In vitro lipase inhibitory effect and kinetic properties of di-terpenoid fraction from Calotropis procera (Aiton)

doi:10.1016/j.bcab.2015.08.014

Biocatalysis and Agricultural Biotechnology

Volume 4, Issue 4, October 2015, Pages 579-585

https://doi.org/10.1016/j.bcab.2015.08.014 Get rights and content

Abstract

Calotropis procera Aiton (Asclepiadaceae) root extract was investigated as an inhibitor of pancreatic lipase (PL) and its kinetic parameters were studied in an attempt to explain its hypolipidemic activity. The purified di-terpenoid fraction showed highest inhibition with an IC₅₀ of 9.47 µg/mL in comparision with a standard inhibitor, Orlistat (IC₅₀ 0.15 µM). In the presence of purified di-terpenoid fraction, the value of apparent K_m (K_mapp) was found to be increased whereas apparent V_max (V_maxapp) remained unchanged. Inhibition constant (K_i) of purified di-terpenoid fraction was found to be significantly lower than the positive control (Orlistat). Moreover, in the presence of purified di-terpenoid fraction, substantial decrease was noticed in specificity constant (K_cat/K_m). Based on kinetic data, the inhibition was found to be of competitive type. The bioactivity was discussed in terms of bioactive metabolites present in the extracts. Our results demonstrated that the antihyperlipidaemic effects of C. procera root extract can be partly attributed to the inhibition of PL by di-terpenoids present in it.

Introduction

There is good evidence of an association between obesity and increased risk of coronary heart diseases (CHDs) in adults. Obesity plays important role in the development of the CHDs and it is a growing threat to the health of populations globally (Mohammad et al., 2013). Sedentary lifestyle and physical inactivity further contribute to obesity and doubles the risk of developing heart diseases, hyperlipidemia, atherosclerosis and type II diabetes (Birari and Bhutani, 2007).

Lipases (E.C. 3.1.1.3) are mostly utilized as biocatalysts in organic industries and thus they found extra applications in organic synthesis, they are involved in many reactions like esterification, alcoholysis, aminolysis and transformation reactions (Ghori et al., 2011). Lipases are responsible for catalysis of triglycerides to di-glycerides, mono-glycerides, glycerol and fatty acids. Increase in the triglyceride level in plasma is a major factor which contributes triglyceride absorption and ultimately increases the risks of CHDs. Moreover, dietary triglycerides do not get absorbed until they are hydrolyzed into free fatty acids by triglyceride lipases (Bustanji et al., 2011a, Bustanji et al., 2011b). The free fatty acids are directly and indirectly associated with increased level of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) in the blood, promoting cardiovascular diseases (Sharma et al., 2005). Further, lipases are known for hydrolyzing high density lipoprotein (HDL) and ultimately reduce the HDL level which is known as 'good cholesterol' (Abdel-magid, 2013). Therefore, selective inhibition of PL can decrease the level of free fatty acids and tryglycerides, and it also upregulates the formation of HDL cholesterol (Jin et al., 2003).

Orlistat, a commercially available PL inhibitor, is widely used as therapeutic agent for curing obesity (Jandacek and Woods, 2004). However, it has side effects like pain in lower back, blood in urine, oily stools, flatulence, fecal urgency, and abdominal cramps (Gupta et al., 2014).

Therefore, there is great demand for new effective, safe and selective PL inhibitors of natural origin. Natural products of plant origin have been widely used from last few decades in the treatment of various diseases worldwide. A number of Indian medicinal plants, such as Eugenia jambolana, Tinospora cardiofolia, Azadirecta indica, Trigonalla foenumgraceum, Camellia sinensis, Cassia mimosoides, Malus domestica, Coffea arabica, Cyclocarya paliurus, Dioscorea nipponica, and Eleutherococcus senticosus have been screened for lipase inhibitory activity (Sukhdev and Singh, 2013).

Plant derived metabolites such as saponins (platycodin saponins, scabio saponins, sessiloside, chiisanoside, chikusetsu saponins, dioscin, escins, tea saponins, and cyclocariosides), polyphenolics and flavones, flavonols, tannins; terpens (carnosic acid, crocin and crocetin) are reported for PL inhibitory effect (Birari and Bhutani, 2007).

Among the plant derived metabolites, terpenoids are considered to be the potent anticancer, anti-inflammatory, and anticarcinogenic compounds. Several reports are available on PL inhibitory effect of plant derived substances, but few have been conducted on terpenes. In the present study, methanol crude extract and purified di-terpenoid fraction of C. procera roots were investigated as inhibitor of PL and kinetic parameters of inhibition were studied in an attempt to explain its hypolipidemic activity. Attempts have also been made to characterize the PL inhibitors using Gas Chromatography High Resolution Mass Spectrometry (GC-HRMS).

Section snippets

Reagents and chemicals

All solvents used for the extraction and chromatographic separation are analytical grades purchased from Merck (Mumbai, India). Porcine pancreatic lipase and its substrate paranitrophenyl butyrate (pNPB) were purchased from Sigma Aldrich (St. Louis, MO, USA).

