Therapeutic effect of Morus alba leaf extract and chlorogenic acid on inhibiting the progression of kidney disease

Figures & data

Figure 1. (A) types of urine sedimentation on all groups n = 5; (B) Kruskal-Wallis’s result on urine crystal formation (Significance *p < 0.05 vs. UUO group, n = 5); (C) Urine crystal image with 40–400× magnification: (i) Calcium oxalate (black arrow) and calcium carbonate (arrowhead) (40×); (ii) calcium oxalate (400×); (iii) calcium carbonate (blue arrowhead) (100×); (iv) triple phosphate (red arrow head) (100×); (v) triple phosphate (red arrow head) (400×); (vi) epithelial squamous (red arrowhead) and triple phosphate (yellow arrowhead) (100×). SO: sham operation; UUO: unilateral urethral obstruction; ENA: enalapril; MLE: Morus alba leaves extract; CGA: chlorogenic acid; Significance: ns: not significant; *p < 0.05 vs. UUO group.

Figure 2. The urine protein creatinine ratio on enalapril, Morus alba leaves extract, and chlorogenic acid administration in the UUO mice. SO: sham operation; UUO: unilateral urethral obstruction; ENA: enalapril; MLE: Morus alba leaves extract; CGA: chlorogenic acid; Significance: ns: not significant; *p < 0.05 vs. UUO group.

Table 1. The urine analysis result of UUO mice administrated with enalapril, Morus alba leaf extract, and chlorogenic acid for 14 days.

Treatment	SG	pH	Nitrite	Ketone (mmol/L)	Protein (g/L)	Leukocyte (leu/µL)	Erythrocyte (Ery/µL)	Glucose (mmol/L)	UBG (µmol/dL)	Bilirubin (µmol/L)
SO	1.017 ± 0.005^a	6.5 ± 0.22^a	0.4 ± 0.24^a	0.0 ± 0.0^b	0.00 ± 0.00^b	0.5 ± 0.50^b	0.0 ± 0.00^b	Normal	Normal	Negative
UUO	1.016 ± 0.003^a	7.2 ± 0.37^a	0.4 ± 0.24^a	0.8 ± 0.3^a	0.50 ± 0.28^a	145 ± 90.96^a	105 ± 59.37^a	Normal	Normal	Negative
UUO + ENA	1.022 ± 0.001^a	6.4 ± 0.19^a	0.4 ± 0.24^a	0.3 ± 0.3^ab	0.20 ± 0.14^ab	5 ± 5.00^b	12 ± 9.69^ab	Normal	Normal	Negative
UUO + MLE	1.018 ± 0.002^a	7.1 ± 0.40^a	0.2 ± 0.20^a	0.2 ± 0.1^ab	0.00 ± 0.00^b	40 ± 24.49^ab	0.00 ± 0.00^b	Normal	Normal	Negative
UUO + CGA	1.025 ± 0.002^a	6.3 ± 0.20^a	0.4 ± 0.24^a	0.0 ± 0.0^b	0.05 ± 0.05^b	15 ± 6.12^ab	5.0 ± 5.0^b	Normal	Normal	Negative

SO: sham operation; UUO: unilateral urethral obstruction; ENA: enalapril; MLE: Morus alba leaves extract; CGA: chlorogenic acid; SG: specific gravity; UBG: urobilinogen.

Data values are presented as mean ± SEM; values with different superscripts in the column differed significantly at p < 0.05 (n = 5).

Table 2. Haematology of the UUO mice administrated with enalapril, Morus alba leaf extract, and chlorogenic acid for 14 days.

Group	RBC (1012/L)	HGB (g/dL)	HCT (%)	MCV (fL)	MCH (pg)	MCHC (g/L)	PLT (103/μL)	WBC (109/L)	Neutrophil (109/L)	Lymphocyte (109/L)	Monocyte (109/L)	Eosinophil (109/L)	Basophil (109/L)
SO	8.99 ± 0.11^a	13.86 ± 0.29^a	39.20 ± 0.64^a	43.62 ± 0.65^a	15.42 ± 0.35^a	353.4 ± 3.20^a	604 ± 99.93^a	4.74 ± 0.61^b	1.53 ± 0.28^a	2.84 ± 0.46^a	0.26 ± 0.07^a	0.10 ± 0.03^a	0.01 ± 0.01^a
UUO	7.42 ± 0.28^b	11.68 ± 0.36^b	33.70 ± 1.13^b	45.44 ± 0.57^a	15.84 ± 0.31^a	348.6 ± 4.59^a	698 ± 52.75^a	6.68 ± 0.48^a	2.28 ± 0.29^a	4.01 ± 0.50^a	0.21 ± 0.06^a	0.19 ± 0.04^a	0.00 ± 0.00^a
UUO + ENA	8.68 ± 0.38^a	13.72 ± 0.51^a	38.32 ± 1.37^a	44.22 ± 0.51^a	15.84 ± 0.22^a	358 ± 3.42^a	753.8 ± 49.96^a	5.60 ± 0.89^ab	1.39 ± 0.24^a	3.70 ± 0.57^a	0.26 ± 0.07^a	0.22 ± 0.11^a	0.00 ± 0.00^a
UUO + MLE	8.63 ± 0.04^a	13.56 ± 0.70^a	38.86 ± 2.06^a	45.00 ± 0.82^a	15.70 ± 0.23^a	349.4 ± 4.97^a	846 ± 61.22^a	4.48 ± 0.09^b	1.58 ± 0.32^a	2.51 ± 0.57^a	0.20 ± 0.05^a	0.18 ± 0.10^a	0.00 ± 0.00^a
UUO + CGA	8.53 ± 0.31^a	13.10 ± 0.37^a	37.20 ± 0.99^ab	43.68 ± 0.56^a	15.38 ± 0.21^a	352 ± 1.52^a	863.6 ± 120.0^a	5.88 ± 0.45^ab	1.86 ± 0.48^a	3.54 ± 0.46^a	0.30 ± 0.05^a	0.19 ± 0.02^a	0.00 ± 0.00^a

