Anti-tumour activity and toxicological studies of combination treatment of Orthosiphon stamineus and gemcitabine on pancreatic xenograft model

Table 1 Treatment conditions for 28 d

No.	Group	The route of treatment and dose
1	Group I (untreated group- distilled water)	Oral (1 mg/kg per day)
2	Group II (chemodrug-gemcitabine)	Intraperitoneal (10 mg/kg per 3 d)
3	Group III (low dose of O.s)	Oral (200 mg/kg per day)
4	Group IV (low dose of O.s -gemcitabine combination)	Oral (200 mg/kg per day) + Intraperitoneal (10 mg/kg per 3 d)
5	Group V (high dose of O.s)	Oral (400 mg/kg per day)
6	Group VI (high dose of O.s-gemcitabine combination)	Oral (400 mg/kg per day) + Intraperitoneal (10 mg/kg per 3 d)

O.s: Orthosiphon stamineus.

Table 2 Effect of C5EOSEW5050ESA and gemcitabine alone or in combination on mouse body weight

No.	Group	Days											P value
		0	2	5	8	11	14	17	20	23	26	28
1	Untreated	24.2 ± 0.8	24.5 ± 1.0	24.7 ± 0.6	24.7 ± 0.9	24.6 ± 1.1	24.8 ± 0.9	25.0 ± 0.3	24.8 ± 0.6	24.6 ± 0.7	24.5 ± 1.1	24.5 ± 0.9	-
2	Gemcitabine (10 mg/kg per 3 d)	24.2 ± 0.7	24.3 ± 1.5	24.1 ± 1.2	23.9 ± 1.7	24.1 ± 1.3	24.0 ± 1.3	24.2 ± 0.5	24.5 ± 0.9	24.6 0 ± .5	24.3 ± 0.7	24.0 ± 0.9	NS
3	O.s (200 mg/kg per day)	23.6 ± 0.6	23.9 ± 0.7	24.9 ± 0.9	23.9 ± 1.1	24.1 ± 1.2	23.8 ± 1.1	24.0 ± 0.7	23.9 ± 9.0	23.8 ± 1.2	24.0 ± 1.5	23.8 ± 0.5	NS
4	O.s (200 mg/kg per day) and gemcitabine (10 mg/kg per 3 d) combination	24.0 ± 0.8	24.0 ± 1.2	23.9 ± 1.0	24.0 ± 1.1	23.4 ± 1.3	23.9 ± 1.5	24.1 ± 0.7	24.2 ± 0.7	23.9 ± 1.3	23.7 ± 1.1	23.5 ± 1.1	NS
5	O.s (400 mg/kg per day)	24.2 ± 0.7	24.4 ± 0.8	24.3 ± 1.0	24.5 ± 1.3	24.1 ± 1.0	24.3 ± 1.0	24.2 ± 1.0	24.4 ± 1.3	24.1 ± 1.4	24.0 ± 1.5	23.8 ± 1.2	NS
6	O.s (400 mg/kg per day) and gemcitabine (10 mg/kg per 3 d) combination	23.9 ± 0.7	24.0 ± 0.2	23.4 ± 0.7	23.4 ± 0.7	23.4 ± 0.5	23.4 ± 0.4	23.3 ± 0.5	23.5 ± 0.5	23.3 ± 0.8	23.3 ± 0.5	23.3 ± 0.3	NS

Each group contains 6 mice. The results showed no significant difference in the reduction of body weight of mice compared to the untreated group. Data are represented as mean ± SD. (NS: Not significant compared to the untreated group, P > 0.05). No statistical difference was also observed among all treatment groups using Two-way ANOVA and Tukey’s honest significant difference posthoc test. O.s: Orthosiphon stamineus.

