to unacceptable mortality rates in the alcohol group and a shorter duration (30 days) at the 2.8 g kg G 1 dose did not result in significant alcohol-induced bone loss (data not shown). As a result, this study selected the 2.8 g kg G 1 alcohol dosage with a 40 days duration for further investigation into the effects of the herbal extract and PCA on alcohol-induced osteopenia This study shows that the herbal extract (Jing extract) effectively mitigates alcohol-induced osteopenia in Balb/c female mice (Fig. 3), which confirmed previous findings in male mice 19 . The three doses of Jing extract, 0.125, 0.25 or 0.5 g kg G 1 , ameliorated the trabecular bone damage caused by chronic alcohol consumption (Fig. 3), implying that the potential of this TCM extract in preventing alcohol-induced osteoporosis faced by long-term drinkers. Xian-Ling-Gu-Bao (XLGB) capsule is an effective traditional Chinese medicine prescription used to prevent and treat osteoporosis in China 31-33 The XLGB was used as the TCM antiosteoporotic medicine control in current experiments. The current study showed that both Jing extract and XLGB significantly prevented alcohol-induced osteopenia in Balb/c female mice and their preventing effects have no significant difference (Fig. 5). These findings highlight the potential of Jing extract as a promising intervention to mitigate the detrimental effects of chronic alcohol consumption on bone health There are several different murine models of alcohol-induced bone loss regarding alcohol administration, including oral alcohol administration by gavage 43,44 , alcohol in drinking water 45,46 , intraperitoneal (IP) administration of alcohol by injection 44 and Liber-DeCarli liquid diet feeding 29,30,47 . While there are both benefits and drawbacks associated with various methods of alcohol administration, utilizing oral alcohol administration via gavage in animals proves to be a superior model for mimicking intermittent drinking patterns observed in humans. In this study, we employed oral alcohol administration via gavage in Balb/c female mice as the murine models for alcohol-induced bone loss, aiming to investigate the impacts of the herbal extract (Fig. 3) and PCA (Fig. 5) on alcoholic osteoporosis. The C 57 BL/6 J mice voluntarily drink alcohol-laden liquids preferentially and are widely used in alcohol-liquid diet experiments 29,48 . In the second model of alcohol-induced bone loss of this study, we used C 57 BL/6 J mice with the Lieber-DeCarli alcohol liquid diet to investigate whether protocatechuic acid (PCA), a component found in the herbal extract, mitigates alcohol-induced osteopenia (Fig. 4). In both oral alcohol administration by gavage model (Fig. 5) and Lieber-DeCarli alcohol liquid diet model (Fig. 4), PCA significantly attenuated trabecular bone loss in mice These findings highlighted PCA as one of the potential phytomedicines within the herbal extract Among the TCM herbs in the herbal extract used in this study, Astragalus , Cistanche deserticola , Dioscorea polystachya , Lycium barbarum , Epimedium , Cinnamomum cassia , Syzygium aromaticum , Angelica sinensis and Curculigo orchioides (Fig. 1). Protocatechuic acid (PCA) was detected in Angelica sinensis 49 , Cinnamomum cassia 50 , Syzygium aromaticum 51 , Dioscorea polystachya 52 and Lycium barbarum 53 . Protocatechuic acid (PCA), a natural phenolic compound in various plant-based foods and traditional herbal medicines, has a wide array of health benefits 25-28 . In an animal model of postmenopausal osteoporosis, oral supplementation with PCA (20 mg/kg/day) significantly ameliorated the ovariectomized-mediated bone loss and changes in bone biomechanical properties in female ICR mice 54 . In a lipopolysaccharide (LPS)-induced mouse model of inflammatory bone loss, oral administered 25 mg kg G 1 of PCA attenuates bone loss in ICR male mice 55 . The PCA significantly increased intracellular mineralization in MSCs in vitro 56 Previous study demonstrated that PCA significantly alleviates the inhibitory effects of alcohol on osteoblastogenesis 19 Further in vivo investigations are needed to explore the mechanism by which PCA ameliorates alcohol-induced bone loss resulting from decreased bone formation or increased bone resorption 237

