Investigating Anti-Obesity Potential of Papaver Somniferum Seeds in Obese Rat Model

Summary

The study investigates the anti-obesity efficacy of Papaver somniferum seeds in obesity-induced Albino rats. Washed poppy seeds effectively reduced weight, lowered glucose levels, and improved lipid profiles without toxicity. In contrast, unwashed seeds altered blood parameters, suggesting potential toxicity and the need for further research.

Abstract

Obesity is a major global health issue, affecting nearly 30% of the population worldwide. Despite the prevalence of obesity, there is currently no data available on the anti-obesity and metabolic effects of Papaver somniferum. The objective of the study was to confirm the anti-obesity and metabolic effects of Papaver somniferum (poppy) seeds in high-fat diet (HFD)-induced obese rats, assessing their impact on weight reduction, lipid profile, and organ toxicity. The experiment was conducted in two phases: a 4 week poppy seed intervention and a 6 week obesity induction trial. Rats were separated into groups and given both washed or unwashed poppy seeds, HFD, and a prescription medication for weight loss. The findings demonstrated that washing poppy seeds significantly decreased weight gain and enhanced lipid profiles, particularly reducing triglycerides, low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL). Additionally, treated groups showed a decrease in glucose levels. However, higher doses of unwashed poppy seeds caused modest liver stress, indicated by raised alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and renal histopathology showed mild inflammation, but hematological parameters were constant. These results suggest that washed poppy seeds may have the potential to reduce obesity and enhance metabolic health without adverse harm, indicating the need for further research to explore their therapeutic potential.

Introduction

Obesity is a medical condition characterized by an excessively high body fat percentage despite a body mass index (BMI) of less than 20^1,2. When the body's controlled system fails to maintain the proper balance between nutrients and energy in the body's regulatory system, it results in the accumulation of excess body fat deposited in the body³. The BMI is the primary diagnostic tool used to determine normal weight, overweight, and obesity status. It is commonly used in clinical research to identify individuals with excess weight or obesity. Research indicates that obesity is a disease in and of itself rather than a major cause of any chronic disease⁴. It is primarily caused by the consumption of excessive calorie-containing food and a sedentary lifestyle⁵. Genetic weight-gaining mechanisms and prolonged and excessive exposure to food high in energy may also contribute to obesity. The global prevalence of obesity has increased rapidly in recent years, with an estimated 2.1 billion people globally, or 30% of the world's population, suffering from obesity and being overweight. This ratio is predicted to reach 40% by 2030 if the current trends continue to rise. Environmental factors like ecosystem and social issues also influence the development of obesity^6,7. Obesity has been linked to several forms of cancer, including uterine, breast, and colon cancer, as well as comorbidities such as dyslipidemia, diabetes, and musculoskeletal disorders (especially osteoarthritis). Furthermore, obesity is also associated with cardiac risk factors, including hyperglycemia and a high body mass index. The most well-known factors that contribute to obesity include hormonal problems and compulsive eating⁸. While traditional treatments for obesity have not been extensively studied, they generally pose minimal risks. On the other hand, western medications often come with costly side effects that can pose significant health risks. Therefore, an alternate approach to creating safe, efficient anti-obesity medications can be to investigate natural products against obesity. The advancement of phytochemical studies supports the traditional use of therapeutic herbs⁹, which may be used as an alternative therapy for obesity. Several scientific investigations have shown the effectiveness of herbal medicines in treating obesity for centuries. Earlier studies have demonstrated that medicinal herbs, which contain a variety of pharmacological components, are consumed as food¹⁰. There has been discussion about the interest in employing natural herbs as medications. Using these herbs has been associated with very few negative consequences¹¹; these plants can improve digestion and accelerate weight reduction¹². Medicinal plants employ a multifaceted approach to address obesity, encompassing five primary mechanisms: appetite suppression, stimulation of thermogenesis and lipid metabolism, inhibition of pancreatic lipase activity, prevention of pathogenesis, and promotion of lipolysis¹³. Furthermore, natural herbs often contain bioactive compounds that act as digestive enzyme inhibitors, thereby hindering the hydrolysis and absorption of dietary carbohydrates and fats¹⁴.

