Poria

LGZG comprises four Chinese herbs: Poria (20 g), Ramulus Cinnamomi (15 g), Rhizoma Atractylodis Macrocephalae (15 g), and Radix Glycyrrhizae (10 g). The dosage is determined according to the text book of The Hndouts of Jingui Yaolue [10 ]. All herbs were purchased from Longhua Hospital affiliated to Shanghai University of TCM. Herbal decoction was prepared in accordance with conventional TCM decocting methods, briefly, (1) place all herbs in a cooking pot (porcelain) with 500 mL water; (2) boil the herbs with highest heat after 30 minutes of soak; (3) reduce heat and simmer for 20 minutes; (4) transfer the liquid by filtration; (5) add water and boil the remaining, and then repeat (3) and (4) one more time to make a second dose of medicine; (6) mix the two doses in a glass pot. The final concentrated decoction is 100 mL (pure solution). Quality was control under high-performance liquid chromatography (HPLC) as previously described [11 ], and details were shown in Supplement 1 (see Supplementary Material available online at http://dx.doi.org/10.1155/2013/429738). LGZG was administered at a dose of 10 mL/kg/d (pure solution), which was approximately 7 times of the standard dose in practice, according to the dose-equivalence equation between rats and humans [12 ]. Meanwhile, the following formulas were prepared based on the herbal combination rule: Lingguizhugan Decoction without Ramulus Cinnamomi (LZG, Poria, Rhizoma Atractylodis Macrocephalae, Radix Glycyrrhizae), Lingguizhugan Decoction without Poria (GZG, Ramulus Cinnamomi, Atractylodis Macrocephalae, Radix Glycyrrhizae), Lingguizhugan Decoction without Rhizoma Atractylodis Macrocephalae and Radix Glycyrrhizae (LG, Poria, Ramulus Cinnamomi). Methods used in decoction and quality control were the same as that used in LGZG, and equal doses were administered. Ordinary diet and HFD (consists of 10% lard oil, 2% cholesterol, and 88% STD) were obtained from Shanghai Si-Lai-Ke Experimental Animal Ltd. (Shanghai, China).

Composition proportions of Xiaoyaosan prescription: Poria : Radix Paeoniae Alba : Radix Et Rhizoma Glycyrrhizae : Radix Bupleuri : Radix Angelicae Sinensis : Rhizoma Atractylodis Macrocephalae : Herba Menthae Haplocalycis : Rhizoma Zingiberis Recens equaled to 3 : 3 : 1.5 : 3 : 3 : 3 : 1 : 1. All 8 crude drugs were purchased from Beijing Tongrentang (Bozhou) Decoction Pieces Limited Company and authenticated by Dr. B. Liu, Department of Botany, and Beijing University of Chinese Medicine. All crude drugs were extracted by the Chinese medicine preparation room of China-Japan Friendship Hospital as previously described [7 (link)]. Extraction steps were performed as follows: Poria, Radix Paeoniae Alba, and Radix Rhizoma Glycyrrhizae were boiled and extracted three times with 10 volumes (2 h), 8 volumes (1 h), and 8 volumes (1 h) of water to obtain the extraction liquid (A). Radix Bupleuri, Radix Angelicae Sinensis, Rhizoma Atractylodis Macrocephalae, Herba Menthae, and Rhizoma Zingiberis Recens were soaked with 10 volumes of water for 12 h to obtain the volatile oil, drug liquid (B), and drug residue (C). Subsequently, C was boiled in 8 volumes of water for 1 h and extracted twice to obtain the extraction solution (D). Extraction solutions A, B, and D were mixed to form the water extraction liquid (E). Next, E was filtered and centrifuged (3000 r/min for 40 min). The supernatant was collected and vacuum dried at 70°C. Then, the dried product and the volatile oil were processed into dry decoction for use. The extraction rate was 18.8%. Xiaoyaosan (was dissolved in deionized water and administered by intragastric injection at a dose of) was 3.854 g/kg·d, and deionized water was used in all groups.

LGQHD is composed of Poria cocos Schw Wolf (Poria coco, 茯苓) 20 g, Cinnamomum cassia Presl (Cassia twig, 桂枝) 15 g, Atractylodes macrocephala Koidz (Atractylodes macrocephala, 白术) 10 g, Paeonia lactiflora Pall., and P. veitchii Lynch (Radices paeoniae rubra, 赤芍) 10 g. They are, respectively, derived from the dried mycorrhizal nucleus of Poria coco, the dried shoots of Cassia twig, the dried rhizome of Atractylodes macrocephala, and the dried roots of Radices paeoniae rubra. All herbs are purchased by Hebei Bai Cao Kang Pharmaceutical Co., Ltd. The preparation process consists of the traditional process of water extraction and volatile oil of the tablets to make β-CD inclusions in a ratio of about 1 : 4, and the aqueous solution is concentrated into an infusion. It was prepared as an extract at a concentration of 2.28 g of raw drug/g, which was provided by the Department of Pharmaceutics at Xiyuan hospital, China Academy of Chinese Medicine. Sacubitril Valsartan Sodium Tablets (trade name: Entresto) were manufactured by Novartis Pharma Schweiz AG with the (approval number H20170344, Switzerland) with the specification of 50 mg/tablet.
Waters (USAXevo)'s G2-S QTOF, quadrupole time of flight high-resolution mass spectrometer, and ACQUITY UPLC H-Class, ultraperformance liquid chromatograph, were used. A guard column and an ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm) were included in the column's setup.
The Traditional Chinese Medicine Preparation Department at Xiyuan hospital, Chinese Academy of Traditional Chinese Medicine, provides LGQHD Infusion. Precisely 90 ml of LGQHD extraction is taken, 5.5 ml of the 70% methanol solution is added, kept at room temperature for 30 minutes, and then it is filtered through a 0.22 μm filter membrane. The LGQHD extract test solution was then made and kept in the refrigerator at a temperature of −20°C. The proper amount of LGQHD extraction solution is taken, it is liquefied with 70% methanol, and the sample for analysis is injected. Chromatographic circumstances mobile phase: gradient elution of water (A)-acetonitrile (B) as described in Table 1. The column temperature was 40°C, the flow rate was 0.4 ml/min, and the injection volume was 2 μl. The ESI source, positive and negative ion scan, and scan range m/z: 50–1200 Da were the mass spectrometry conditions.
Data were acquired using Masslynx V4.1 software, and the resulting data were processed using Progenesis QI software and calibrated and peak aligned by peak detection and calibration processing algorithms, respectively. Meanwhile, the chemical formulae of the possible target compounds were imported into the software, and the following main components of the LGQHD were identified by qualitative analysis of the components through accurate molecular weight comparison.

According to the weight, the compatibility ratio of the four medicines of Poria coco, Cassia twig, Atractylodes macrocephala, and Radices paeoniae rubra in LGQHD is 4 : 3 : 2 : 2. The drug was decocted three times with water and filtered through the process each time, and the filtrate to be combined finally. The extract was then further filtered to remove solid impurities and finally condensed into granules. According to the table of equivalent dose ratios converted by body surface area between humans and animals [15 ], based on a standard adult body mass of 70 kg, the dose for rats = standard adult clinical dose mg/kg × 70 kg × 0.018/200. The LGQH-H group was administered 9.92 g/kg/d, which is 2 times the clinically equivalent dosage. The LGQH-L group was administered 4.96 g/kg/d, which is the clinical equivalent dosage.