Pharmacokinetic and Phytochemical Appraisal of Trigonella foenum and Andrographis lineata in Dimethylnitrosamine Induced Injury in Albino Rats

Trigonelline is a plant alkaloid and Andrographolide is a diterpene lactone, both exhibiting antiinflammatory, antioxidant and neuroprotective activities. The present study was designed to evaluate the antioxidative and anti-inflammatory activity of the above said compounds in Dimethylnitrosamine induced toxicity in albino rats. Extraction of Trigonella foenum and Andrographis lineata was carried out by using methanol, petroleum ether, ethyl acetate and ethanol assisted with suitable temperature, followed by DPPH scavenging activity (IC-50) of these extracts.Cmax, Tmax, t1/2, CL, Vd and AUC were evaluated as pharmacokinetic parameters by using calibration curves of Andrographolide and Trigonelline. Extracts of T. foenum-graecum and A. lineate have antioxidant activity by inhibiting DPPH (IC50 value was 69.04±3.65% and 71.76±6.99%, respectively) comparable with ascorbic acid (53.99±4.88%). Phytochemical analysis of T. foenum-graecum and A. lineate was found with maximum number of phenols in them, with least recovery in the ethyl acetate extract while maximum phenols were found in Original Research Article Zahid et al.; JPRI, 33(46B): 73-79, 2021; Article no.JPRI.75341 74 T. foenum graecum. All the pharmacokinetic parameters of trigonelline and andrographolide administered in low dose (200 mg/kg and 50 mg/kg respectively) confirmed the better antioxidative activity than that of ascorbic acid used as a potent antioxidant.


INTRODUCTION
The genomic technology has ability to give insight into the investigations in altering the gene expression caused by acute effect with Dimethylnitrosamine. Its acute administration in rats exhibits overall significant changes in the profile of gene expression [1]. The most considerable changes were examined and were related to genes involving DNA damage, stress, cell proliferation, and alteration in metabolic enzymes [2]. The gene expression profiles have catalogued the molecular responses to acute Dimethylnitrosamine toxicity and revealed the genetic basis of hepatic toxicity [3]. Nature has always been explored by mankind since ancient times and plants have always been explored in search of new drugs [4]. Plants secondary metabolites have medicinal values and show therapeutic potential like anti-viral, antiinflammatory and immune-modulatory effects on hepatocytes proved to be crucial in chronic hepatitis [5]. Trigonella Foenum-Graecum (Trigonelline) has antioxidative, anti-lipidemic, anti-fibrotic, anti-inflammatory, membrane stabilizing, immunomodulatory and liver regenerating properties [6]. Trigonelline, which is the bioactive secondary metabolite of Trigonella Foenum protected completely against significant increase in the membrane ratios of cholesterol [7], phospholipids and sphingomyelin, phosphatidylcholine in rats with carbon tetrachloride induced cirrhosis [8]. Andrographolide, the major component of Andrographis Lineata has been reported to possess the strong anti-inflammatory activity [9]. and is a lactone diterpene that gives the plant a bitter taste [10]. Enormous literature has focused on the anti-viral, immunomodulatory, hepatoprotective and anticancer potential of andrographolide [11]. The purpose of this study was first to analyze the anti-inflammatory potential of Andrographis lineata and Trigonella Foenum along with their bioactive secondary metabolites Andrographolide and Trigonelline through its pharmacokinetic parameters [12].

MATERIALS AND METHODS
All chemicals and reagents used were of analytical grades and were purchased from

Antioxidative and Phytochemical Analysis of Plant Extracts
Antioxidative and scavenging activity of Andrographis Lineata and Trigonella Foenum was analyzed by DPHH assay [13]. Various phytochemicals tannins, alkaloids, phenols, flavonoids and carotenoids were also analyzed [14].

Pharmacokinetic Analysis for Trigonelline and Andrographolide
Kinetic version 5.5 was used to study the various pharmacokinetic aspects like peak serum concentration (C max ), time to reach peak concentration (T max ), elimination half life (t 1/2 ), total body clearance (CL), volume of distribution (V d ) and area under curve (AUC) were evaluated for Trigonelline and andrographolide. Various pharmacokinetic parameters including peak serum concentration (Cmax), time to reach peak concentration (Tmax) were evaluated from concentration-time data. Moreover, elimination half life (t1/2), total body clearance (CL), volume of distribution (Vd) and area under curve (AUC) were also evaluated by.non-compartmental method bypharmacokinetic software Kinetica-Version 5.0 and calculated using the trapezoidal rule.

Statistical Analysis
Results has been expressed as MEAN±SD while P-values less than 0.05 were considered as significant.

