Introduction

Nowadays, obesity is considered as one of the most prevalent health problems among the world population. Hyperlipidemia caused by several factors such as excessive food, lack of exercise, leads typically to different health risks like stroke, Diabetics, cancer, various metabolic syndromes and coronary heart diseases (Ankur et al., 2012; Lloyd, Jones, Hong, & Labartha, 2010). Globally hyperlipidemia causes 2.6 million death and 29.7 million disability-adjusted life years. Cardiovascular diseases (CVDs) have significantly contributed to the number of global deaths, whereas, by the year 2020, it is expected that CVDs will become 650 million were obese.

The treatment of obesity or hyperlipidemia is very challenging because of the scarcity of proper therapeutic agents and their toxic side effects (WHO, 2020). At present statin remains the first choice for antilipidemic therapy, but due to adherence to statin therapy or resistance, many patients do not reach LDL Cholesterol target levels. It is also reported that under optimum statin therapy patients with familial hypercholesterolemia which result in a high level of LDL Cholesterol. The most commonly reported side effects of statins are myalgia and myositis. At present, no convincing clinical data is indicating nonstatin drugs like omega-3 fatty acids, niacin or fibrates either as monotherapy or combination, in addition to high-intensity statins, provide extra benefit for secondary prevention of cardiovascular diseases with an acceptable margin of safety beyond the high-intensity statin derivatives (Keene, Price, Shun-Shin, & Francis, 2014; Zodda, Giammona, & Schifilliti, 2018). Generally, obesity can be managed by a controlled diet and physical exercise regimen. In recent years the use of dietary food supplements rich with fibres and anti-oxidants has become an effective strategy to prevent hyperlipidemia (Swapnil, Galib, & Pradeep, 2017). In the past few decades, there has been an increase in the screening of herbal medicine for hyperlipidemia therapy to reduce the risk of heart disease. Plenty of herbal medicine is reported in Ayurveda for the treatment of hyperlipidemia, without side effects and well-tolerated. The toxic side effects and high cost of hyperlipidemia medications also led to the invention of other alternative therapies (Parasuraman, Kumar, Kumar, & Emerson, 2010). Herbal drugs are the part of traditional Ayurveda system recently, the interest in medicinal plants increased among the people in the present study highlighted the concept of Polyherbalisam (Chatterjee, Pancholi, & Biswas, 2012).

Here drugs are collected in two different areas (Malappuram district of Kerala and Nilgiris district of Tamil Nadu) in two different seasons (summer and rainy) to compare the effectiveness of drugs. The four herbal drugs used were Gymnema (Gymnema sylvestra family-Asclepiadaceae), Amla ( Emblica officinalis Family -Euphorbiaceae), Acacia(Acacia catechu family-Leguminosae) and Curry leaves (Murraya koenigii Family-Rutaceae). Acacia bark contains polyphenols that are responsible for the antilipidemic effect and have anti-oxidant action also (Amish, Natvarlal, Amit, & Jitendra, 2011). Gymnema leaves the active constituent gymnemic acid has antiobesity action and antidiabetic effect (Kapoor, 1990; Kirtikar & Basu, 1998). Curry leaves lower LDL and increases HDL curry leaves contains carbazole alkaloids; they possess antilipidemic effect and anti-oxidant action (Dinesh, Kumar, Analava, & Manjunatha, 2010). Amla is known for its anti-oxidant and anticholesterolemic effect, its rich with polyphenols and Vit C (Biswa, Jagatkumar, Bhatt, Kovur, & Hemavathi, 2012; Lama & Saikia, 2013).In the traditional alternative system of medicine, it is common to use different plants and combined extracts as a drug of choice rather than single ones to get the benefit of a synergistic effect of polyherbal formulations (Spinella, 2002; Srivastava, Lal, & Pant, 2012).

the leading cause of death and disability world-wide (WHO, 2020). CVDs are the number one cause of death globally; it estimated that 17.9 million people died due to CVDs in 2016, representing 31% of all global death (WHO, 2020). World-wide obesity has nearly tripled since 1975. In 2016, more than 1.9 billion adults were overweight, out of this over