Papaver somniferum, commonly known as the opium poppy or Khashkhash in the subcontinent, is a globally recognized traditional plant with a rich history of use. Various phytochemicals, including alkaloids such as morphine, codeine, noscapine, papaverine, and thebaine, have been isolated from opium¹⁵. While the poppy plant is primarily associated with its psychoactive and analgesic properties, its seeds are increasingly recognized for their potential health benefits. Poppy seeds are a rich source of polyunsaturated fatty acids, particularly omega-3 fatty acids, which have been linked to weight management¹⁶. Notably, poppy seeds contain α-linolenic acid, an omega-3 fatty acid whose anti-obesity potential has garnered considerable attention. The 10 and 12 isomers of α-linolenic acid have been specifically implicated in weight loss. Numerous human studies have demonstrated that supplementation with a combination of these isomers can decrease body fat percentage¹⁷. The primary objective of this investigation was to evaluate the effects of P. somniferum on weight reduction in an animal model. Additionally, the study aimed to assess its impact on lipid profile, hematological parameters, kidney and liver function, and adipose tissue histology.

Protocol

All procedures were conducted after the ethical committee of the University of Lahore Pakistan approved them in the meeting held on 21-04-2021 with Registration No: REG. # EPZOOL02193026

1. Housing of animals

1. House 35 male Wistar albino rats (3 weeks old) individually in standard laboratory cages. Provide ad libitum access to rodent chow and water throughout the experiment.
2. Maintain the vivarium environment at a constant temperature of 22 ± 1 °C and a relative humidity of 50% ± 10%. Implement a 12 h:12 h light-dark cycle using artificial illumination.

2. Grouping of animals

1. At the start of the experiment, divide animals into 2 groups: obese and non-obese. Provide the obese group with a high-fat diet in addition to the normal diet. Feed the non-obese group a normal diet and use it as the control group (control negative) in the experiment.

3. Preparation of high-fat diet (HFD)

1. Formulate a high-fat diet (HFD) by increasing the proportion of fats derived from both plant and animal sources. Combine the HFD with the standard chow to prepare pellets.
2. Set the fat content in the HFD at 4 g per 30 g of feed. Place the pellets in the animal cages for 24 h. Weigh and record any remaining pellet after 24 h, then remove it. Provide fresh pellets each day. Maintain this HFD feeding regimen for 6 weeks to induce obesity.
3. Monitor the body weight of the animals and record every week to check if it is increasing or not. To measure the body weight, put the jar on the weight machine and cover it. Add the rat inside the jar to check the weight.
4. Supplement the same pellets with poppy seeds for the remaining 4 weeks to investigate their potential weight-loss effects.

4. Grouping of obese animals

1. After 6 weeks, further divide obese animals into four groups: Group 1 is the obese control (control positive), which continued feeding on HFD but was not provided with any treatment. Group 2 (Standard) is treated with commercially available medicine (see Table of Materials) for obesity control. Group 3 (unwashed) and Group 4 (Washed) are fed with washed and unwashed poppy seeds, respectively.

5. Preparation of poppy seeds

1. Obtain poppy seeds (P. somniferum) from a local commercial supplier as it is easily available in stores. Sun-dry the seeds for a few hours. Divide the seeds into two groups: unwashed and washed.
2. Wash the washed group poppy seeds 7x with distilled water, followed by sun drying. Add 500 g of poppy seed to a 1 L beaker, add water and mix with hands. Then discard the water and repeat the procedure 7x to make sure to remove all the impurities, dust, and other particles from the seeds. After the washing, place the seeds in sunlight to dry.

6. Calculation of dosage and preparation of feed

1. Determine the dosage based on the animal's feed intake, standardized at 30 g per individual. Administer poppy seeds at a concentration of 0.5 g per 30 g of feed.
2. Incorporate the poppy seeds into the feed by mixing them with distilled water to form a pellet. Prepare the pellets fresh daily throughout the experimental period of 4 weeks.