Table 3 Effect of C5EOSEW5050ESA and gemcitabine alone or in combination on mouse key organ weight

No.	Group	Organ weight, g			P value
		Liver	Kidney	Spleen
1	Untreated	1.7 ± 0.22	0.47 ± 0.06	0.11 ± 0.03	-
2	Gemcitabine (10 mg/kg per 3 d)	1.7 ± 0.23	0.46 ± 0.06	0.10 ± 0.03	NS
3	O.s (200 mg/kg per day)	1.67 ± 0.23	0.47 ± 0.06	0.11 ± 0.03	NS
4	O.s (200 mg/kg per day) and gemcitabine (10 mg/kg per 3 d) combination	1.31 ± 0.25	0.41 ± 0.08	0.07 ± 0.03	NS
5	O.s (400 mg/kg per day)	1.37 ± 0.35	0.41 ± 0.09	0.24 ± 0.02	NS
6	O.s (400 mg/kg per day) and gemcitabine (10 mg/kg per 3 d) combination	1.43 ± 0.19	0.43 ± 0.09	0.08 ± 0.02	NS

Data are presented as mean ± SD (n = 6) (NS: Not significant compared to the untreated group, P > 0.05). No statistical difference was also observed among all treatment groups using two-way ANOVA and Tukey’s honest significant difference posthoc test. O.s: Orthosiphon stamineus.

Table 4 Haematological parameters in different treatment groups

No.	Group	Hb,g/L	Total RBC as 1012/L	PCV, L/L	MCV, fL	MCH, pg	MCHC, g/L	RDW, %	Total WBC as 109/L	N, %	L, %	M, %	E, %	B, %	Plts as 109/L
1	Untreated	121 ± 1.0	7.91 ± 0.9	0.36 ± 0.0	46 ± 1.5	15 ± 0.7	332 ± 1.1	20.6 ± 1.0	5.8 ± 1.3	37 ± 1.0	55 ± 1.1	5 ± 0.8	2 ± 0.1	1 ± 0.0	790 ± 1.2
2	Gemcitabine (10 mg/kg per 3 d)	118 ± 1.4	7.60 ± 1.1	0.38 ± 0.0	50.0 ± 1.8	16.0 ± 0.6	313 ± 1.8	22.1 ± 0.7	6.0 ± 1.2	33.0 ± 1.2	60.0 ± 0.7	6.0 ± 1.3	1.0 ± 0.1	0.0 ± 0.1	720 ± 1.4
3	O.s (200 mg/kg per day)	118 ± 1.5	8.0 ± 0.8	0.38 ± 0.0	48.0 ± 2.0	15.0 ± 1.1	307 ± 0.9	22.3 ± 1.6	6.10 ± 0.7	35.0 ± 0.8	53.0 ± 0.6	9.0 ± 1.0	1.0 ± 0.1	2.0 ± 0.0	801 ± 0.8
4	O.s (200 mg/kg per day) and gemcitabine (10 mg/kg per 3 d) combination	115 ± 1.7	7.1 ± 1.4	0.35 ± 0.0	50.0 ± 1.6	16.0 ± 0.7	321 ± 1.5	21.5 ± 0.8	5.70 ± 1.5	32.0 ± 0.9	60.0 ± 1.1	5.0 ± 0.7	2.0 ± 0.1	1.0 ± 0.1	760 ± 0.9
5	O.s (400 mg/kg per day)	123 ± 1.6	7.9 ± 0.7	0.36 ± 0.0	46.0 ± 1.5	16.0 ± 1.6	335 ± 2.0	21.0 ± 0.9	5.70 ± 1.8	33.0 ± 1.2	61.0 ± 1.2	4.0 ± 0.6	2.0 ± 0.1	0.0 ± 0.0	740 ± 1.0
6	O.s (400 mg/kg per day) and gemcitabine (10 mg/kg per 3 d) combination	123 ± 1.1	7.70 ± 0.9	0.36 ± 0.0	47.0 ± 1.2	16.0 ± 0.4	331 ± 1.2	22.6 ± 1.3	6.0 ± 1.5	30.0 ± 0.9	60.0 ± 1.1	8.0 ± 1.3	1.0 ± 0.1	1.0 ± 0.0	810 ± 1.3

Results are expressed as mean ± SD (n = 6). The P values in all treated groups were insignificant compared to the untreated group. No statistical difference was also observed among all treatment groups using two-way ANOVA and Tukey’s honest significant difference posthoc test. B: Basophile; E: Eosinophile; Hb: Haemoglobin; L: Lymphocyte; M: Monocyte; MCH: Mean cell haemoglobin; MCHC: Mean cell haemoglobin concentration; MCV: Mean cell volume; N: Neutrophile; O.s: Orthosiphon stamineus; PCV: Packed cell volume; Plts: Platelets; RBC: Red blood cells; RDW: Red cell distribution width; WBC: White blood cells.

