Background
Liver, a vital organ (organ of metabolism and excretion) in the human body, plays an astounding array of functions vital for the maintenance as well as performance of the body. Unfortunately, liver is exposed to a variety of xenobiotics, chemotherapeutic agents, drug-drug interactions and environmental pollutants which weaken and damage the liver leading to hazardous liver diseases such as Hepatitis, Cirrhosis and Cancer etc. [
1]. LDs and their complications are often linked with imbalance between the production of free radicals (ROS) and body’s antioxidant defense mechanism that result into increased oxidative stress. These ROS have an important role in the etiology of LDs andthe antioxidant therapy is expected to impart beneficial effects in treating these. Liver disease (LD), a multi-factorial disease remains one of the most serious health problems and millions of people world-wide are suffering from one form or the other. High cost of treatment and adverse effects are the disadvantages associated with synthetic drugs when used for prolonged periods [
2]. Therefore, it is logical to think of herbal remedies for the treatment of LD. Silymarin, a known hepatoprotective drug has well defined hepatoprotective, free radical scavenging and antioxidant properties, that improves the antioxidant defense by preventing the glutathione depletion as well as antifibrotic activity. It has been investigated through in-vitro and in-vivo experimental studies by Radko and Cybulski 2007 [
3]. Although, clinical trials suggested the safety of silymarin at higher doses (up to 1500 mg/day) in humans, but the pharmacokinetic studies have revealed poor absorption, rapid metabolism and excretion in bile and urine and all these ultimately results in poor oral bioavailability of silymarin [
4].
Generally, all pharmacokinetic parameters of silymarin are referred to, and standardized as, silybin. According to Wu et al. 2007, silymarin (silybin), when administered orally, is rapidly absorbed with a T
max (2–4 h) and t
1/2 (6 h). Due to extensive enterohepatic circulation, only 20–50% of oral silymarin is absorbed from the gastrointestinal tract and 0.73% oral bioavailability of silymarin (silybin) in rat plasma was reported [
5,
6].
Silybum marianum (milk thistle, family: Asteraceae), is one of the oldest and thoroughly researched plants of ancient times used in the treatment of liver and gall bladder disorders, including jaundice, cirrhosis and hepatitis and Silymarin is the active constituent of this plant which is a 70–80% standardized extract consisting of silymarin flavonolignans (silybin A & B, isosilybin A & B, silydianin, and silychristin) and flavonoids (taxifolin and quercetin), and the remaining 20–30% consists of chemically undefined fraction comprising polymeric and oxidized polyphenolic compounds [
7]. There are as many as 75 brands of silymarin available in market in different dosage forms such as tablets, capsules, syrups, etc. Some of the important brands are Legalon capsules, Carsil Tablets and Alrin-B syrup etc. An array of methods are available in the literature that can improve the bioavailability of silymarin like formation of microparticles, nanoparticles, self- emulsifying drug delivery systems, phytosomes, liposomes and micelles as summarized by Javed et al. 2012 [
7]. But they suffer from disadvantages of using a large amount of surfactants, co-surfactants, exogenous compounds as these cause irritation to patients suffering from gastric disorders and ulcers and thus leading to abdominal discomfort [
8]. The concept of using the bioenhancers to increase the drug bioavailability is one of the newest approaches. The discovery of first bioenhancer piperine in 1979 by scientists in RRL, Jammu, India introduced a new concept in science [
9]. Non-toxicity, effectiveness at low concentrations, easy to formulate with the drug, enhanced uptake and absorption of drug and lastly, synergizing the activity of the drug are the advantages associated with the bioenhancers. Bioenhancers increase in the bioavailability of nutraceuticals by acting through several mechanisms, which include acting on gastrointestinal tract to enhance absorption, or by altering the drug metabolism process [
10].
