• YANMEI CAO^1*,
• et al.: Therapeutic Effects of Cyathula officinalis Kuan on Acute Blood Stasis Rat Model¹,
• Its Constituents¹

1. ¹  Pharmacognosy Magazine, Volume 13, Issue 52, October-December 2017 product confessed by the Ministry of Health of the People’s Republic of China. The aqueous extract of C. officinalis Kuan (CO) has been reported to exhibit anti‑inflammatory activity through inhibiting the mice auricle tumefaction degree induced by xylol and rats’ feet swelling caused by egg white.[2] Various components, such as phytoecdysteroids, saponins, flavones, and polysaccharides, have been identified from C. officinalis Kuan. Modern pharmacological studies are mostly focused on the activities of polysaccharides from C. officinalis Kuan. For example, the polysaccharides of C. officinalis Kuan have been reported to possess immune enhancement activity by upregulating humoral and cellular immune responses.[3] Besides, a water‑soluble polysaccharide has been reported to have significant antioxidant activity, and a fructan CoPS3 could effectively inhibit the growth of Lewis pulmonary carcinoma implanted in mice.[4,5] At present, there is no systematic research conducted on C. officinalis Kuan, especially the therapeutic effects on blood stasis model and correlative components. The active fraction of C. officinalis Kuan (ACO) responsible for anti‑inflammatory and protective effects on endothelial cells (human umbilical vein endothelial cells [HUVECs]) injury has been investigated previously.[6] In this study, the effects of CO and ACO on improving blood rheology and anti‑inflammatory properties were examined in acute blood stasis model rats, and the major constituents of ACO were identified by HPLC‑QTOF/MS/MS method. Besides, the effects of two representative components (cyasterone and chikusetsusaponin IV) from ACO on thrombin‑induced HUVECs damage model were also assessed by levels of thromboxane A2 (TXA2), endothelin (ET), malondialdehyde (MDA), cyclooxygenase‑2 (COX‑2), endothelial nitric oxide synthase (eNOS), and superoxide dismutase (SOD). MATERIALS AND METHODS Chemicals and reagents Chloral hydrate was purchased from Sinopharm Chemical Reagent Beijing Co., Ltd; adrenaline hydrochloride injection was purchased from Shanghai Harvest Pharmaceutical Co., Ltd.; cyasterone (C29H44O8, MW 520.65) and chikusetsusaponin IV (C47H74O18, MW 927.08) with a purity of 98% as determined by HPLC were purchased from SenBeijia Biological Technology Co., Ltd. (Nanjing, China). Ethanol, petroleum ether, dichloromethane and n‑butanol (chemical pure), methanol (HPLC grade) were supplied by Tedia Company Inc. (Fairfield, USA). HPLC‑grade formic acid was purchased from Merck Company (Darmstadt, Germany). Enzyme‑linked immunosorbent assay (ELISA) kits for interleukin‑6 (IL‑6), nitric oxide (NO), tumor necrosis factor alpha (TNF‑α), COX‑2, TXA2, ET, MDA, eNOS, and SOD were obtained from Roche Diagnostics (Mannheim, Germany). HUVECs and thrombin were purchased from Yi Fei Xue Biotechnology (Nanjing, China). Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum were obtained from Invitrogen (Life Technologies Corporation, Carlsbad, CA, USA). All other reagents were of analytical grade, and distilled water was used to extract and prepare the samples. Plant material C. officinalis Kuan was obtained in Sichuan Province, in December 2015 (No. 151201). It was identified and authenticated by Prof. Wu‑De Kang of the School of Pharmacy of the Nanjing University of Chinese Medicine. A voucher specimen was deposited at the Nanjing University of Chinese Medicine, Nanjing 210023, PR, China. Extraction procedures The dried C. officinalis Kuan (1.6 kg) was turned into powder form and refluxed with the 85% ethanol for 10 times (3 times × 1.5 h). The extracts were concentrated under vacuum and dried, obtaining CO (79.2 g, with respect to 1.6 kg of the plant material). Then, CO (47 g, with respect to 950 g of the plant material) was resuspended in distilled water and submitted to sequential extraction with petroleum ether, dichloromethane, and n‑butanol (5 times), obtaining n‑butanol fraction. Then, n‑butanol extract was concentrated under vacuum and dried, obtaining the ACO (33.5 g, with respect to 950 g of the plant material). The samples were stored at − 4°C until used. Animals Male Wistar rats (200 ± 30 g) were provided by the Shanghai Jiesijie Experimental Animal Center with animal license: Certificate No. SCXK (Hu) 2013‑0006. Animals were housed in a standard breeding room (25°C, relative humidity, 12 h dark‑light cycle). All animals were acclimated in the laboratory for at least 7 days before the experiment. The animals were given chow and water ad libitum but fasted overnight before the operation. The experiment was approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine and was conducted in accordance with the international guidelines for laboratory animal use and care published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). Experimental model and drug administration Dosage of C. officinalis Kuan was determined based on the conversions from clinical adult dosage. Thus, the oral dosage in a rat of C. officinalis Kuan may be designed to be 5.3 g/kg (crude herbal dose). In the present study, the yields of CO and ACO were 4.95% and 3.53%, respectively. Meanwhile, the oral dosages of CO in the previous studies were taken into consideration in our present study. Therefore, we used three doses of CO at 0.26, 0.53, 1.04 g/kg (equivalent to crude herbal doses 5.3, 10.8, 21.3 g/kg, respectively) and ACO at 0.38, 0.75, 1.5 g/kg (equivalent to crude herbal doses 10.8, 21.3, 42.5 g/kg, respectively) in this study. Seventy‑two male Wistar rats approximately 7–8 weeks of age were randomly divided into nine groups (n = 8), including blank group, model group, aspirin group (0.1 g/kg), CO (0.26 g/kg), CO (0.53 g/kg), CO (1.04 g/kg), and ACO (0.38 g/kg), ACO (0.75 g/kg), ACO (1.5 g/kg) groups. All drug treatments were administered, respectively, by gavage for 3 days. Rats of blank group and model group were only orally administered normal saline. The day before taking blood, rats except the blank group were hypodermically injected with adrenaline hydrochloride (0.8 mL/kg) twice at an interval of 4 h. Two hours after the first injection of adrenaline hydrochloride, the rats were kept in ice‑cold water (0–2°C) to swim for 5 min.[7] Measurement of whole blood viscosity and plasma viscosity All animals were anesthetized with 10% chloral hydrate before surgery. Blood (3 mL) was immediately collected in heparinized tubes from the abdominal aorta. Blood samples (1 mL) were used for measuring the whole blood viscosity. Plasma samples were prepared by centrifuging at 3000 rpm for 15 min. Whole blood viscosity and plasma viscosity were measured as soon as possible by a LG‑R‑80 B automated viscometer. Measurement of plasma interleukin‑6, nitric oxide, tumor necrosis factor‑α, and cyclooxygenase‑2 values Plasma samples were prepared by centrifuging at 3000 rpm for 15 min and the levels of IL‑6, NO, TNF‑α, and COX‑2 were measured by ELISA kits (R and D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Briefly, ELISA plates were coated with specific IL‑6, NO, TNF‑α, or COX‑2 antibody. Diluted plasma samples were, China.

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