Int. J. Pharmacol., 20 (2): 229-240, 2024 Based on Lipinskiʼs drugability in which poor absorption or permeation is more likely when there are more than 5 H-bond donors, 10 H-bond acceptors, the molecular weight is greater than 500 and the calculated Log P (Partition coefficient) is greater than 5 57 , the drug-like property parameters of PCA include 3 H-bond donors, 4 H-bond acceptors, 154.1 of molecular weight and 0.903 of calculated Log P, suggested that PCA is a promising drug candidate. Botanical-based natural products are an important resource for medicinal drug discovery and continue to provide diverse pharmacophores with therapeutic potential against human diseases 58 . In this study, current findings highlight PCA as one of the potential phytomedicines within the herbal extract. However, further investigations are warranted to ascertain whether the remaining natural products identified in the herbal extract possess similar potential as phytomedicines in our mouse model of osteopenia CONCLUSION This study demonstrates that a TCM extract and protocatechuic acid, an emerging phytomedicine within the herbal extract, effectively mitigate alcoholic osteoporosis in murine models. Natural products derived from TCM herbs have made substantial contributions to pharmacotherapies and remain an invaluable resource for drug discovery. This study demonstrates the potential of therapeutic natural products in preventing alcohol-induced osteoporosis faced by long-term drinkers SIGNIFICANCE STATEMENT Traditional Chinese Medicine (TCM) has treated various diseases, including bone diseases and alcoholism. Chronic excessive drinking results in alcohol-induced bone diseases, including osteoporosis, which increases fracture risk. Current study showed that a TCM herbal extract and protocatechuic acid (PCA), a phytomedicine within the TCM herbal extract, effectively mitigate chronic excessive alcohol consumption-induced osteoporosis in mice, implying that traditional medicinal plants have the therapeutic potential of preventing chronic excessive drinking-induced bone diseases ACKNOWLEDGMENTS The authors appreciate the researchers and workers at Jing Brand Research Institute for collecting the traditional Chinese medicine herbs for this study and depositing them at the Herbarium of Jing Brand Research Institute, located in Daye, Hubei, China. This work was supported by a grant (2018 A 007061) from Hubei State Guoqiang Baojianjiu Center for Engineering and Technology Research REFERENCES 1 Howard, A.A., J.H. Arnsten and M.N. Gourevitch, 2004. Effect of alcohol consumption on diabetes mellitus: A systematic review. Ann. Intern. Med., 140: 211-219 2 Karlamangla, A.S., C.A. Sarkisian, D.M. Kado, H. Dedes and D.H. Liao et al ., 2009. Light to moderate alcohol consumption and disability: Variable benefits by health status Am. J. Epidemiol., 169: 96-104 3 Mostofsky, E., K.J. Mukamal, E.L. Giovannucci, M.J. Stampfer and E.B. Rimm, 2016. Key findings on alcohol consumption and a variety of health outcomes from the nursesʼ health study. Am. J. Public Health, 106: 1586-1591 4 Kunzmann, A.T., H.G. Coleman, W.Y. Huang and S.I. Berndt, 2018. The association of lifetime alcohol use with mortality and cancer risk in older adults: A cohort study. PLoS Med., Vol. 15. 