Evaluation of antioxidant, anti-inflammatory, and cytotoxic activities of Crotalaria pallida Aiton leaves

• Cite this article as: Sato VH^1*,
• Chewchinda S¹,
• Nuamnaichati N¹,
• Mangmool S¹,
• Sungthong B¹,
• Lertsatitthanakorn P¹,
• et al. Pharmacological mechanisms of the¹

1. ¹ water leaves extract of Lysiphyllum strychnifolium for its anti-inflammatory and anti-hyperuricemic actions for gout treatment. Phcog Mag 2019;15:98-106. VILASINEE HIRUNPANICH SATO, et al.: Anti‑inflammatory and Anti‑hyperuricemic Activities of Lysiphyllum strychnifolium Extract Pharmacognosy Magazine, Volume 15, Issue 60, January-March 2019 99 joints and tissues can induce an inflammatory response. The treatment of hyperuricemia, therefore, focuses on decreasing the uric acid level and suppressing inflammation.[1] Current anti‑hyperuricemic agents employed in clinical settings are xanthine oxidase (XO) inhibitors, such as allopurinol and febuxostat, as well as uricosuric agents. Recently, it has been found that urate reabsorption at the proximal tubules plays a key role in the renal urate excretion.[2] Multiple lines of evidence indicated that some urate transporters in the proximal tubule, such as solute carrier family 22 member 12 (SLC22A12) or urate-anion transporter 1 [URAT1] and SLC2A9 or glucose transporter 9 [GLUT9], mediate the renal urate reabsorption.[1] Uricosuric agents such as probenecid and benzbromarone act on the urate transports and inhibit the urate reabsorption at the renal proximal tubules. Colchicine has been used to treat acute inflammation in gout. However, these drugs are commonly associated with adverse effects, such as fever, skin rashes, allergic reactions, hepatitis, and nephropathy, which may limit their clinical uses.[1] Recent studies have hypothesized that the production of uric acid catalyzed by XO generates free radicals which might adversely affect mitochondrial function and ATP production, leading to cardiovascular diseases.[2] Therefore, alternative medicines which exhibit urate‑lowering, anti‑inflammation, and antioxidant effects have been extensively explored in recent studies,[2,3] and most such candidates are natural products which have already been used as a folk remedy. Lysiphyllum strychnifolium is classified as a family of Leguminosae and has been known as “Khayan” or “Ya Nang Dang” in North East of Thailand. Since it was formerly classified in the genus Bauhinia, the synonym of Bauhinia strychnifolium has been also used.[4,5] In Thai traditional medicine, the stems and roots of L. strychnifolium have been used to treat cancer, fever, and allergy.[4] Boiling of its leaves and stems with water has been used to detoxify heavy metals, pesticides, and alcohol. Nowadays, dried stems or leaves of L. strychnifolium are distributed as commercial products in the form of tea or dried powder for infusion. Several bioactive compounds including trilobatin, quercetin, 3,5,6,3’,5’‑pen tahydroxy‑flavanonol‑3‑O‑α‑L‑rhamnopyranoside, 3,5,7‑trihydroxy‑c hromoone‑3‑O‑α‑L‑rhamnopyranoside, β‑sitosterol, stigma sterol and gallic acid have been observed in L. strychnifolium.[6] Previous studies revealed that the extract of L. strychnifolium stems exhibited anti‑cancer activity against cancer cell lines and possessed a greater antioxidant activity than that of green tea.[7,8] Recently, the hypouricemic and anti‑inflammatory properties have been claimed by Thai hyperuricemic patients who often drink fresh L. strychnifolium leaves boiled in water. However, experimental evidence for the anti‑hyperuricemic and anti‑inflammatory activities of L. strychnifolium have been lacking so far. Therefore, the present study was conducted to evaluate the hypouricemic effect of L. strychnifolium leaves extract by determining the inhibitory activity on XO in vitro and by measuring the uric acid‑lowering activity using potassium oxonate (PO)‑induced hyperuricemic mice in vivo. Moreover, the effect of L. strychnifolium leaves extract on the protein expression of