Evaluation of Antioxidant, Anti-inflammatory, and Cytotoxic Activities of Crotalaria pallida Aiton Leaves

• Cite this article as: Lim C^1*,
• Lee B¹,
• Lim S¹,
• Lee SE¹,
• Cho S. Astragali radix reduces¹

1. ¹ ischemia-induced brain injury by inhibiting edema and expression of Aquaporin-4 in mice. Phcog Mag 2019;15:335-41. CHIYEON LIM, et al.: Astragali Radix Reduces Ischemia‑Induced Brain Injury in Mice 336 Pharmacognosy Magazine, Volume 15, Issue 61, April-June 2019 limited.[1‑3] Therefore, safer and more effective therapeutic strategies are required. Astragali Radix, the dried root of Astragalus membranaceus (Fisch.) Bunge, is one of the most widely used herbal medicines and is commercially cultivated in China and Korea.[4,5] Astragali Radix (known as Hwang‑gi in Korean) is a primary energizing medicinal herb that is used to invigorate vital energy. It is frequently used to treat various age‑related symptoms, such as immune system disorders, menopause, neuropathy, nephropathy, fatigue, hepatic injury, and cancer.[5‑10] The chemical constituents of Astragali Radix include astragalosides, calycosin, kumatakenin, formononetin, and their respective glycosides and malonates.[11] Astragaloside IV, a major active triterpenoid in Astragali Radix, has anticancer and neuroprotective effects.[12,13] Li et al. reported that astragaloside IV protected blood–brain barrier (BBB) integrity by activating the nuclear factor erythroid 2‑related factor 2 signaling pathway.[14] Astragaloside IV has also been reported to alleviate behavioral deficiencies in an animal model of Parkinson’s disease.[15] Calycosin is known to exert anticancer effects which are thought to occur by targeting the forkhead box P3 (Foxp3)‑mediated expression of vascular endothelial growth factor and matrix metallopeptidase‑9 (MMP‑9).[16] Recently, calycosin was reported to exhibit neuroprotective effects against ischemia‑induced cerebral injury by increasing the expression of transient receptor potential cation channel subfamily C member 6 and phospho‑cyclic AMP response element‑binding protein and by inhibiting calpain activation.[3] Song et al. reported that calycosin improved cognitive function by mitigating oxidative stress and inflammatory responses in a mouse model of Alzheimer’s disease.[17] Fei et al. reported that formononetin ameliorated learning and memory deficits by suppressing the amyloid‑beta clearance pathway.[18] Liu et al. reported that Astragali Radix extract administered intraperitoneally to rats with ischemic injury suppressed c‑Jun N‑terminal kinase 3 gene expression, resulting in reduced infarct volumes and less neurobehavioral deficits.[19] Thus, Astragali Radix has therapeutic potential for the treatment of ischemic stroke. However, mechanisms responsible for the therapeutic effects of Amex are not fully explained by its neuroprotective properties. Further evaluation of the protective mechanisms of orally administered Amex is required. In our preliminary study, pretreatment with methanol extracts of Astragali Radix (Amex) protected mice against ischemia‑induced brain damage and brain edema. Thus, this study was conducted to investigate the therapeutic effects of Astragali Radix administered after ischemia‑induced brain injury in a mouse model. MATERIALS AND METHODS Ischemic stroke model and Astragali Radix treatment Male adult specific‑pathogen‑free C57BL/6 mice (22–25 g) were obtained from Daehan Biolink Co. (Chungbuk, Korea). Animals were housed in a temperature/humidity‑controlled animal facility under a 12‑h light/dark cycle and provided food and water ad libitum. All animal experiments were approved and regulated by the Ethics Committee of Pusan National University (PNU) (approval number PNU‑2016‑1087; date of approval, February 26, 2016). Ischemic stroke was induced by middle cerebral artery occlusion (MCAO) as previously described.[20] Briefly, mice were anesthetized with 2% isoflurane and maintained using 1.5% isoflurane in a 70% N2O and 30% O2 mixture. The left common carotid artery was isolated, and an 11‑mm‑long 8‑0 monofilament nylon suture (Ethicon Inc., NJ, USA) was inserted and advanced into the internal carotid artery (ICA) to occlude the MCA. The suture was removed after 2 h of MCAO to allow for reperfusion [Figure.
2. ² S]. Mice in the normal group and the control group were administered the same volume of normal saline. Body weight and physiological parameter measurements Mice were weighed daily during the experimental period, and blood was collected by cardiac puncture under deep anesthesia 3 days after MCAO. To obtain serum, blood samples were centrifuged at 1500 ×g for 15 min at 4°C. Serum concentration of electrolytes, such as sodium (Na+), potassium (K+), and chloride (Cl−), was measured using an electrolyte analyzer (Dri‑Chem 3500i, Fuji, Japan) to monitor and exclude animals with potential electrolyte imbalances that might affect results. Infarct volume measurement and brain edema calculation After ischemia for 2 h and reperfusion for 24 h, the brains were removed, sliced into ten coronal sections (1 mm thick), stained with 2% 2,3,5‑triphenyltetrazolium chloride (TTC) for 17 min at room temperature (25°C), and immersed in 10% neutral buffered formalin for 2 h. Total infarct volumes were calculated using infarct areas in these sections as previously described[20] using a digital camera (Canon.
3. ³ S]. Amex were prepared for administration to mice by dissolution in dimethyl sulfoxide (DMSO). This solution was diluted with 0.9% normal saline, passed through a 0.45‑μm pore size syringe filter, and adjusted to concentrations of 100, 300, and 1000 mg/kg. Mice were divided into five groups of six mice: sham‑operated (normal) group, MCAO‑operated (control) group, and three MCAO‑operated Amex‑treated groups with Amex concentrations of 100, 300, and 1000 mg/kg (titled Amex 100, 300, and 1000, respectively). Mice in the Amex‑treated groups received oral Amex at the designated concentrations at 2, 26, and 50 h after reperfusion [Figure.
4. ⁴ D, Canon Korea, Korea) and ImageJ software. Brain edema indices were calculated by dividing injured brain hemisphere areas by normal brain hemisphere areas using TTC‑stained brain sections. Whole‑brain water content was determined using a previously described vacuum drying method[21] to evaluate edema formation and was calculated by dividing brain wet weights by dry weights. Neurological deficit scores Neurological deficit (ND) scores were quantified at 3, 24, 48, and 72 h after reperfusion in a blinded manner using a five‑point scale. • Grade 0: No ND • Grade 1: Failure or incomplete extension of right forepaw • Grade 2: Reduced voluntary movement in all directions and circling to the right when the tail was pulled.

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