Pharmacokinetic Studies in Rats after Oral / Intragastric Administration of Trigonelline Andandrographolide
The oral administration of trigonelline and andrographolide attains a therapeutic use as an antioxidative agent. The pharmacokinetic parameters of these two respective drugs are compartmentalized and mostly dose dependent. The absorption of trigonelline administered by oral route and observed after 24 hours from rat bile is decreased to 2-3%. The Table 1 shows that half-life of trigonelline is 4.14 hours which indicates it as a short acting drug. Other parameters calculated are C max , t max , AUC, Ke, Vd and Cl. Results of all these parameters link together to show that trigonelline is rich in antioxidative phytochemicals i-e phenols and their scavenging activity against hepatic injury induced by dimethylnitrosamine is higher as compared to that of ascorbic acid. Less half-life of trigonelline metabolite results in a mean residence time of 9 hours and an elimination rate constant of 0.43 hr -1 indicating a short duration of time in blood plasma. The less volume of distribution of trigonelline (1.63 L) in albino rats results in a C max of 3.7 to reach the optimum therapeutic response with a good bioavailability relative to that of standard ascorbic acid and the graph shows time approaching the respective C max to be 5 hours. The area under curve evaluated by using trapezoidal rule is calculated as 217.25 with a clearance rate of 155.12. The results of all the pharmacokinetic parameters of trigonelline administered in low dose (200 mg/kg) confirmed the better antioxidative activity than that of ascorbic acid used as a potent antioxidant ( Table 1).The increased rate of absorption in case of andrographolide caused a valuable and abrupt change in the results obtained by statistical approach. The pharmacokinetic variability of andrographolide and trigonelline was found valuable in diseased state of hepatic injured rats. The dose adjustment at a t max of 5 hours (similar to that of trigonelline) to meet the effective C max is 1.9 which is approximately half the optimum dose required for trigonelline. Due to very less plasma peak concentration of andrographolide, there is a prominent change in pharmacokinetic parameters as compared to optimum dose of ascorbic acid required to reach the effective therapeutic response. As the results indicate that half-life of andrographolide is 12.54 hours therefore less dose is required to keep a constant and steady state plasma drug concentration. The results show the values of AUC and Vd as 525.001 and 2.3 respectively. Higher value of AUC in case of andrographolide indicates its longer stay in plasma which results in higher scavenging activity despite of very less Cmax. An increased value of MRT (19.58 hours) and decreased value of Ke (0.41) with a clearance rate of 205.66 provides an easy compliance for administration of a prolonged and steady state concentration of under discussed drug molecules. T. foenum-graecum absorption is very low through oral route about 2-3%, observed after 24hrs from rat bile. The excreted form of T. foenum-graecum through bile is in the form of glucuronidase conjugates and sulphates in case of human beings. Peak plasma concentration could be seen in 6 to 8hrs and elimination half-life was found to be6hrs. Pharmacokinetic analysis of T. foenum-graecum has revealed that it is eliminated through bile as metabolites because it is taken through oral route and distributed in various organs and seems to be circulated in enterohepatic system. Moreover, the complex of T. foenum-graecum and phosphatidylcholine has shown an increased bioavailability by oral route in healthy volunteers may be due to the facilitating ole of drug complex through the alimentary canal (Ghosh et al.,  2010).The data regarding plasma concentration of T. foenum-graecum versus time profile in rats is presented (Fig. 1). The results of the present pharmacokinetic studies (Table 1) shows that C max , T max , t 1/2 , AUC, MRT, Vd, C1T and Kcof T. foenum-graecumafter a single dose administration in rats was 3.3 μg ml

Secondary
Metabolites and Antioxidative Characterization of

T. foenum-graecum and A. lineata
In the first phase of the experiment the crude extract of T. foenum-graecumn and A.lineata was extracted in different solvent systems e.g., methanol, ethanol, chloroform, ethyl acetate, petroleum ether. The results (Fig. 2) shows that the maximum extraction was observed in petroleum ether (11.67%) followed by chloroform, ethanol, ethyl acetate and methanol (5.78%, 5.76%, 4.87% and5.78% respectively). Results has been expressed as MEAN±SD   Fig. 4 petroleum ether extracts of T. foenum-graecum and A. lineatahas antiradical activity by inhibiting of DPPH radical (IC 50 value of 69.04±3.65% and 71.76±6.99% respectively), as compared to ascorbic acid standard (IC 50 = 53.99±4.88%). IC 50 value is the effective concentration at which the antioxidant activity is 50%. This means these extracts were able to reduce the stable radical of DPPH to yellow colored diphenyl picrylhydrazine. It has been found that cysteine, glutathione, ascorbic acid, tocopherol, poly-hydroxy aromatic compounds (hydroquinone, pyrogallol, gallic acid, etc.) reduce and decolorize DPPH by their hydrogen donating ability. It appears that extracts of T. foenum-graecum and A. lineata possess hydrogen donating abilities to act as an antioxidant. Fig. 4 shows the highest DPPH radical scavenging effect in petroleum ether extracts of A.lineate (71.76±6.99%) and T. foenum-graecum (69.04±3.65%). The data were even superior to standard antioxidant ascorbic acid (53.99±4.88%).

CONCLUSION
The present study concluded that maximum recovery of the active compounds was obtained in petroleum ether, then in chloroform, ethanol and methanol. However, least recovery was observed with ethyl acetate. Both trigonelline and andrographolide possess therapeutic antiinflammatory and antioxidative properties at their specific dose regimen.

CONSENT
It is not applicable.