10.1371/journal.pmed.1002585 5 Garaycoechea, J.I., G.P. Crossan, F. Langevin, L. Mulderrig and S. Louzada et al ., 2018. Alcohol and endogenous aldehydes damage chromosomes and mutate stem cells. Nature, 553: 171-177 6 Wood, A.M., S. Kaptoge, A.S. Butterworth, P. Willeit and S. Warnakula et al ., 2018. Risk thresholds for alcohol consumption: Combined analysis of individual-participant data for 599 912 current drinkers in 83 prospective studies Lancet, 391: 1513-1523 7 Degenhardt, L., F. Charlson, A. Ferrari, D. Santomauro and H. Erskine et al ., 2018. The global burden of disease attributable to alcohol and drug use in 195 countries and territories, 1990-2016: A systematic analysis for the Global Burden of Disease Study 2016. Lancet Psychiatry, 5: 987-1012 8 Cosman, F., S.J. de Beur, M.S. LeBoff, E.M. Lewiecki, B. Tanner, S. Randall and R. Lindsay, 2014. Clinician's guide to prevention and treatment of osteoporosis. Osteoporosis Int., 25: 2359-2381 9 Wright, N.C., A.C. Looker, K.G. Saag, J.R. Curtis, E.S. Delzell, S. Randall and B. Dawson-Hughes, 2014. The recent prevalence of osteoporosis and low bone mass in the United States based on bone mineral density at the femoral neck or lumbar spine. J. Bone Mineral Res., 29: 2520-2526 10. Burge, R., B. Dawson-Hughes, D.H. Solomon, J.B. Wong, A. King and A. Tosteson, 2007. Incidence and economic burden of osteoporosis-related fractures in the United States, 2005-2025. J. Bone Mineral Res., 22: 465-475 11. Langdahl, B.L. and J.D. Andersen, 2018. Treatment of osteoporosis: Unmet needs and emerging solutions. J. Bone Metab., 25: 133-140 238

Int. J. Pharmacol., 20 (2): 229-240, 2024 12. Compston, J.E., M.R. McClung and W.D. Leslie, 2019 Osteoporosis. Lancet, 393: 364-376 13. Mikosch, P., 2014. Alcohol and bone. Wien. Med Wochenschr., 164: 15-24 14. Gaddini, G.W., R.T. Turner, K.A. Grant and U.T. Iwaniec, 2016 Alcohol: A simple nutrient with complex actions on bone in the adult skeleton. Alcohol Clin. Exp. Res., 40: 657-671 15. Maurel, D.B., N. Boisseau, C.L. Benhamou and C. Jaffre, 2012 Alcohol and bone: Review of dose effects and mechanisms Osteoporosis Int., 23: 1-16 16. Turner, R.T., 2000. Skeletal response to alcohol. Alcohol. Clin Exp. Res., 24: 1693-1701 17. Chakkalakal, D.A., 2005. Alcohol-induced bone loss and deficient bone repair. Alcohol Clin. Exp. Res., 29: 2077-2090 18. Sommer, I., A.T. Erkkilä, R. Järvinen, J. Mursu and J. Sirola et al ., 2013. Alcohol consumption and bone mineral density in elderly women. Public Health Nutr., 16: 704-712 19. Qian, D., H. Zhou, P. Fan, T. Yu and A. Patel et al ., 2021 A traditional Chinese medicine plant extract prevents alcohol-induced osteopenia. Front. Pharmacol., Vol. 12 10.3389/fphar.2021.754088 20. Shen, B., 2015. A new golden age of natural products drug discovery. Cell, 163: 1297-1300 21. Atanasov, A.G., S.B. Zotchev, V.M. Dirsch, I.N.P.S.T. and C.T. Supuran, 2021. Natural products in drug discovery: Advances and opportunities. Nat. Rev. Drug Discovery, 20: 200-216 22. Cai, Y.S., J. Xu, M. Chen, D. Wang and Y. Yang et al ., 2019 Compound analysis of Jing Liqueur and nrf 2 activation by Jing Liqueur-one of the most popular beverages in China Beverages, Vol. 6. 