SLC22A12 in the kidney of hyperuricemic mice was evaluated to explore the anti‑hyperuricemic effect on renal transporters. The antioxidant effect of L. strychnifolium leaves extract on free radicals was also assessed. Furthermore, the mRNA expression of inflammatory markers, i.e., cyclooxygenase‑II (COX‑II), inducible nitric oxide synthase (iNOS), transforming growth factor‑β (TGF‑β) and tumor necrosis factor‑α (TNF‑α) was quantitated in lipopolysaccharide (LPS)‑stimulated RAW 264.7 macrophage cells. MATERIALS AND METHODS Chemicals Allopurinol, acetonitrile, gallic acid, Folin‑Ciocalteu reagent, carboxymethylcellulose‑sodium (CMC‑Na), quercetin, Trolox, sodium acetate trihydrate, sodium octane sulfonate, uric acid, XO enzyme from bovine milk, 2,2′‑azinobis‑(3‑ethylbenzothiazoline‑6‑sulfonic acid) (ABTS), 2,2‑diphenyl‑1‑picrylhydrazyl (DPPH), PO were purchased from Sigma‑Aldrich Chemical Co. (St. Louis, MO, USA). Aluminum chloride, ascorbic acid, potassium persulfate, and sodium bicarbonate were obtained from Ajax Finechem, Australia. All other chemical reagents were of analytical grade and used without further purification. Plant material L. strychnifolium leaves were harvested from Mahasarakham Province, in February ‑ April 2016. They were identified by Assist. Prof. Wanida Caichompoo, Faculty of Pharmacy, Mahasarakham University, with the voucher number monosodium urate (MSU). PH‑LEG‑BS01 and its reference specimen have been deposited at the herbarium of the institute. L. strychnifolium leaves were collected, cleaned, dried at 50°C for 48 h, and cut into small pieces. Water extraction was carried out by an infusion method. Dried leaves (100 g) were extracted in boiling water (1 L) for 15 min. The pooled extract was filtered through a Whatman filter paper (No. 1), and then concentrated using a freeze dryer (Labconco, Kansas, MO, USA). The residue was lyophilized into powder and stored in a sealed container protected from light at −20°C until used for assays. Determination of gallic acid content in Lysiphyllum strychnifolium extract by a high‑performance liquid chromatography Gallic acid was used as a chemical marker of L. strychnifolium leaves extract. The content of gallic acid in the extract was determined using a previously reported high‑performance liquid chromatography (HPLC) method with some modifications.[9] Methanol solution containing known concentrations of gallic acid (1.56–50 μg/ml) were prepared and used for calibration. L. strychnifolium (100 mg) leaves were mixed with 20 ml of 12% hydrochloric acid for 30 min under reflux on a water bath. The mixture was extracted by 25 ml of diethyl ether 3 times, evaporated to dryness, reconstituted in 5 ml of methanol, and filtered through a 0.45 µm nylon membrane. The obtained filtrate was injected into the HPLC in triplicate. The HPLC system consisted of a system controller SCL‑10AVP, a detector ultraviolet (UV)‑visible SPD‑10A, a dual piston solvent delivery pump LC‑10AD and an auto‑injector SIL‑10A (Shimadzu, Kyoto, Japan). The analytical column used was a BDS Hypersil C18 column (150 mm × 4.6 mm, i.d. 5 μm) (Thermo Fisher Scientific, Waltham, MA, USA), connected with a BDS Hypersil C18 guard column (10 mm × 4 mm, i.d. 5 μm) (Thermo Fisher Scientific). The mobile phase was a mixture of 0.05% phosphoric acid (solvent A) and methanol (solvent B). The gradient elution, with the total run‑time of 30 min, was set as follows: 10% B for 10 min, 10%–30% B (3 min), 30%–60% B (5 min), 60%–20% B (3 min), 20%–10% B (3 min) and 10% B (6 min). The flow rate was 1.0 ml/min, UV detection at 271 nm, and the volume of injection 20 µl. Anti‑inflammatory effect of Lysiphyllum strychnifolium extract in lipopolysaccharide‑stimulated macrophages Cell culture RAW 264.7 macrophage cells were provided by Dr. Primchanien Moongkarndi, Faculty of Pharmacy, Mahidol University in Thailand, and cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1.0% penicillin/, Thailand.

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