Many antilipidemic drugs are available in modern system of medicine. The commonly prescribed drug are statins, fibrates, bile acid sequestrants, niacin and ezetimibe etc. Statins have side effects like myalgia and myositis. Fibrates are having severe side effects like myopathy, Gastric irritation, Utricaria. Niacin causes hepatotoxicity, renal failure and atrial fibrillation. Antilipidemic drug ezetimibe exhibit side effects like arthralgia and abdominal pain (Keene et al., 2014; Zodda et al., 2018). Plenty of herbal medicines are reported in the Ayurveda literature for the treatment of hyperlipidemia without side effects and well-tolerated, The main aim of the study, is to evaluate the antilipidemic potential of this polyherbal combination which is used as a traditional medicine in Malabar area, Kerala and also we are comparing the effectiveness of formulations as the crude drugs collected from different regions and seasons. The antilipidemic activity was evaluated by in vitro anti-oxidant, In vitro antilipidemic and In vivo high diet-induced hyperlipidemia model.

Materials and Methods

Collection and authentication

Ingredients of Polyherbal formulation were collected from Malappuram dist. Kerala, and Nilgiri District of Tamilnadu, during summer (March-April) and monsoon (July-august) season and were authenticated by Dr.Sreekala, senior scientist, drug standardization division, Arya Vidhya Sala Kottakkal, Centre for medicinal plant research, Malappuram District. Kerala.

Preparation of formulation

Antilipidemic herbal drugs used were described in Table 1.

Ingredients of the formulation are Murraya koenigii leaves(from 4 to 5 years old plant), Acacia catechu bark(from 18 to 20 old tree), Emblica officinalis fruits ( from more than 15 years old tree) and leaves of Gymnema sylvestra(from more than four years old plant). All the ingredients are collected, shade dried and powdered separately and passed through 80 mesh size sieve and are mixed with equal proportion. The powdered Polyherbal formulation is stored in an airtight container.

Preliminary phytochemical screening

Preliminary phytochemical analysis of four churnas was carried out for identification of the active constituents using standard procedures (Khandelwal, 2004).

In-vitro Antilipidemic assay

Cholesterol enzymatic endpoint method

In vitro, the antilipidemic assay was carried out for four different samples by Cholestrol enzymatic endpoint method for evaluating the antilipidemic potential of churnas.

An anti-cholesterol assay is carried out by cholesterol enzymatic endpoint method as described; cholesterol was dissolved in chloroform solution at a concentration of 2.5mg/mL^–1. Then 10µl water extracts of the polyherbal formulations (PHF1, PHF2, PHF3 and PHF4) were pipetted into microtitre plate followed by addition of 2000 µl of randox reagent and 10 µl of cholesterol as a sample. 20 µl of distilled water and 2000 µl of randox reagent were used as blank. Negative control contains 20 µl of cholesterol and2000 µl of randox reagent, and standard contains 20 µl of simvastatin and 2000 µl of randox reagent. All the content incubated at for 0-30 minutes at room temperature and absorbance was taken at 500nm at UV-Visible spectrophotometer against blank. The result of the assay was calculated using the following formulae (Pant, Simaria, Varsi, Bhan, & Sibi, 2015).

$\% inhibition = \frac{Negative control – Sample }{Negative control}\times 100$

In-vitro anti-oxidant assay

DPPH radical scavenging assay

Different volumes of extracts 1.25μl - 20μl (12.5 - 200µg/ml) from a stock concentration 10mg/ml were made up to a final volume of 20µl with DMSO and 1.48ml DPPH (0.1mm) solution was added. Control without the test compound, but an equivalent amount of distilled water was taken. The reaction mixture incubated in dark condition at room temperature for 20 minutes. After 20 minutes, the absorbance of the mixture was read at 517nm. 3ml of DPPH was taken as control (Singh, Murthy, & Jayaprakasha, 2002).

