Enhancing oral bioavailability of andrographolide using solubilizing agents and bioenhancer: comparative pharmacokinetics of Andrographis paniculata formulations in beagle dogs

Figures & data

Table 1. Formulation development of A. paniculata capsules.

Formulations	Dosage of AP	Route of administration	% of mixing agents in A. paniculata capsules
			β-cyclodextrin	SDS	Piperine
A. paniculata alone	3 mg/kg	p.o.	–	–	–
A. paniculata + β-cyclodextrin	3 mg/kg	p.o.	50% w/w	–	–
A. paniculata + SDS	3 mg/kg	p.o.	–	1% w/w	–
A. paniculata + piperine + SDS	3 mg/kg	p.o.	–	1% w/w	10% w/w

Abbreviations: SDS, sodium dodecyl sulfate; AP, andrographolide; p.o., oral administration.

Table 2. Andrographolide contents in A. paniculata capsules (data are expressed as means ± SD, n = 10).

Ingredients	Weight (mg/capsule) mean ± SD (n = 10)	Andrographolide (mg/capsule) mean ± SD (n = 10)
A. paniculata alone	210.89 ± 10.41	5.10 ± 0.05
A. paniculata + β-cyclodextrin	209.45 ± 22.69	5.29 ± 0.10
A. paniculata + SDS	217.95 ± 10.64	5.62 ± 0.05
A. paniculata + piperine + SDS	243.90 ± 17.55	6.13 ± 0.05

Abbreviations: SDS, sodium dodecyl sulfate.

Figure 1. Chromatographic profiling and tentative compound identification of A. paniculata powder.

Table 3. Characterization of the phytochemical constituents of A. paniculata powder.

Compound	Rt (min)	Molecular ion (m/z)	Adduct	MS/MS Fragments (m/z)	Tentative Identification	Formula	Error (ppm)	Group of compounds
M1	4.286	355.1016	[M + H]^+	163.0379,145.0273,135.0429,117.0328, 89.0374	Caffeoylquinic acid	C16H18O9	2.14	Phenolic acid
M2	5.972	333.2052	[M + H-H2O]^+	315.1941,297.1839,257.1525,205.1571, 175.1468,145.0997,111.0797	14-Deoxy-11-hydroxyandrographolide	C20H30O5	1.61	Diterpenoid
M3	6.157	787.1926	[M + H]^+	625.1387,463.0856,287.0539,145.0463	Kaempferol 3-O-sophoroside 7-O-glucuronide	C33H38O22	0.19	Flavonoid
M4	6.288	701.4252	[2M + H]^+	333.205, 315.1946	Isoandrographolide	C20H30O5	2.76	Diterpenoid
M5	6.580	447.0913	[M + H]^+	271.0593,153.0169	Apigenin 7-glucuronide	C21H18O11	1.99	Flavonoid
M6	6.647	463.1225	[M + H]^+	407.2371,301.0695	Kaempferide 7-glucoside	C22H22O11	2.13	Flavonoid
M7	7.148	463.0863	[M + H]^+	287.0542	Kaempferol-3-glucuronide	C21H18O12	1.73	Flavonoid
M8	7.591	477.1385	[M + H]^+	315.0858,145.0485,85.0275	4’,5-Dihydroxy-7,8-dimethoxyflavone glucoside	C23H24O11	1.34	Flavonoid
M9	7.684	351.2158	[M + H]^+	333.2049,315.1949,297.1837,257.1526,207.1736	Andrographolide isomer	C20H30O5	2.28	Diterpenoid
M10	8.572	333.2053	[M + H-H2O]^+	315.1948,297.1845,257.1529,205.1581, 111.0797,81.0696,55.0536	Andrographolide isomer	C20H20O5	2.21	Diterpenoid
M11	8.853	701.4259	[2M + H]^+	351.2165,333.2058,315.1953,297.1844	Andrographolide	C20H30O5	0.04	Diterpenoid
M12	9.633	498.3055	[M + NH4]^+	319.2263,301.2163,289.2156	Neoanhydrographolide	C26H40O8	1.29	Diterpenoid
M13	11.602	669.4357	[2M + H]^+	317.2111,299.2003,287.2003,259.1689, 229.1216,205.1218,179.1057,153.0906, 133.1001,111.0798,81.0690	Dehydropinifolic acid	C20H30O4	0.59	Diterpenoid

Table 4. Tolerability profile after oral administration of A. paniculata preparations (equivalent to 3 mg/kg of andrographolide).