Table 5 Blood biochemical parameters and electrolyte profiles in different treatment groups

No.	Group	Creatinine, mmol/L	Urea,mmol/L	Uric acid, mmol/L	AKT, U/L	AST, U/L	ALT, U/L	GGT, U/L	Total protein, g/L	Albumin, g/L	Globulin, g/L	Albumin/ Globulin ratio	Total Bilirubin, µmol/L	Na	K	CL
1	Untreated	28.1 ± 1.5	8.80 ± 0.9	0.20 ± 0.0	96.0 ± 0.5	195 ± 0.7	73.0 ± 0.7	< 3.0 ± 0.0	50.0 ± 0.5	30.0 ± 1.1	23.0 ± 0.5	1.40 ± 0.3	< 2.0 ± 0.0	178 ± 0.3	8.00 ± 0.7	124 ± 1.1
2	Gemcitabine (10 mg/kg per 3 d)	28.0 ± 1.9	9.00 ± 1.1	0.16 ± 0.1	93.0 ± 1.1	190 ± 0.9	72.0 ± 0.9	< 3.0 ± 0.0	52.0 ± 1.2	32.0 ± 0.7	24.0 ± 0.7	1.40 ± 0.2	< 2.0 ± 0.0	173 ± 0.5	9.30 ± 0.4	123 ± 0.2
3	O.s (200 mg/kg per day)	29.0 ± 1.5	9.20 ± 1.3	0.19 ± 0.0	98.0 ± 1.2	201 ± 1.1	80.0 ± 1.1	< 3.0 ± 0.0	50.5 ± 0.7	33.0 ± 0.3	23.0 ± 0.7	1.30 ± 0.3	< 2.0 ± 0.0	174 ± 0.8	9.00 ± 0.8	128 ± 0.9
4	O.s (200 mg/kg per day) and gemcitabine (10 mg/kg per 3 d) combination	27.5 ± 1.1	9.50 ± 0.9	0.21 ± 0.1	101 ± 0.9	210 ± 0.7	77.0 ± 0.7	< 3.0 ± 0.0	51.3 ± 1.1	29.9 ± 0.9	22.5 ± 0.9	1.30 ± 0.2	< 2.0 ± 0.0	186 ± 0.9	8.60 ± 0.6	130 ± 1.1
5	O.s (400 mg/kg per day)	28.5 ± 0.9	8.80 ± 0.7	0.22 ± 0.1	101 ± 0.7	199 ± 0.9	66.0 ± 0.5	< 3.0 ± 0.0	51.9 ± 0.5	32.4 ± 0.8	25.0 ± 0.9	1.40 ± 0.2	< 2.0 ± 0.0	190 ± 0.4	10.0 ± 0.5	133 ± 0.9
6	O.s (400 mg/kg per day) and gemcitabine (10 mg/kg per 3 d) combination	27.0 ± 0.8	8.50 ± 1.3	0.21 ± 0.0	103 ± 0.8	190 ± 0.7	70.0 ± 0.9	< 3.0 ± 0.0	52.0 ± 0.7	30.5 ± 0.6	24.0 ± 0.7	1.40 ± 0.1	< 2.0 ± 0.0	185 ± 0.6	9.80 ± 0.9	123 ± 0.5

Results are expressed as the mean ± SD (n = 6). The P values in all treated groups were insignificant compared to the untreated group. No statistical difference was also observed among all treatment groups using two-way ANOVA and Tukey’s honest significant difference posthoc test. AKT: Alkaline phosphatase; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; CL: Chloride; GGT: Gamma-glutamyl transferase; K: Potassium; Na: Sodium; O.s: Orthosiphon stamineus.