$\% inhibition = \frac{Negative control – Sample }{Negative control}\times 100$

Nitric oxide radical assay

Sodium nitroprusside (5mmolL^-1)in phosphate-buffered saline pH 7.4, was mixed with different concentration of the extracts 125-2000μg /ml from a stock concentration of 10mg/ml and incubated at 25°C for 30minutes. Control without the test compound, but an equivalent amount of distilled water was taken. After 30minutes, 1.5mL of the incubated solution was removed and diluted with 1.5mL of Griess reagent (1% sulphanilamide, 2% phosphoric acid and 0.1% N-1-naphthyl ethylene diamine dihydrochloride). The absorbance of the chromophore formed was measured at 546nm, and the percentage scavenging activity was measured regarding the standard (Garret, 1964).

$\% inhibition =\frac{Control - Test }{Control}\times 100$

Pharmacological screening

The approval of the institutional animal ethics committee (IAEC), with CPCSEA Reg no-1195/Re/S/08/CPCSEA of Al Shifa College of Pharmacy, Perinthalmanna, Kerala, India was taken before experiments.

Evaluation of acute oral toxicity

In acute toxicity study, the limit test dose, i.e. 2000mg/kg body weight was used as per OECD guidelines, no apparent signs of toxicity were visible, at the dose of 2000mg/kg body weight, so one-tenth of upper limit dose were selected for the studies (OECD, 2001).

Animals

Adult male albino rats of Wistar strain weighing in between 150-160gms, aged 12 weeks were purchase from small animal breeding unit, college of veterinary animal sciences, Mannuthy, Thrissur, Kerala, India. All animals were kept in Polypropylene cages (three rats in each cage) at an ambient temperature condition of 25+ ^o C with a relative humidity of 50%-60%. 12 h light and 12 h dark cycles were maintained in the animal house till animals were acclimatized to the laboratory condition. The animals were fed with standard pellets purchased from Hindustan Lever LTD, Bangalore India with free access of water. Test groups fed with high-fat diet (41.5%lipids, 40.2% carbohydrates and 18.3%proteins (kcal)) purchased from Hindustan Lever LTD along with animal feed after one week of acclimatization. The entire experiments were designed per institutional animal ethics committee (IAEC), with CPCSEA Reg no-1195/Re/S/08/CPCSEA of Al Shifa College of Pharmacy, Perinthalmanna, Kerala, India.

Evaluation of Antilipidemic Activity (High-fat diet-induced model)

Study design-The Wister albino rats were divided into five different groups, i.e. six animals in each group.

Group 1: Served as control, provided with regular diet and administered with vehicle (1%CMC) orally.

Group 2: Served as obese control administered with vehicle (1%CMC) orally.

Group 3: Treated with formulation (200mg/kg) in 1% CMC orally via gastric intubation.

Group 4: Treated with formulation (400mg/kg) 1% CMC orally via gastric incubation.

Group 5: Treated with standard drug Atorvastatin (10mg/kg) 1% CMC orally via gastric intubation.

The animals were marked for individual identification. Groups 2-5 were fed with a high-fat diet for 1 to 4 weeks to induce hyperlipidemia.Throughout the experiment, the body weights of the animals were recorded. Polyherbal formulation with two different doses (200mg/kg, 400mg/kg) was used as test drug while Atorvastatin purchased from (Dr Reddy's Laboratories. Hyderabad) (10mg/kg) was used as standard drug. At the end of the 30^th day, the animals were used for the study of various biochemical parameters (Rahul, Vishal, Kamlesh, Kumar, & Bhutani, 2010; Shrivastava et al., 2009).

Body Weight evaluation

During the experimental period of 30 days. The mice were weighed every day using an electronic balance, and food intake was monitored in every two days.

Evaluation of biochemical parameters

After 30 days of treatments, the animals were starved for 16 hrs. After that, blood samples were collected from retinol-orbital plexus by using microcapillary tubes. Then serum was separated for the analysis of TG, TC, LDL, VLDL and HDL.

Atherogenic Index

The atherogenic index was calculated by using the formula (Singh et al., 2002).

$AI =\frac{Total Cholesterol - HDL}{HDL}$

Statistical analysis

Data were analyzed by using one way ANOVA followed by students T-test, and p<0.05 was significant. Values are expressed as Mean ±SEM, six animals in each group.