Biochemical parameters	Experimental group (oral administration of A. paniculata capsule equivalent to 3 mg/kg of andrographolide)
	Pre-treatment	A. paniculata alone p.o. Day 1	A. paniculata alone p.o. Day 7	A. paniculata + β-cyclodextrin p.o. Day 1	A. paniculata + β-cyclodextrin p.o. Day 7	A. paniculata + SDS p.o. Day 1	A. paniculata + SDS p.o. Day 7	A. paniculata + piperine + SDS p.o. Day 1	A. paniculata + piperine + SDS p.o. Day 7
Physical appearance	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal
Body weight	12.25 ± 1.21	13.11 ± 1.34	12.85 ± 1.43	12.89 ± 1.73	12.53 ± 1.18	13.09 ± 1.52	12.79 ± 1.02	13.18 ± 1.27	12.63 ± 1.25
White blood cell (x 10^3/µL)	9.19 ± 1.16	9.49 ± 1.38	10.11 ± 2.47	10.13 ± 0.26	9.59 ± 0.87	8.61 ± 2.12	8.22 ± 1.07	8.07 ± 1.51	8.63 ± 1.71
Red blood cell (x 10^6/µL)	6.93 ± 0.27	7.55 ± 0.58	7.36 ± 0.18	7.21 ± 0.31	7.04 ± 0.56	7.28 ± 0.39	7.15 ± 0.95	7.26 ± 0.28	7.35 ± 0.51
Hemoglobin (g/dL)	14..4 ± 0.5	15.50 ± 1.21	15.0 ± 0.5	15.0 ± 0.4	14.6 ± 1.2	15.1 ± 0.6	14.7 ± 1.7	15.0 ± 0.4	15.2 ± 0.8
Platelet (x 10^3/µL)	336 ± 43	280 ± 47	269 ± 41	294 ± 30	280 ± 42	310 ± 61	300 ± 39	303 ± 32	275 ± 46
Blood urea nitrogen (mg/dL)	12.8 ± 0.4	11.4 ± 1.3	11.5 ± 0.7	12.1 ± 1.1	13.1 ± 1.9	11.7 ± 1.6	11.1 ± 0.9	12.5 ± 2.3	11.9 ± 1.0
Creatinine (mg/dL)	0.9 ± 0.2	0.5 ± 0.2	0.6 ± 0.1	0.6 ± 0.1	0.6 ± 0.1	0.6 ± 0.1	0.6 ± 0.1	0.6 ± 0.1	0.7 ± 0.1
AST (U/L)	26 ± 5	35 ± 3	31 ± 1	22 ± 3	26 ± 6	27 ± 2	32 ± 3	31 ± 1	32 ± 4
ALT (U/L)	33 ± 8	39 ± 8	38 ± 10	29 ± 5	29 ± 10	37 ± 5	37 ± 7	38 ± 8	36 ± 9
Alkaline phosphatase (U/L)	39 ± 22	39 ± 4	40 ± 8	35 ± 10	37 ± 21	34 ± 5	36 ± 5	35 ± 5	41 ± 5

AST: aspartate transaminase; ALT: alanine transaminase; SDS: sodium dodecyl sulfate. Data are expressed as mean ± SD, (n = 4). Decimal numbers were reported according to the laboratory standard of the Small Animal Hospital, Faculty of Veterinary Science, Chulalongkorn University.

p.o., oral administration.

Figure 2. (a–d). Comparative mean plasma concentration versus time profiles of andrographolide after single and multiple oral administration of A. paniculata alone and in different combination formulations (equivalent to 3 mg/kg of andrographolide) in beagle dogs. Data are presented as means ± SD (n = 4).

Table 5. Comparative pharmacokinetic parameters of andrographolide after single and multiple oral administration of A. paniculata capsules (equivalent to 3 mg/kg of andrographolide) in beagle dogs.