10.3390/beverages 6010001 23. Hu, Y., Z. Wang, F. Xia, W. Yang, Y.C. Liu and J.B. Wan, 2021 Simultaneous quantification of bioactive components in Chinese herbal spirits by ultra-high performance liquid chromatography coupled to triple-quadrupole mass spectrometry (UHPLC-QQQ-MS/MS). Chin. Med., Vol. 16 10.1186/s 13020-021-00435-0 24. Zhou, J., F.J. Liu, X.X. Li, P. Li and H. Yang et al ., 2021 A strategy for rapid discovery of traceable chemical markers in herbal products using MZmine 2 data processing toolbox: A case of Jing Liqueur. Chin. Herb. Med., 13: 430-438 25. Masella, R., C. Santangelo, M. DʼArchivio, G. LiVolti, C. Giovannini and F. Galvano, 2012. Protocatechuic acid and human disease prevention: Biological activities and molecular mechanisms. Curr. Med. Chem., 19: 2901-2917 26. Song, J., Y. He, C. Luo, B. Feng and F. Ran et al ., 2020 New progress in the pharmacology of protocatechuic acid: A compound ingested in daily foods and herbs frequently and heavily. Pharmacol. Res., Vol. 161 10.1016/j.phrs.2020.105109 27. Khan, A.K., R. Rashid, N. Fatima, S. Mahmood and S. Mir et al ., 2015. Pharmacological activities of protocatechuic acid Acta Pol. Pharm., 72: 643-650 28. Semaming, Y., P. Pannengpetch, S.C. Chattipakorn and N. Chattipakorn, 2015. Pharmacological properties of protocatechuic acid and its potential roles as complementary medicine. Evidence-Based Complementary Altern. Med., Vol. 2015. 10.1155/2015/593902 29. Bertola, A., S. Mathews, S.H. Ki, H. Wang and B. Gao, 2013 Mouse model of chronic and binge ethanol feeding (the NIAAA model). Nat. Protoc., 8: 627-637 30. Mercer, K.E., R.A. Wynne, O.P. Lazarenko, C.K. Lumpkin and W.R. Hogue et al ., 2012. Vitamin D supplementation protects against bone loss associated with chronic alcohol administration in female mice. J. Pharmacol. Exp. Ther., 343: 401-412 31. Wu, Z.H., X. Zhu, C.K. Xu, Y.J. Chen, L. Zhang and C.L. Zhang, 2017. Effect of Xianling Gubao capsules on bone mineral density in osteoporosis patients. J. Biol. Regulators Homeostatic Agents, 31: 359-364 32. Tang, X.Y., Z.Q. Dai, Q.C. Wu, J.X. Zeng and M.X. Gao et al ., 2020. Simultaneous determination of multiple components in rat plasma and pharmacokinetic studies at a pharmacodynamic dose of Xian-Ling-Gu-Bao capsule by UPLC-MS/MS. J. Pharm. Biomed. Anal., Vol. 177 10.1016/j.jpba.2019.112836 33. Ding, Y., H. Ma, Y. Xu, F. Yang, Y. Li, F. Shi and Y. Lu, 2021 Potentiation of flutamide-induced hepatotoxicity in mice by Xian-Ling-Gu-Bao through induction of CYP 1 A 2 J. Ethnopharmacol., Vol. 278. 10.1016/j.jep.2021.114299 34. Nair, A.B. and S. Jacob, 2016. A simple practice guide for dose conversion between animals and human. J. Basic Clin. Pharm., 7: 27-31 35. Wojcikowski, K. and G. Gobe, 2014. Animal studies on medicinal herbs: Predictability, dose conversion and potential value. Phytother. Res., 28: 22-27 36. Watt, J., A.W. Alund, C.F. Pulliam, K.E. Mercer, L.J. Suva, J.R. Chen and M.J.J. Ronis, 2018. NOX 4 deletion in male mice exacerbates the effect of ethanol on trabecular bone and osteoblastogenesis. J. Pharmacol. Exp. Ther., 366: 46-57 37. Chen, J.R., R.L. Haley, M. Hidestrand, K. Shankar and X. Liu et al ., 2006. Estradiol protects against ethanol-induced bone loss by inhibiting up-regulation of receptor activator of nuclear factor- 6 B ligand in osteoblasts. J. Pharmacol. Exp Ther., 319: 1182-1190 38. Xia, X.L., 2013. History of Chinese medicinal wine Chin. J. Integr. Med., 19: 549-555 39. Cheung, H., H. Doughty, A. Hinsley, E. Hsu and T.M. Lee et al ., 2021. Understanding traditional Chinese medicine to strengthen conservation outcomes. People Nat., 3: 115-128 239

