**2. Materials and Methods**

## *2.1. Extraction of Lespedeza bicolor*

Aerial parts of LB were obtained from Jayeonchunsa Co. (Damyang, Korea). LBE was extracted with 70% ethanol at room temperature overnight. Then, the extract was filtered, evaporated, and dry frozen. The obtained hydroalcoholic extract of LBE was kept at −20 ◦C until it was used. The extract was dissolved in distilled water at 25 mg/mL (LL) and 62.5 mg/mL (HL) independently.

### *2.2. Animals and Study Design*

Male 4-week-old C57BL/6 mice (*n* = 50) were purchased from Raon Bio (Gyeonggi-do, South Korea) and were housed in 2–3 per cage in a 12 h light/12 h dark cycle under controlled temperature (22 ± 1 ◦C) and humidity (50 ± 5%). After 1 week for acclimation, mice were randomly grouped into 2 groups: a normal control group (NC; *n* = 10) which was fed a rodent diet (10% kcal fat, Research Diets, New Brunswick, NJ, USA), and a diabetic group (DM; *n* = 30) which was fed with a high-fat-containing rodent diet (40% kcal fat, Research Diets, New Brunswick, NJ, USA). Food and distilled water were supplied ad libitium.

After 4 weeks of diet treatment, diabetic groups were injected twice with streptozotocin (30 mg/kg body weight, Sigma Aldrich, St. Louis, MO, USA) into peritoneum by a 1 week interval in citrate buffer (pH 4.5) to induce T2DM. Simultaneously, the NC mice were injected with only citric acid buffer. Fasting blood glucose (FBG) levels were measured every week from the tail vein using OneTouch Select blood glucometer (LifeScan Inc., Milpitas, CA, USA) until 5 weeks from the last injection. Mice measured at FBG > 140.4 mg/dL (7.8 mmol/L) at least twice were considered as being in a diabetic state. Diabetes was induced in 30 out of 40 mice. The induction protocol of diabetes was in reference to a previous study by Zhang et al. [32].

Mice considered in a diabetic state were divided into three groups, and all groups (*n* = 10 per group) were differently treated as follows: (A) the NC group, 10% kcal control diet-fed non-diabetic mice group, was supplemented with distilled water; (B) the diabetic control (DMC) group, 40% kcal high fat diet (HFD)-fed diabetic mice group, was supplemented with distilled water; (C) the LL group, HFD-fed diabetic mice group, was supplemented with a low dosage of LBE (100mg/kg vody weight (BW)); and (D) the HL group, HFD-fed diabetic mice group, was supplemented with a high dosage of LBE (250mg/kg BW). Distilled water or LBE freshly dissolved in distilled water was administrated by oral gavage every day for 12 weeks, and 10 h fasting blood glucose level from tail vein was monitored once a week during all supplementation.

At the end of the supplementation period, mice were anesthetized by inhalation with diethyl ether (Duksan, Seoul, Korea). The blood samples were collected by cardiac puncture in heparin-coated tubes (Sigma Aldrich, St. Louis, MO, USA), and were centrifuged at 850× *g* at 4 ◦C for 15 min to obtain plasma. The kidney was removed from each mouse, weighed, and washed by saline. The kidney tissues were frozen in liquid nitrogen, and were stored at −80 ◦C before the experiment. Other portions of the kidney were fixed with 10% formaldehyde for paraffin embedding. All experiment protocol was approved by the Institutional Animal Care and Use Committee of Kyung Hee University (KHUASP(SE)-19-076 on 06/14/2019).
