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. 2019 Sep;14(3):254–259. doi: 10.26574/maedica.2019.14.3.254

Prominent Pancreatic Lipase Inhibition and Free Radical Scavenging Activity of a Myristica fragrans Ethanolic Extract in vitro. Potential Role in Obesity Treatment

Yakaiah VANGOORI 1, Anusha DAKSHINAMOORTHI 2, S KAVIMANI 3
PMCID: PMC6861708  PMID: 31798741

Abstract

Objective:The objective of the present study was to evaluate the antioxidant and lipase inhibitory potential of various extracts of Myristica fragrans (in vitro).

Material and methods:Ethanolic extracts of Myristica fragrans were studied for their free radical scavenging and lipase inhibitory potentials by using porcine lipase, PNPB and DPPH. All results were obtained by applying active formulas and calculating the percentage of inhibition.

Results:Among all extracts, Myristica fragrans ethanolic extract has shown the strongest pancreatic lipase inhibitory activity at 100 ìg/mL (66.24%), with the closest potency to tthat of the standard drug, Orlistat (81.57%). This extract has also exhibited a potent antioxidant activity. The findings of the present study clearly showed that DPPH free radical scavenging activity of MFE produced 88% inhibition at 5 mg/mL as compared to standard ascorbic acid, which was 90%.

Conclusions:Ethanolic extracts of Myristica fragrans had a marked PL inhibitory action and antioxidant effect. Therefore, based on this research evidence, they could be aternatively used for obesity treatment.


Keywords:pancreatic lipase inhibition, Orlistat, antioxidant property, Myristica fragrans, DPPH, ascorbic acid.

INTRODUCTION

Overweight and obesity are defined as abnormal or excessive fat accumulation. The fundamental cause of obesity and overweight is an energy imba lance between calories consumed and calories expended. Obesity is a chronic multi-factorial disease and one of the most important causes of morbidity and premature mortality worldwide. It has been termed as the “New world syndrome” (1). Body mass index (BMI) is a simple index of weight-for-height that is commonly used to classify overweight and obesity in adults (2). Raised BMI (> 25 for overweight and >30 for obesity) is a major risk factor for non-communicable diseases such as cardiovascular diseases (mainly heart disease and stroke), which were the lea_ ding cause of death in 2012; diabetes, musculoskeletal disorders (especially osteoarthritis – a highly disabling degenerative disease of the joints); some cancers (including endometrial, breast, ovarian, prostate, liver, gallbladder, kidney, and colon). Obesity linked hyperlipidemia can increase systemic oxidative stress (3). The risk for these non-communicable diseases increases, with increases in BMI. As per the WHO report, nearly 2.8 million people are dying every year because of overweight and obesity (4). Even in developed countries, obesity treatment is still a big challenging task due to its multiple factors (5)

Many scientific trails have been made for the treatment of obesity to evaluate new targeted mechanisms such as pancreatic lipase enzyme inhibition, inhibitory action on hunger sensory mechanism, provoking actions on energy loss, free radicals scavenging effect, suppression of adipocyte growth and stirring effect on lipid metabolism (6). In all these different innovative approaches, pancreatic lipase inhibitory effect and free radical scavenging activity evaluation methods were studied for anti obesity effect of mace.

In the last few decades, plant medication has been getting more importance in the field of herbal research, and the usage of plant derived medicine has been increasing for the treatment of chronic diseases globally. Herbal medicine interventions being prepared for the treatment of obesity and weight reduction (7). Currently, obesity treatment with herbal medicine is the best alternative treatment strategy with fewer side effects and low cost (8). Myristica fragrans (mace), which is commonly known as nutmeg, belongs to the Myristicaceae family and is an evergreen aromatic tree (9). It grows in India, South East Asia, and North Australia and Pacific islands. The seed (nutmeg) and its fleshy aril (mace) are used as spices. It contains 4% myristicin. The nutmeg spice has been recognized in Europe since the 12th century, when it was used as a condiment and fumigant (10). Medicinally, it is used to support digestion and treat rheumatism. Myristica fragrans seed is also used for diarrhoea, sore mouth and insomnia. It has an anti-obesity effect by both inhibiting pancreatic lipase (11) and exerting an antioxidant effect (12). In the present study, Myristica fragrans was selected to evaluate its potential pancreatic lipase inhibitory action and antioxidant properties (in vitro).

MATERIAL AND METHOD

Chemicals: Orlistat (Sigma Aldrich, St. Louis, USA), DPPH (Himedia). Plant materials and extract preparation: Fresh and dried mace was purchased from a wholesale grocery store for extract preparation. Authentication was done by Dr. K. Venkata Ratnam, M. Phil., PhD, Assistant Professor of Botany, Rayalaseema University, Kurnool, A.P. Dried mace was ground to a fine powder and extract was prepared by using a Soxhlet apparatus with three different solvents (petroleum ether, chloroform, and ethanol) (13).

