INTRODUCTION:

Gmelina arborea Roxb (Family Verbenaceae) fruits are oval in shape, ¾ inches in length and are yellow. The fruits are sweet in taste and sometimes astringent^1-3. The plant, G. arborea was reported to have several medicinal properties such as aphrodisiac, astringent, analgesic⁴, antipyretic⁵, anthelmintic⁶, antidiabetic^7,8, antioxidant⁸, diuretic⁹, antiepileptic¹⁰, anti-inflammatory¹¹, and tonic characteristics.

The G. arborea plant materials are the rich source of flavonoid compounds¹² and are responsible for various pharmacological activities especially diuretic¹³, antihypertensive and anti-inflammatory¹⁴. The isolation of phytoconstituent by the chromatographic method and as physical, chemical test and spectroscopic evidences such as IR, UV, H¹ NMR and C¹³NMR and MS, the structure of the isolated compound was a flavonoidal structure that is acacetin (5,7-dihydroxy-4 methoxy flavone) which may be responsible for various pharmacological activities of the plants. The yellow colored compound which solubilizes in methanol, chloroform and n-hexane and water having a melting point was 176 to 178°C and optical rotation was³⁰[α]_n– 32.5° (C=0.84 in methanol)^12-14.

Biopharmaceutics determines the interrelationship of the physical/chemical properties of the drug, the dosage form (drug product) in which the drug is given, and the route of administration on the rate and extent of systemic drug absorption¹⁵. Pharmacokinetics refers to the study of the time course of a drug within the body (extent and duration of systemic exposure to the drug) and also incorporates the process about the drug’s absorption, distribution, metabolism, and excretion (ADME) pattern. Pharmacokinetics has emerged as an integral part of drug development, especially when identifying a drug’s biological properties. Pharmacokinetic analysis is performed by Noncompartmental or compartmental methods^16,17. Noncompartmental methods estimate the exposure to a drug by estimating the area under the curve of a concentration-time graph. Compartmental methods estimate the concentration-time graph using kinetic models¹⁸.

LC-MS is a technique routinely used in sample analysis that combines liquid chromatography (LC) with mass spectrometry (MS). Extremely sensitive modern MS has helped LC-MS replace several immunoassays. LC-MS has helped improve the efficiency of drug discovery due to its excellent sensitivity and specificity. The technique can be combined with stable isotope dilution for precise and reproducible assays¹⁹.

The objective of the present study to carry out pharmacokinetic evaluation of isolated flavonoidal compound acacetin to access therapeutic effectiveness.

MATERIALS AND METHODOLOGY:

Acacetin, from fruits of Gmelina arborea, was used as test drug to determine its pharmacokinetic parameters. Artificial plasma was obtained from Sigma-Aldrich, New Delhi. Methyl cyanide and formic acid were purchased from S.D. Fine Chem., Mumbai. All other chemicals and reagents were of analytical grade and procured from an authorized dealer.

Animal model selection:

Healthy male Wister rats (weighing 180 to 200 g) were used as model animals. The animals were kept in the standard polypropylene cages at 25±2°C/ 60% relative humidity in normal day and night photocycle (12: 12 h). The animals were acclimatized for a period of 14 days before performing the experiments. They were fed with a standard pellet diet and water ad libitum. Prior to the study, the experimental protocols were approved by the Institutional Animal Ethics Committee of Gayatri College of Pharmacy, Gayatri Vihar, Jamadarpali, Sambalpur, Odisha (Ethical committee letter No. 1339/ac/10/CPCSEA).

Drug sample preparation and treatment:

The solution of sample, acacetin was prepared using normal saline water as a vehicle. Acacetin was dissolved in water using ethanol as co-solvent. The sample was made completely soluble by sonication method. The drug solution was administered orally at a dose of 10 mg/Kg body weight of rat, whose absorption of the drug may be following one compartmental open model with first-order absorption kinetics. Another dose (2 mg/Kg body weight of rat) of drug solution was injected as an intravenous bolus dose, which was considered as standard²⁰.

Blood sample collection:

The test drug sample solution was administered orally to three rats and blood samples (2.5ml) were collected at 0.5, 1, 2, 4, 6 and 8 h. In case of intravenous injection, blood samples volume 2.5 ml were collected at time intervals of 0.08, 0.25, 0.5, 1, 2, 4 and 6 h respectively. All study was done over three replicates (n = 3).

Purification of plasma samples:

The collected blood samples were processed to separate plasma from other blood components. The plasma samples were pre-cleaned by protein precipitation method. The plasma samples (Initial standard) were treated with 0.5 ml of acetonitrile and mixture was incubated for 10 min. The mixture was centrifuged at 5000 rpm for 2 min. The proteins, amino acids, salts, and lipids will be settled to the bottom. A known volume (0.4 ml) of supernatant liquid that was separated in pure form was collected²¹.