7. Dissection of rats and blood collection

1. Fast the rats for 24 h. Sacrifice the animal by using chloroform and collect the blood and organ samples for further procedures as described below.
2. Anesthetize the rats using chloroform. Administer chloroform by a trained lab technician at the rate of 1% inhaled chloroform (0.05 mL/L) and ensure the rat before is properly anesthetized by pinching the foot of the rat.
3. Perform dissection using standard dissecting box instruments. Disinfect a sharp knife, scissors, and forceps prior to starting the dissection. During dissection remove kidney, liver, and adipose tissue samples. Preserve the excised samples in formalin-filled centrifuge tubes.
4. Collect blood samples in EDTA-coated vials. Extract 12 mL of blood from each animal and then further divide 3 mL each to test kidney function, liver functioning test, and lipid profiling.
5. Centrifuge the collected blood samples at 1,957 x g for 5 min. Separate blood serum from the rest as a clear or yellowish liquid. Separate the serum and aliquot it into micro centrifuge tubes for further biochemical analyses.

8. Sample analysis

1. Determine the total lipid profile, including total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides, and VLDL, using standard enzymatic colorimetric methods¹⁸. Base the determination of HDL cholesterol on a time-consuming precipitation procedure¹⁸.
2. Monitor rat glucose levels regularly during the whole experiment using a blood glucose monitor GL-110¹⁹.

9. Evaluation of toxicity

1. Assess the toxicological impact of Papaver somniferum on the parameters ALT, AST, ALP, TP, and bilirubin using liver function tests²⁰. Measure serum creatinine levels following the methodology outlined in²¹.
2. Determine hematological parameters, including red blood cell (RBC) and white blood cell (WBC) counts, using a Neubauer hemocytometer. Assess hemoglobin concentration using the cyanmethemoglobin method.
3. Conduct a histopathological examination of the collected tissue samples. Stain sections with hematoxylin and eosin (H&E) for microscopic evaluation.

10. Statistical analysis

1. Use SPSS software version 16 for statistical analysis. Apply an independent t-test to the first month's data for the obesity induction trial.
2. Utilize repeated measures ANOVA with LSD post-hoc test for the second phase. Employ one-way ANOVA with LSD to analyze blood parameters for toxicity assessment
3. Define statistical significance as p ≤ 0.05, with p ≤ 0.001 considered highly significant.

Results

Rats with an initial body weight of 40-45 g (weaning stage) were selected for the 70 day experiment, which was divided into two phases. The first phase, lasting 6 weeks, involved inducing obesity in the experimental group by supplementing their standard chow with a high-fat diet (HFD; Figure 1). In the subsequent 4 week phase, the obese rats were administered Papaver somniferum. Body weight was measured at the beginning and end of the experiment. Repeated measures A...

Discussion

The protocol has the following critical steps to be taken care of. The first critical step was to induce obesity in the animals by using High fat diet. The second step was to monitor animals which are on high fat diet that they do not develop diabetes or any other disease due to HFD.

The protocol also has the following limitations. Due to academic restrictions, the experimental time to study the exact effect of poppy seeds was only 10 weeks. A more detailed study of poppy seeds with different ...

Materials

Name	Company	Catalog Number	Comments
Blood glucose monitor GL-110	Certeza	GL-110	A portable device used to measure blood glucose levels.
Centrifuge	Eppendorf	22620005	A laboratory equipment that spins samples at high speeds to separate components based on density.
EDTA-coated vials	BD Vacutainer	367861	Tubes used for collecting blood samples, preventing clotting by binding calcium ions.
Eppendorf tubes	Eppendorf	0030 120.094	Small plastic tubes used for storing and handling small volumes of liquid.
Falcon tubes	Corning	352059	Conical tubes used for centrifugation and various laboratory applications.
Microscope	Olympus	CX23RF100	An optical instrument used for viewing small objects, typically magnified through lenses.
Neubauer hemocytometer	Hawksley	H.H1	A specialized slide used for counting cells under a microscope.
Orlistat120 mg	Windlas Biotech Ltd
Pipette tips	Eppendorf	0030 073.435	Disposable tips used with pipettes for transferring liquids accurately.
Serological pipettes	Falcon	357551	Graduated pipettes used for transferring liquids in larger volumes.
SPSS software version 16	IBM	N/A (software, not a physical product)	A statistical software package used for data analysis.
Standard laboratory cage (Super Mouse 750)	Lab Products, Inc.	10021	A cage designed to house laboratory mice, providing a controlled environment.