Pancreatic lipase inhibition assay (in vitro)

Porcine pancreatic lipase (PPL) activity was measured by using p-nitrophenyl butyrate (PNPB) as a substrate. The method used for measuring pancreatic lipase activity was modified from that previously described by Kim et al (2010). Thus, a solution of 0.1 mM potassium phosphate buffer (pH 6.0) and PNPB was prepared from stock solution by acetonitrile up to 10 mL.

Porcine pancreatic lipase (PPL type II-Sigma) solution was prepared by dissolving 10 mg of the enzyme in 10 mL of buffer solution (1 mg/mL) by gentle mixing, just before use.

Varying concentrations (25, 50, and 100 ìg/mL) of positive control, Orlistat, were prepared with DMSO. The same concentrations were prepared for different extracts of Myristica fragrans (14).

Procedure description (15). Porcine pancreatic lipase activity was measured by using PNPB as a substrate; PPL stock solutions (1 mg/mL) were prepared in a 0.1 mM potassium phosphate buffer (pH 6.0) and the solutions were stored at -20 °C. To determine lipase inhibitory activity, extracts (final concentrations 25, 50, 100 ƒÊg/mL) or Orlistat (similar concentrations) as a positive control were preincubated with PPL for 1 h in a potassium phosphate buffer (0.1 mM, pH 7.2, 0.1% Tween 80) at 30 °C before assaying PPL activity. The reaction was then started by adding 0.1 µL pNPB as a substrate, all in a final volume of 100 µL. After incubation at 30 °C for 5 min, the amount of p-nitrophenol released in the reaction was measured at 405 nm using a UV-Visible spectrophotometer. The activity of the negative control was also examined with and without inhibitor. The inhibitory activity (I) was calculated according to the following formula: Inhibitory activity (I %) = 100 - [(B - b)/(A - a) x 100], where A=activity without inhibitor; a=negative control without inhibitor; B=activity with inhibitor; b=negative control with inhibitor. DMSO was used as negative control and its activity was examined too.

Antioxidant effect of Myristica fragrans (in vitro)

Natural free radical scavengers are very important in the body protective mechanism against reactive oxygen species (ROS), which are harmful products released during normal cell aerobic respiration (16). Free radicals may cause oxidative stress by oxidizing biomolecules and results in tissue damage. Atherosclerosis, cancer, emphysema, cirrhosis, obesity and arthritis are some abnormal conditions linked with oxidative stress. Natural radical scavengers in herbal products are related to three major groups: carotenoids, vitamins and phenols. Phenolic compounds are plant derived antioxidants that possess metalchelating capabilities and radical scavenging properties. Reports on the antioxidant properties of Indian mace are very limited. Hence, the present study was undertaken to investigate the antioxidant properties of different extracts of mace using various in vitro procedures (17).

DPPH assay (18)

DPPH (1, 1-Diphenyl-2-picrylhydrazyl) is a stable free radical with red colour (maximum absorbance at 517 nm); if free radicals have been scavenged, DPPH would generate yellow colour. This characteristic feature is used to evaluate free radical scavenging activity. DPPH radical reacts with an antioxidant compound that can donate hydrogen, and gets reduced. When acted upon by an antioxidant, DPPH is converted into diphenyl- picryl hydrazine. This can be identified by the conversion of purple to light yellow colour (18). DPPH, ethanol, and ascorbic acid (Std) were used as reagents.

The DPPH scavenging activity of Myristica fragrans extracts (petroleum ether, chloroform, and ethanolic extracts) were determined by an earlier reported assay method (15). Solution of DPPH in 95% ethanol (0.1 mM) was prepared and 1 mL of this solution was added to 3 mL of various concentrations of plant extract as well as to standard compound (ascorbic acid 1-5 mg). After 30 min, absorbance was measured at 517 nm. The percentage of inhibition was calculated by comparing the absorbance values of control and samples, according to the following formula: % Inhibition = [(AB-AA)/AB] x 100, where AB=absorbance of blank DPPH solution, and AA=absorbance of tested extract.

RESULTS

All results were measured by using appropriate formulas and the percentage of inhibition was calcuated.

Pancreatic lipase inhibition assay

Anti-lipase activity was investigated at a concentration of 25, 50, 100 ìg/mL for PPL inhibition. The inhibitory activities towards pancreatic lipase are reported in Table 1. Among various extracts (petroleum ether, chloroform, ethanol) of Myristica fragrans, ethanolic extract at a concentration of 100 ìg/mL significantly inhibited PPL (66.24%) in vitro activity when compared with other extracts. Treatment with the well-known anti-lipase agent Orlistat as a positive control (at final concentration 100 ìg/mL) significantly inhibited (81.57%) PPL activity. Orlistat, a hydrogenated derivative of lipstatin, is the only pancreatic lipase inhibitor currently approved for a long-term treatment of obesity. Results are shown in Figure 1. Among various extracts (petroleum ether, chloroform, and ethanol) of Myristica fragrans, the ethanolic one at a concentration of 100 ìg/mL significantly inhibited PPL (66.24%), being closer to Orlistat (81.57%).