Internal standard for acacetin:

The internal standard for pharmacokinetic study in rats was carried out using acacetin as test sample at a concentration of 1 µM using the following specifications such as post oral sample with six-time points and intravenous sample with seven-time point. Water was used as blank with internal standard and acetonitrile was used as a solvent without the internal standard.

Calibration standard preparation of acacetin:

A calibration curve was prepared to consist of a blank sample, a zero sample (Matrix sample processed with internal standard) and five none zero samples. The known concentration of test sample was added to the blank plasma sample. Five standard samples were prepared at various strength ranging from 0.01 µM to 10 µM at the rate of 300 ng/mole correspond to 3 to 3000 ng/ml. These samples were treated identically with collected plasma samples. These samples were analyzed using LC-MS and data are used to develop a calibration curve.

Bioanalysis by LC-MS study:

The plasma samples collected as internal standards, calibration standards and compound F test samples as administered orally and intravenously were analyzed for quantification of drug (compound F) by using LC-MS with the following specifications: Column was Agilent Zorbax SB – C18, 2.1 × 5 mm (Bruker EASY-nLC II, Bruker India Scientific Pvt. Ltd., Mumbai, India). Acetonitrile with 0.1% formic acid was used as the mobile phase. The gradient was initial 100 % A for 2 min, 0 to 100 % B for 15 min, hold 100 % B for 8 min and return to initial 100 % A. Flow rate was maintained at 0.25 ml/min. The column temperature was maintained at 25°C. The pH of the samples was maintained at 3. The samples were injected at a volume of 5 µL. Each run required 20 min^20-22.

RESULTS AND DISCUSSIONS:

The calibration curve of the testing sample, acacetin in blank plasma was determined. Regression coefficient was found to be 0.9918, with regression equation, y = 1768.7x + 0.0018 (Table 1). The results indicated (Tables 2 to 5) that with an increase in time course of the test sample in animal body, the concentration of sample decreases linearly irrespective of time, signifying that the sample gets rapidly eliminated from the body, that is elimination half of the sample, acacetin may be less as evident from data shown in Fig 1 to 4.

Fig 1. Drug plasma level time profile curve of acacetin by I.V. administration.

Fig 2. Drug plasma level time profile curve of acacetin by oral administration.

Fig 3. Logarithm curve of drug plasma level time profile curve of acacetin by I.V. administration.

Fig 4. Logarithm curve of drug plasma level time profile curve of acacetin by oral administration.

Table 1: Calibration linear fit curve of the test sample (acacetin) using blank plasma.

Sample code & strength (µM)	Type	RT (min)	Area	Response	Conc. (µg/ml)	Recovery (%)
Std 01	Standard	2.13	35	21	0.0119	101
Std 02	Standard	2.11	583	157	0.0887	89
Std 03	Standard	2.09	6730	1735	0.9811	98
Std 04	Standard	1.97	32268	9482	5.3611	107
Std 05	Standard	2.13	53002	17904	10.1224	101

RT–Retention time.

Table 2: Internal standard data of pharmacokinetic study in rats with acacetin at 1 µM.

Sample code	Type	RT (min)	Area	Response	Conc. (µg/ml)	Recovery (%)
Std 01 (µM)	QC	3.55	797	796.9	0.4506	45
Std 02 (µM)	QC	3.57	1068	1067.9	0.6038	60
Std 03 (µM)	QC	3.55	1281	1281.1	0.7243	72
Solvent 1	Blank	3.43	1	1	0.0006	0
Solvent 2	Blank	3.42	1	1	0.0005	0

RT – Retention time. QC – Quality control.

Table 3: Bioanalytical data of sample (acacetin) by IV administration.

Code	Name	Type	RT (min)	Area	Response	Conc. (µg/ml)	Recovery (%)
1	IV/0.08	Analyte	3.56	1891	1890.8	1.069	107
2	IV/0.25	Analyte	3.55	1348	1347.7	0.762	76
3	IV/0.5	Analyte	3.57	1150	1149.7	0.650	65
4	IV/01	Analyte	3.55	967	967.4	0.547	55
5	IV/02	Analyte	3.55	456	456.3	0.258	26
6	IV/04	Analyte	3.57	196	196.3	0.111	11
7	IV/06	Analyte	3.54	50	49.52	0.028	3

Table 4: Plasma level data of the sample (Acacetin) by IV administration.

Time (h)	Avg. Conc. (ng/ml) (n = 3)	S.D. (ng/ml)	% C.V.
0.08	1069	48	4
0.25	762	176	23
0.5	650	170	26
1.0	547	135	25
2.0	258	75	29
4.0	111	33	30
6.0	28	8	29

S.D. – Standard deviation. C.V. – Calculated variance.