Int. J. Pharmacol., 20 (2): 229-240, 2024 40. Zhang, N.D., T. Han, B.K. Huang, K. Rahman and Y.P. Jiang et al ., 2016. Traditional Chinese medicine formulas for the treatment of osteoporosis: Implication for antiosteoporotic drug discovery. J. Ethnopharmacol., 189: 61-80 41. Lin, J., J. Zhu, Y. Wang, N. Zhang and H.J. Gober et al ., 2017 Chinese single herbs and active ingredients for postmenopausal osteoporosis: From preclinical evidence to action mechanism. BioSci. Trends, 11: 496-506 42. An, J., H. Yang, Q. Zhang, C. Liu, J. Zhao, L. Zhang and B. Chen, 2016. Natural products for treatment of osteoporosis: The effects and mechanisms on promoting osteoblast-mediated bone formation. Life Sci., 147: 46-58 43. Martiniakova, M., A. Sarocka, R. Babosova, B. Grosskopf and E. Kapusta et al ., 2018. Changes in the microstructure of compact and trabecular bone tissues of mice subchronically exposed to alcohol. J. Biol. Res. Thessaloniki, Vol. 25. 10.1186/s 40709-018-0079-1 44. Iwaniec, U.T. and R.T. Turner, 2013. Intraperitoneal injection of ethanol results in drastic changes in bone metabolism not observed when ethanol is administered by oral gavage. Alcohol. Clin. Exp. Res., 37: 1271-1277 45. Liu, Y., X. Kou, C. Chen, W. Yu and Y. Su et al ., 2016. Chronic high dose alcohol induces osteopenia via activation of mTOR signaling in bone marrow mesenchymal stem cells. Stem Cells, 34: 2157-2168 46. Okuda, T., T. Haseba, M. Katsuyama, M. Maruyama, T. Akimoto, T. Igarashi and Y. Ohno, 2018. Metabolic pharmacokinetics of early chronic alcohol consumption mediated by liver alcohol dehydrogenases 1 and 3 in mice. J. Gastroenterol. Hepatol., 33: 1912-1919 47. Dai, J., D. Lin, J. Zhang, P. Habib and P. Smith et al ., 2000 Chronic alcohol ingestion induces osteoclastogenesis and bone loss through IL-6 in mice. J. Clin. Invest., 106: 887-895 48. Belknap, J.K., J.C. Crabbe and E.R. Young, 1993. Voluntary consumption of ethanol in 15 inbred mouse strains. Psychopharmacology, 112: 503-510 49. Huang, W.Y. and S.J. Sheu, 2006. Separation and identification of the organic acids in Angelicae Radix and Ligustici Rhizoma by HPLC and CE. J. Sep. Sci., 29: 2616-2624 50. Tao, Y., X. Xu, J. Yan and B. Cai, 2019. A sensitive UPLC-MS/MS method for simultaneous determination of polyphenols in rat plasma: Application to a pharmacokinetic study of dispensing granules and standard decoction of Cinnamomum cassia twigs. Biomed. Chromatogr., Vol. 33 10.1002/bmc.4534 51. Xie, L., J. Guo, Y. Zhang, Y. Hu, Q. You and S. Shi, 2015. Novel molecular imprinted polymers over magnetic mesoporous silica microspheres for selective and efficient determination of protocatechuic acid in Syzygium aromaticum . Food Chem., 178: 18-25 52. Renuka and Nishtha, 2022. Dioscorea bulbifera medicinal plant: Phytochemistry and salutary potential. Mod. Appl Pharm. Pharmacol., Vol. 2. 10.31031/MAPP.2022.02.000550 53. Gao, H., X. Yuan, Z. Wang, Q. Gao and J. Yang, 2020. Profiles and neuroprotective effects of Lycium ruthenicum polyphenols against oxidative stress-induced cytotoxicity in PC 12 cells. J. Food Biochem., Vol. 44. 10.1111/jfbc.13112 54. Jang, S.A., H.S. Song, J.E. Kwon, H.J. Baek and H.J. Koo et al ., 2018. Protocatechuic acid attenuates trabecular bone loss in ovariectomized mice. Oxid. Med. Cell. Longevity, Vol. 2018 10.1155/2018/7280342 55. Park, S.H., J.Y. Kim, Y.H. Cheon, J.M. Baek and S.J. Ahn et al ., 2016. Protocatechuic acid attenuates osteoclastogenesis by downregulating JNK/c Fos/NFATc 1 signaling and prevents inflammatory bone loss in mice. Phytother. Res., 30: 604-612 56. Rivera-Piza, A., Y.J. An, D.K. Kim, S.H. Lee, J.B. Kim, J.S. Choi and S.J. Lee, 2017. Protocatechuic acid enhances osteogenesis, but inhibits adipogenesis in C 3 H 10 T 1/2 and 3 T 3-L 1 cells J. Med. Food, 20: 309-319 57. Benet, L.Z., C.M. Hosey, O. Ursu and T.I. Oprea, 2016 BDDCS, the rule of 5 and drugability. Adv. Drug Delivery Rev., 101: 89-98 58. He, M., T. Grkovic, J.R. Evans, C.C. Thornburg and R.K. Akee et al ., 2019. The NCI library of traditional Chinese medicinal plant extracts-Preliminary assessment of the NCI-60 activity and chemical profiling of selected species. Fitoterapia, Vol. 137. 10.1016/j.fitote.2019.104285 240

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