SO: sham operation; UUO: unilateral urethral obstruction; ENA: enalapril; MLE: Morus alba leaves extract; CGA: chlorogenic acid; RBC: red blood cell; HGB: haemoglobin; HCT: haematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular haemoglobin; MCHC: mean corpuscular haemoglobin concentration; PLT: platelets.

Data values are presented as mean ± SEM; values with different superscripts in the column differed significantly at p < 0.05 (n = 5).

Figure 3. Biochemistry analyses of the UUO Mice treated with enalapril, Morus alba Leaf extract and chlorogenic acid. Data values are presented as mean ± SEM; Asterisks symbol (a–c) shows statistical significance between the groups (n = 5). SO: sham operation; UUO: unilateral urethral obstruction; ENA: enalapril; MA: Morus alba; CGA: chlorogenic acid; AST: aspartate aminotransferase; ALT: alanine aminotransferase; CK: creatine kinase; MLE: Morus alba leaves extract.

Figure 4. Gross anatomy of mice kidneys treated with enalapril, Morus alba leaves extract, and chlorogenic acid for 14 days in the UUO mice. (A) Sham operation (negative control), (B) UUO (positive control), (C) UUO treated with enalapril, (D) UUO treated with Morus alba leaves extract, (E) UUO treated with chlorogenic acid, (F) UUO Kidney to body weight ratio in Morus alba and chlorogenic acid treatment in the mice. (A,B) Collateral unobstructed kidney to body weight ratio in Morus alba and chlorogenic acid treatment in the mice. SO: sham operation; UUO: unilateral urethral obstruction; ENA: enalapril; MLE: Morus alba leaves extract; CGA: chlorogenic acid; Significance: ns: not significant; *p < 0.05; ^****p < 0.0001.

Figure 5. Histology of kidney mice administrated with enalapril, Morus alba leaves extract, and chlorogenic acid in Haematoxylin and Eosin (H&E) staining. (A) Sham operation group (10 µm scale bar) shows the normal structure of glomerulus and tubules; (B) UUO group depicts interstitial fibrosis, Bowman’s space dilatation, and tubular epithelial cell loss with the intralobular eosinophilic cast (50 µm scale bar); (C) UUO group with higher magnification of group figure B (10 µm scale bar); (D) UUO group shows interstitial fibrosis, dilatation of tubules and infiltration of the inflammatory cell (10 µm scale bar); (E) UUO treated with enalapril group (10 µm scale bar); (F) UUO treated with Morus alba leaves extract (10 µm scale bar); (G) UUO treated with chlorogenic acid (10 µm scale bar). G: glomerulus; P: proximal tubule; D: distal tubule; yellow arrow: intralobular cast; blue arrow: fibrosis; red arrow: cellular inflammatory; black arrow: tubular dilation.

Table 3. Kidney histology scoring of the UUO mice administrated with enalapril, Morus alba leaf extract, and chlorogenic acid for 14 days.

Group	Bowman’s space dilatation	Tubular dilatation	Tubular epithelial lost	Cellular inflammatory	Eosinophilic intralobular cast
Sham operation	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0
UUO	3.6 ± 0.24	4.0 ± 0.0	3.8 ± 0.2	4.0 ± 0.0	3.8 ± 0.2
UUO + ENA	2.0 ± 0.3*	2.6 ± 0.4	2.6 ± 0.2	1.0 ± 0.0**	1.4 ± 0.2**
UUO + MLE	2.4 ± 0.2	2.6 ± 0.2*	2.8 ± 0.2	2.0 ± 0.4	2.0 ± 0.3
UUO + CGA	1.8 ± 0.2**	2.2 ± 0.2**	1.8 ± 0.2**	1.2 ± 0.2*	1.6 ± 0.2*

SO: sham operation; UUO: unilateral urethral obstruction; ENA: enalapril; MLE: Morus alba leaves extract; CGA: chlorogenic acid.

Significance *p < 0.05; **p < 0.01 vs. UUO group, n = 5.

Data availability statement

Data will be made available on request.