Table 5: Bioanalytical data of sample (Acacetin) plasma by oral administration.

Code	Name	Type	RT (min)	Area	Response	Conc. (µg/ml)	Recovery (%)
1	OR/0.5	Analyte	3.57	2462	2462.1	1.392	139
2	OR/01	Analyte	3.55	2950	2950.2	1.668	167
3	OR/02	Analyte	3.54	2506	2506.3	1.417	142
4	OR/04	Analyte	3.56	1371	1370.8	0.775	76
5	OR/06	Analyte	3.55	594	594.3	0.336	34
6	OR/08	Analyte	3.57	251	251	0.142	14

The intravenous administration of acacetin solution at a dose of 2 mg/Kg of body weight of rat simulates with intravenous bolus dose administration, which follows linear (First-order) kinetics. The first order drug elimination constant (K) can be computed from the slope of the graph, which is obtained by plotting Log of plasma drug concentration verses time (Log C V/s. T) [23, 24] and its value K is 0.423 h^-1 (Fig 3). The half life of the sample, T_1/2 = 1.64 h ≈ 2 h. A total amount of the sample (acacetin) available to systemic calculation was calculated as the Area under curve (AUC) of plasma drug time profile curve. The AUC was calculated as Trapezoidal rule ^{23, 24} that is a summation of areas of all triangles and trapeziums which come under plasma drug time profile curve (Fig 1, 3) was calculated and AUC was found to be 1.542 µg h/ml.

Table 6: Plasma level data of sample (Acacetin) by oral administration.

Time (h)	Avg. Conc. (ng/ml) (n = 3)	S.D. (ng/ml)	% C.V.
0.5	1392	236	17
1.0	1668	432	26
2.0	1417	214	15
4.0	775	40	5
6.0	336	94	28
8.0	142	40	28

S.D.–Standard deviation. C.V.–Calculated variance.

Table 7: Pharmacokinetic data of acacetin from drug plasma data administered intravenously and orally.

Sl. No.	Pharmacokinetic parameters	IV administration	Oral administration
01	C_max (µg/ml)	-	1.668
02	T_max (h)	-	1
03	AUC (µg h/ml)	1.542	6.44
04	K_E (h^-1)	0.423	0.416
05	T_1/2(h)	2	2
06	K_a (h^-1)	-	1.6
07	F (%)	-	84

The oral administration of acacetin solution at a dose of 10 mg/Kg of body weight of rat simulates with oral administration of drug following one compartment open model with first-order drug absorption kinetics. The results obtained in the graph of plasma drug time profile curve after oral administration of drug, it was confirmed that the drug movements in the body first follows absorption phase followed by elimination phase. The results obtained in Fig 50 were correlated with plasma drug time profile curve (Fig 2).

The plasma drug time profile curve in Fig 2, explained that peak plasma concentration (C_max) of acacetin was 1.668 µg/ml. The time of peak plasma concentration of acacetin in rat body was 1 h. The total amount of the sample (acacetin) available to systemic calculation was calculated as Area under curve (AUC) of plasma drug time profile curve (Fig 4). The AUC was calculated as Trapezoidal rule that is a summation of areas of all triangles and trapeziums comes under plasma drug time profile curve (Fig 2, 4) was calculated and AUC was found to be 6.44 µg h/ml. The bioavailability of the sample, acacetin was found to be 84 %. First order drug elimination rate constant can be computed from the slope of the graph, which is obtained by plotting Log of plasma drug concentration verses time (Log C V/s. T) as follows (Fig 4). The elimination rate constant was found to be 0.416 h^-1. A half life of sample (Acacetin) was 2 h. The first order absorption rate constant was calculated by using the method of residual ²⁵ and the first order absorption rate constant (K_a) was found to be 1.6 h^-1.

CONCLUSION:

The study concluded that the flavonoid compound, acacetin being present in fruits of G. arborea, possessed satisfactory bioavailability, with the significant pharmacokinetic profile which would be helpful for successful designing of a suitable dosage form for any drug delivery system for effective management of hypertension. Research study to be extended in pre-formulation and formulation study of an active moiety with clinical evaluations.

ACKNOWLEDGMENT:

Authors wish to thanks Department of Life Sciences, Howrah, Calcutta, W.B., for providing the facility for blood sample analysis by LC-MS.

AUTHOR CONTRIBUTION:

BSN prepared the manuscript and involved in experimental work. HKR interpretated the result and PE contributed grammatical correction. The planning and methodology was carried out by SCD.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 27.01.2019 Modified on 25.02.2019

Research J. Pharm. and Tech 2019; 12(8):3659-3663.

DOI: 10.5958/0974-360X.2019.00624.3