Results and Discussion

Preliminary phytochemical screening

Preliminary phytochemical analysis of four churnas was carried out result are shown in Table 2.

In vitro Antilipidemic assay-By Cholesterol enzymatic endpoint method

Polyherbal formulations were prepared and analyzed for Anti-cholesterol assay. As per the result obtained, increasing activity is observed up to 20 minutes, and a maximum % of inhibition was found at 92.66±0.57% (PHF1). Simvastatin was found as positive control and 94.33±0.57 % of inhibition observed after 20minutes. At the end of the study, the maximum% inhibition for various polyherbal formulations was found to be 92.66±0.57, 88.66±1.52, 84.33±0.57 and 85.66±1.15 for PHF1, PHF2, PHF3 and PHF4 respectively Table 3.

In-vitro anti-oxidant assay

In-vitro anti-oxidant activity is carried out to find out which of the four formulation shows maximum anti-oxidant power. The anti-oxidant activity was determined by two different methods (Nitric oxide and DPPH method). From this IC₅₀ value was calculated, it is shown that there is no marked difference in activity in the samples collected from two different regions and two different climatic conditions. The results are tabulated and shown in graphs. (Table 5; Table 4, Figure 2; Figure 1) Out of four formulations, PHF 1 shows low IC₅₀ values in DPPH and Nitric oxide methods (250.45± 0.60, 985.40±5.59), respectively (Table 7; Table 6).

DPPH method

In vivo Antilipidemic activity

Based on the results of in vitro anti-oxidant and antilipidemic study Polyherbal formulation 1

Observation of animal health and body weight

No death was observed in any group over the administration period. Compare with the control group test group exhibited an overall reduction in movement and daily activities. Concerning food intake, all rats showed a significant increase in body weight compared to the control group. The study shows that the reduction in body weight of animals for the sample formulations was as equal to that of a standard drug (Atorvastatin) and reached the same weight as that of non-treated animal control without fatty diet after the study. The results are shown in the Figure 3.

(PHF 1) selected for in vivo evaluation. High diet-induced hyperlipidemia model carried out the pharmacological analysis. There was a significant increase noticed in serum cholesterol, triglyceride, LDL, VLDL and decrease in HDL in High diet-induced control model when compared to standard groups.

After treatment with a polyherbal formulation in two different doses, i.e., 200 mg/kg, 400 mg/kg body weight. It shows a decrease in serum cholesterol, triglycerides, LDL, VLDL and increases in HDL in a dose-dependent manner. Polyherbal formulation at 400mg/kg dose shows the almost equal effect (32.18±1.17) as that of Atorvastatin(33.87±0.85), this result supports the pharmacological effect of polyherbal formulation as an antilipidemic drug Table 8. The results were graphically represented ( Figure 9; Figure 8; Figure 7; Figure 6; Figure 5; Figure 4).

Conclusions

This study was undertaken to assess in vitro Antioxidant, Antilipidemic and in vivo Antilipidemic activity of Polyherbal formulations, in comparison with its geographical and seasonal variations. In-vitro anti-oxidant evaluation by DPPH and Nitric oxide method shows that Polyherbal formulation 1(PHF-1) have maximum anti-oxidant power as well as the antilipidemic effect in vitro. By in vivo analysis, the formulation showed a marked decrease in the serum Total Cholesterol, Triglyceride, VLDL, LDL levels and increase in HDL level in diet-induced hyperlipidemia model in a dose-dependent manner. The study also highlights the herbal drugs collected in summer season may have more therapeutic activity than the herbals collected in the rainy season. This study leads to the conclusion that this Polyherbal formulation can be utilized for its Antilipidemic activity and Antiobesity action in future, this work has been extended by including more hyperlipidemia models for a meaningful and tangible conclusion.

Acknowledgment

The authors are thankful to Vellore institute of technology, India, for their support and encouragement for this work.

Conflict Of Interest

The authors declare no conflict of interest, financial or otherwise.

Funding Support

The authors declare that they have no funding support for this study.