In our study, three natural products as bioenhancers were selected based on their mechanism of action: first, Fulvic acid (FA) – a water soluble carrier for increasing the solubility of silymarin by complex formation [
11], second piperine (P) – a known inhibitor of hepatic and intestinal glucoronidation inhibitor [
12] and third, lysergol (L) – a permeability enhancer of drugs across intestinal epithelial cells for better absorption and efficacy [
13]. All the formulations no. 1–5 were subjected to accelerated stability studies as per ICH guidelines Q1A. The carbon tetrachloride induced hepatotoxicity study in rats was performed to evaluate the effect of silymarin alone and with bioenhancers in all the tablet formulations. Previously, researchers from all over the world have demonstrated the hepatoprotective activity of silymarin against various toxic models and partial hepatectomy models in experimental animals by using chemical toxins such as carbon tetrachloride (CCl
4), acetaminophen, D-galactosamine, ethanol, and
Amanita phalloides toxin [
14]. In cellular events that modulate hepatotoxicity, CCl
4 is metabolized by CYP450 enzymes in liver endoplasmic reticulum in reactive trichloromethyl free radicals which in turn react with oxygen and form trichloromethylperoxy radicals. These radicals attack lipids on endoplasmic reticulum of liver cells and leads to elevation of liver enzymes and ultimately cell death. CCl
4 interferes with the transport function of the liver cells, leading to leakage of SGOT and SGPT from the cell cytoplasm into the serum, thereby increasing their levels in serum and reduces the capacity of liver to synthesize albumin, leading to decreased serum levels [
15].
Recent studies conducted in the past decade have shown the hepatoprotective potential of silymarin against CCl
4 induced liver injury. Silymarin and garlic oil were reported as highly promising compounds in protecting the hepatic tissue against oxidative damage and preventing hepatic dysfunction due to CCl
4 induced hepatotoxicity in rats [
16]. In another study, the restoration of the CCl
4–induced hepatic fibrosis was reported due to high doses of silymarin in rats [
17]. The biochemical parameters returned to normal values in CCl
4 intoxicated rats after treating with silymarin and/or ginger for one month [
18]. A significant reduction in enzyme levels in silymarin lipid microspheres treated group was reported by Abrol et al. 2005 when compared to toxic control, normal control (plain lipid microspheres) as well as groups treated with silymarin solution [
19]. In another study conducted by El-Samaligy et al. 2006, silymarin hybrid liposomes produced a significant decrease in both the transaminase levels (SGOT and SGPT) when challenged with intraperitonial CCl
4 () in comparison to the orally administered silymarin suspension [
20]. Synergistic effects of silymarin and standardized extract of
Phyllanthus amarus against CCl
4 induced hepatotoxicity in rat model was also reported previously [
21].
Discussion
In recent times, several studies have been carried out to demonstrate the efficacy of herbal drugs and nutraceuticals in LDs and most of these studies showed significant hepatoprotectivity with lesser side effects and good efficacy [
33].
By now, it is well understood that silymarin has significant antioxidant and hepatoprotective potential if it is bioavailable [
34]. The only limitation in its use is its poor bioavailability that leads to higher daily doses in order to observe some of its pharmacological activity. If by some approach, the bioavailability increases, it would lead to lesser amount and frequency of dosing and better pharmacological activity of silymarin. The aim of our work was to use natural products as bioenhancers along with silymarin in order to increase its bioavailability either by increasing its water solubility, increasing its permeability or by inhibiting its metabolism. Silymarin can also modify the plasma membrane phospholipid content therefore, protects against the CCl
4 induced alterations of the liver plasma membrane through its antioxidant properties [
35].
Our systematic study brought up the results that if the bioavailability of silymarin is increased with the help of bioenhancers like FA and P, together these three compounds may act as strong antioxidants and provide synergistic and additive hepatoprotective effects. So we suggest, a formulation with good anti–inflammatory and antioxidant potential and is anticipated to show good hepatoprotective activity if used properly. We hypothesized that silymarin along with FA and P in a definite concentration in a pharmaceutical dosage form would provide much better hepatoprotection because of two reasons: Firstly, with their bioenhancing effects on silymarin and secondly, together with silymarin they proved to be a good antioxidant combination which is important for the protection against the injury caused by CCl4.
The results obtained from the present study indicated that SGOT, SGPT, ALKP and Total Bilirubin levels were markedly increased in toxic group after CCl4 treatment as compared to the normal group signifying the induction of liver injury in rats (p < 0.05). Silymarin along with bioenhancers ameliorated the hepatotoxic effect of CCl4 and exhibited significant hepatoprotective activity against CCl4-induced liver injury in the following order: silymarin- FA- P formulation, > silymarin – FA > silymarin – P > silymarin – Lformulation by normalizing the elevated levels of hepatic enzymes when compared to plain silymarin formulation. A novel treatment of LDs by the use of a strong antioxidant silymarin in combination with FA and P in a tablet dosage form is anticipated. To further prove this point and idea, the safety as well as the efficacy must be evaluated in pre-clinical and clinical studies.