Rhubarb

All data were pre-processed with Profinder (version B.06.00, Agilent Technologies, USA). For molecular feature extraction, up to 2000 compounds with a peak height above 300 counts were extracted. The initial and final retention times were set for data collection. The missing value estimation, data filtering and data normalization were achieved by the MetaboAlalyst 3.0 online software. The resultant data matrices were introduced into the SIMCA-P+ 13.0 (Umetrics, Umeå, Sweden) software for multivariate statistical analysis including principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA). Prior to PCA, all variables obtained from the data matrix were mean-centered and scaled to the pareto variance. The PCA score plot was used to present the natural interrelationship among observations. To find potential biomarkers, the OPLS-DA model was used to explore deep differences between the control, model and rhubarb-treated groups. Variables with a VIP value (VIP ≥1.0) and |p(corr)| ≥ 0.5 (Wheelock and Wheelock, 2013 (link)) in the OPLS-DA model were selected as potential biomarkers.

Both TSF (Lot number0606320) and the placebo were prepared and standardized by an established company recognized for high quality control standards: Jiangyin Tianjiang Pharmaceutical, Jiangsu, China (http://www.tianjiang.com). TSF consists of seven natural herbs: astragalus (A.membranaceus (Fisch.) Bge.), burning bush (E.alatus (Thunb.) Sieb.), rehmannia (R.glutinosa Libosch), bitter orange (C.aurantium L.), cornus (C.officinalis Sieb. Et Zuce), rhubarb (R.palmatum L.) and notoginseng (P.notoginseng (Burk.) F.H. Chen) (Table 1). Each component in TSF was produced by soaking in distilled water for 30 minutes, boiling in 10 volumes of water (v/w) for 1 hour, extracting with water twice, filtrating and condensing to the concentration of 1 g/ml and processed to fine granular by spray drying. The final product was made by combining the individual herbal granules in the proportions indicated in Table 1.TSF is a dispensing formula in its scientific research stage and has not yet been licensed for clinical use in China.
Ingredients of the placebo were lactose (78.43%), maltodextrin (14.88%), tartrazine (0.07%), sunset yellow (0.026%), caramel (6.5%), picric acid (0.026%), and sucralose (0.07%).These were prepared by the same company as TSF. Based on our knowledge, none of the ingredients in the placebo at this dosage has been reported to have physiological effects.

Eight-week-old C57Bl6/J male mice (n = 75; Janvier, France) were housed in pairs under specific conditions, i.e., specific and opportunistic pathogen-free conditions (SOPF) and in a controlled environment (12 h daylight cycle, temperature of 22 ± 2°C) with food and water ad libitum. After being acclimatized during one week with a control diet (CTRL) (D12450H, Research diet) and matched according to body weight and fat mass, mice were divided into 5 distinct dietary groups (n = 15/group): (1) control group of mice fed a control diet (D12450H, Research diets) containing 10% calories from fat (CTRL group); (2) mice fed a high-fat and high-sucrose diet (HFHS diet, D12451, Research diets) containing 45% calories from fat and 35% calories from carbohydrates (HFHS group); (3) mice fed a HFHS diet supplemented with rhubarb extract (0.3% w/w in the diet, Ortis, Belgium) (RHUB group), (4) mice fed a HFHS diet supplemented with inulin, a chicory root extract (20% food intake in water, Fibruline, Cosucra, Belgium) (ITF group); (5) mice fed a HFHS supplemented with rhubarb and inulin (0.3% rhubarb in the diet and 20% inulin in the drinking water) (RHUB+ITF group). Supplementation with either rhubarb, inulin or both started concomitantly with the introduction of HFHS diet. Water containing inulin was replaced every two days and the concentration of inulin was adapted depending on food intake for each cage. Body weight, food intake and water intake were recorded every week for the duration of the experiment (6 to 9 weeks). Body composition was assessed once a week by using 7,5-MHz time-domain nuclear magnetic resonance (LF50 minispec; Bruker; Rheinstetten, Germany). All mouse experiments were approved by and performed in accordance with the guideline of the local ethics committee (the ethics Committee of the Université catholique de Louvain for Animal Experiments specifically approved this study, agreement number 2017/UCL/MD/005).
At the end of the experiment and after 3 hours of fasting, all mice were anesthetized with isoflurane (Forene, Abbott, Queenborough, Ken, UK) and blood was sampled. After exsanguination, mice were euthanized by cervical dislocation. Organs and tissues were dissected, weighted, and directly immersed in liquid nitrogen before storage at −80°C for further analysis.

Tea leaves, rhubarb rhizome, white mustard, and black seeds were purchased as dried material, and aerial parts of R. graveolens, E. vesicaria, and A. officinalis, grapefruit peels, and grapes were dried in a vacuum-heated oven at 40 °C. All dried materials were powdered, and 50 g of each dried powder was repeatedly extracted with methanol (Adwic Co., Cairo, Egypt) until exhaustion (200 mL × 4) to obtain the methanolic extract. The fruits were peeled, thinly sliced, and dried in a vacuum oven for avocado. The dried fruits were powdered and then extracted with n-hexane (200 mL × 4, Adwic Co.) to obtain the fixed oil. Solvents were removed using a rotary evaporator, and the obtained residues were stored in amber-colored glass at 4 °C until use.

Aerial parts of common rue (Ruta graveolens Linn., Rutaceae), Arugula (Eruca vesicaria (L.) Cav., Brassicaceae) garden asparagus (Asparagus officinalis L., Asparagaceae) were obtained from and authenticated at the medicinal, aromatic, and poisonous plants experimental station (MAPPES), Faculty of Pharmacy, Cairo University. Seeds of black seed (Nigella sativa L., Ranunculaceae) and white mustard (Sinapis alba L., Brassicaceae) were obtained from and authenticated by Agricultural Research Center, Giza, Egypt. Avocado fruits (Persea Americana Mill, Lauraceae, Duke cultivar), red grapes (Vitis vinifera L., Vitaceae, seedless variety), and grapefruits (Citrus paradisi Macfad, Rutaceae) were obtained from a private garden along the Cairo-Alexandria desert road. Dr. Ayman Elkafrawy authenticated plants at the Agricultural Research Center, Giza, Egypt. Voucher specimens are kept at the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Cairo University. The Dried rhubarb rhizome (Rheum officinale Baill, Polygonaceae) and dried green tea leaves (Camellia sinensis (L.) Kuntze, Theaceae) were obtained from a local herbal market in Cairo, Egypt. Dr. Reem Samir Hamdy authenticated them, Lecturer of Plant Taxonomy, Botany Department, Faculty of Science, Cairo University, Giza, Egypt. Plant material was further evaluated for possible contamination by pharmacognostic examination of their morphological and microscopical characters.