		Oral administration (p.o.) equivalent to 3 mg/kg of andrographolide
		Single dose (p.o.)				Multiple doses (p.o.)
Pharmacokinetic parameters		A. paniculata alone	A. paniculata + β-cyclodextrin	A. paniculata + SDS	A. paniculata + piperine + SDS	A. paniculata alone	A. paniculata + β-cyclodextrin	A. paniculata + SDS	A. paniculata + piperine + SDS
Half-life ^a	h	2.90 [2.83]	4.80 [1.62]	3.64 [4.63]	6.96 [0.80]	7.56 [3.17]	8.58 [1.29]	9.30 [1.61]	9.11 [3.41]
Cmax (obs)^b	µg/L	108.07 ± 20.49	150.54 ± 28.13	200.91 ± 34.74	224.93 ± 79.16	116.52 ± 30.62	142.84 ± 26.42	182.23 ± 57.88	245.73 ± 76.73*
Tmax (obs)^a	h	1.00 [0.00]	0.50 [0.00]	1.00 [0.00]	0.50 [0.13]	0.75 [0.50]	1.00 [0.00]	1.00 [0.13]	1.00 [0.00]
AUC(0-t)^b	µg-h/L	393.28 ± 87.94	506.80 ± 114.54	519.80 ± 59.22	565.02 ± 96.77	431.81 ± 68.21	738.14 ± 134.73	737.11 ± 131.40	830.68 ± 202.70
AUC(0-∞)^b	µg-h/L	398.59 ± 93.85	522.22 ± 107.46	526.95 ± 54.91	593.45 ± 102.09	472.01 ± 84.93	836.73 ± 194.30	853.73 ± 190.10	925.37 ± 188.27*
Vd^b	L/kg	32.18 ± 10.57	42.38 ± 23.61	46.46 ± 27.97	62.51 ± 10.39	64.88 ± 40.22	37.49 ± 3.43	53.62 ± 9.39	52.45 ± 20.09
Cl ^b	L/h/kg	7.61 ± 2.42	5.13 ± 1.47	5.94 ± 0.35	5.88 ± 0.73	6.21 ± 1.26	3.16 ± 0.68	3.83 ± 0.92	3.83 ± 0.85
Relative oral bioavailability	(%)	N/A	131.01	132.20	148.89	N/A	177.27	180.87	196.05
AR	(ratio)	N/A	N/A	N/A	N/A	1.18	1.84	1.62	1.56

Abbreviations: ^a data are expressed as median [IQR]; ^b data are expressed as mean ± SD, * p < 0.05 for significant difference.

SDS, sodium dodecyl sulfate; p.o., oral administration.

Half-life, elimination half-life; C_max, maximum plasma concentration; T_max, time to reach maximum concentration; AUC_(0-t), area under the curve from time zero to the last sampling time; AUC_(0-∞), area under the curve that extrapolated to time infinity; V_d/F, apparent volume of distribution; Cl/F, apparent clearance; AR, accumulation ratio.

Figure 3. Cumulative amount of andrographolide in urine after multiple oral administration of A. paniculata formulations (equivalent to 3 mg/kg of andrographolide) in beagle dogs. Data are presented as means ± SD (n = 4).

Table 6. Percent recovery of andrographolide elimination in urine and feces after single and multiple oral administration of A. paniculata capsules (equivalent to 3 mg/kg of andrographolide) in beagle dogs (data are expressed as means ± SD, n = 4).

		% Recovery
		Oral administration (p.o.) equivalent to 3 mg/kg of andrographolide
		A. paniculata alone	A. paniculata + β-cyclodextrin	A. paniculata + SDS	A. paniculata + piperine + SDS
Single dose	Urine
	T 0-24 h	0.800 ± 0.675	0.214 ± 0.087	0.501 ± 0.556	0.668 ± 0.334
	Feces
	T 0-24 h	0.307 ± 0.179	0.154 ± 0.272	0.290 ± 0.242	0.331 ± 0.404
Multiple doses	Urine
	T 0-24 h	0.162 ± 0.174	0.190 ± 0.072	0.264 ± 0.171	0.381 ± 0.224
	T 24-48 h	0.026 ± 0.043	0.097 ± 0.031	0.027 ± 0.019	0.015 ± 0.014
	Feces
	T 0-24 h	0.138 ± 0.084	0.186 ± 0.100	0.107 ± 0.076	0.122 ± 0.129
	T 24-48 h	0.026 ± 0.032	0.042 ± 0.013	0.019 ± 0.025	0.014 ± 0.004

Abbreviations: SDS, sodium dodecyl sulfate; p.o., oral administration.

Figure 4. Quantification of parent compound and conjugated glucuronide metabolites in urine and feces after oral administration of the different formulations of A. paniculata capsules in beagle dogs (T_0-24h).

Data availability statement

The datasets generated and/or analyzed during the present study are not publicly available but are available from the corresponding author on reasonable request that needs a consensus from colleagues.