Result of antioxidant effect of Myristica fragrans

DPPH (1, 1-Diphenyl-2-picrylhydrazyl) assay evaluates the ability of antioxidants to scavenge free radicals. The reduction in DPPH absorption indicates the capacity of the extract to scavenge free radicals. In the present study, the antioxidant activity of various Myristica fragrans extracts was determined and compared to the ascorbic acid standard. The responses to various extract concentrations are shown in Figure 2.

Among all tested extracts with concentrations ranging from 1 to 5 mg/mL, the ethanolic extract exhibited the highest radical scavenging activity (76-88%), being very close to the ascorbic acid standard (84-90%). Among the three solvent (petroleum ether, chloroform, and ethanol) extracts, ethanolic extract contained high levels of polyphenols, tannins, alkaloids, and triterphenoids. Similar results were reported for heart wood of T. paniculata (19). in vitro DPPH radical scavenging activity of MFE showed that, ethanolic extract of Myristica fragrans possessed scavenging activity very near to the standard ascorbic acid.

DISCUSSION

Pancreatic lipase is an important digestive enzyme for both metabolism and absorption of triglycerides to monoglycerides and free fatty acids. If lipase enzyme is inhibited, total cholesterol concentration is reduced in the body. This is one of the possible mechanisms for the treatment of obesity. For weight loss treatment, Orlistat is the standard product which is available as an over-the-counter drug on the market. It is an irreversible lipase inhibitor that covalently binds to Serine 152 residue of lipase (20), which causes a pronounced in vitro and in vivo inhibition of gastric and pancreatic lipases (21, 22). Despite its effectiveness for obesity treatment, it has some GIT side effects such as flatulence, liquid stools, diarrhea, oily spotting, incontinence or fecal urgency, and abdominal cramping (23). These side effects are due to its mode of action and it may not be well tolerated. Hence, it is crucial to discover novel inhibitors, derived from natural sources, particularly plants, that are not associated with such serious side effects.

In the present study, different extracts (petroleum ether, chloroform, and ethanol) of Myristica fragrans were selected to evaluate their pancreatic lipase inhibitory effect and antioxidant property when compared to the standard drug Orlistat. The highest inhibitory activity was shown by the ethanolic extract of Myristica fragrans (66.24%), which was comparable (p<0.05) to that of the reference Orlistat (81.57%), at 100 mirograms/mL against pancreatic lipase. Orlistat was used as a positive control.

The strong PL inhibition may be due to its chemical components occurring in Myristica fragrans. Based on the present study findings, the prominent pancreatic lipase inhibitory effect is explained by the presence of phenolic, flavonoid and alkaloid compounds, which is in accordance with previously reported data (24, 25). Cellular damage is caused by increased ROS production through the oxidation of membrane lipids, proteins, and DNA.

Obesity is one of the conditions which can reduce antioxidant capacity by a decreased activity of antioxidant enzymes (CAT, GPx, and GR), oxidative stress and mitochondrial dysfunction (26). Oxidative stress can be counteracted by using potent antioxidant compounds found in Myristica fragrans. Flavonoids are a group of chemicals occurring in varying amounts in Myristica fragrans, which have been shown to exert potent antioxidant activity against the superoxide radicals (27). According to the present study, the ethanolic extract of Myristica fragrans has shown an anti-lipase activity and antioxidant property, which makes it a promising candidate for obesity treatment through its capacity to reduce oxidative stress.

CONCLUSION

Based on the results obtained from our in vitro study of pancreatic lipase inhibition assay and antioxidant assay (DDPH free radical scavenging assays), we state that the ethanolic extract of Myristica fragrans has a potent PL inhibitory effect and a marked antioxidant activity. These two characteristics may be due to its phytochemical constituents such as phenols, flavonoids, alkaloids. We conclude that Myristica fragrans can be used to treat obesity by reducing oxidative stress and pancreatic lipase activity.

Acknowledgments: The author Yakaiah Vangoori expresses his gratitude to his guide, Dr. D. Anusha, and the staff of Sri Ramachandra Institute of Higher Education and Research (SRIHER), Chennai and Santhiram Medical College, Nandyal, Andhra Pradesh, for their support and guidance throughout this research work.

Conflict of interests: none declared

Financial support none declared.

TABLE 1.

TABLE 1.

Pancreatic lipase inhibitory activity of different extracts and standard drug

FIGURE 1.

FIGURE 1.

Anti-lipase activity of Myristica fragrans extracts and Orlistat

TABLE 2.

TABLE 2.

DPPH free radical scavenging activity of various Myristica fragrans extracts

FIGURE 2.

FIGURE 2.

DPPH free radical scavenging activity of various Myristica fragrans extracts

Contributor Information

Yakaiah VANGOORI, Sri Ramachandra Institute of Higher Education and Research (SRIHER)-Chennai & Santhiram Medical College, Nandyal, AP, India.

Anusha DAKSHINAMOORTHI, Sri Ramachandra Institute of Higher Education and Research (SRIHER)-Chennai, Tamilnadu, India.

S. KAVIMANI, Mother Theresa Post Graduate and Research Institute of Health Sciences, Pondicherry, India

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