Plant material and extraction

Roots of C. procera was collected in the region of Toranmal hill station area (MS, India). For identification purpose, plant specimen in flowering condition was collected in the month of March and it was identified by expert taxonomist.

Screening of lipase inhibitory activity using chromogenic polysorbate plate assay

Inhibition of PL by qualitative plate assay using phenol red, indicated that C. procera root crude extract, purified di-terpenoid fraction and Orlistat standard showed complete inhibition of PL (Fig. 1). Due to change in pH, the diameter of yellow halo in control was observed to be 5.23±1.2 mm, whereas the yellow halo was not observed in case of C. procera root crude extract, purified di-terpenoid fraction and Orlistat standard indicating that complete inhibition of PL.

Estimation of kinetic parameters

For estimation of kinetics

Discussion

Increased level of free fatty acids and triglyceride in plasma is the major factor which contributes development of obesity and CHDs. PL inhibitor like Orlistat is a widely used drug, but has several undesirable side effects (Bustanji et al., 2011a, Bustanji et al., 2011b, Drent and Veen, 1993, Gupta et al., 2014). Therefore, there is a great need of exploring plant derived metabolites as new potent and selective PL inhibitors. Medicinal plants have been the economical source of natural

Conclusion

The results of this study clearly proved that di-terpenoids from C. procera root extract has potent PL inhibition activity. The kinetic studies show that the crude extract as well as the di-terpenoids of C. procera inhibits PL competitively. Cyclic di-terpenoids namely – phenol, 2,4 bis(1, 1-dimethylethyl) ester and 1,2 benzedenedicarboxylic acid, bis(2-methylpropyl) ester were identified as main constituents using GC-HRMS. Investigating and understanding the mechanism of enzyme inhibition

Acknowledgment

This research was supported by the University Grant Commission (UGC File no. 42-456/2013 SR) New Delhi, India.

References (22)

R.B. Birari et al.
Pancreatic lipase inhibitors from natural sources: unexplored potential
Drug. Discov. Today
(2007)
R.J. Jandacek et al.
Pharmaceutical approaches to the treatment of obesity
Drug Discov. Today
(2004)
K. Ninomiya et al.
Carnosic acid, a new class of lipid absorption inhibitor from sage
Bioorg. Med. Chem. Lett.
(2004)
V. Patil et al.
Chemistry of ayurvedic crude drugs-I guggulu (resin from cummiphora mukul) steroidal constituents
Tetrahedron
(1972)
N. Sharma et al.
Screening of some medicinal plants for anti-lipase activity
J. Ethnopharmacol.
(2005)
S. Velmurugan et al.
Antimicrobial effect of Calotropis procera active principles against aquatic microbial pathogens isolated from shrimp and fishes
Asian Pac. J. Trop. Biomed.
(2012)
T. Yang et al.
Preparation of andrographolide-loaded solid lipid nanoparticles and their in vitro and in vivo evaluations: characteristics, release, absorption, transports, pharmacokinetics, and antihyperlipidemic activity
J. Pharm. Sci.
(2013)
A.F. Abdel-magid
Endothelial lipase inhibitors for the treatment of atherosclerosis and cardiovascular disorders
ACS Med. Chem. Lett.
(2013)
M. Benguechoua et al.
Inhibition of candida rugosa lipase by different extracts of five algerian plants and their antioxidant activities
Curr. Enzym. Inhib.
(2014)
Y. Bustanji et al.
Pancreatic lipase inhibition activity of trilactone terpenes of Ginkgo biloba
J. Enzym. Inhib. Med. Chem.
(2011)

Y. Bustanji et al.

Screening of some medicinal plants for their pancreatic lipase inhibitory potential

Jordan J. Pharma. Sci.

(2011)

Cited by (17)

Hydroxylated polymethoxyflavones reduce the activity of pancreatic lipase, inhibit adipogenesis and enhance lipolysis in 3T3-L1 mouse embryonic fibroblast cells
2023, Chemico-Biological Interactions
Hydroxylated polymethoxyflavones (HPMFs) have been shown to possess various anti-disease effects, including against obesity. This study investigates the anti-obesity effects of HPMFs in further detail, aiming to gain understanding of their mechanism of action in this context. The current study demonstrates that two HPMFs; 3′-hydroxy-5,7,4′,5′-tetramethoxyflavone (3′OH-TetMF) and 4′-hydroxy-5,7,3′,5′-tetramethoxyflavone (4′OH-TetMF) possess anti-obesity effects. They both significantly reduced pancreatic lipase activity in a competitive manner as demonstrated by molecular docking and kinetic studies. In cell studies, it was revealed that both of the HPMFs suppress differentiation of 3T3-L1 mouse embryonic fibroblast cells during the early stages of adipogenesis. They also reduced expression of key adipogenic and lipogenic marker genes, namely peroxisome proliferator-activated receptor-gamma (PPARγ), CCAAT/enhancer-binding protein α and β (C/EBP α and β), adipocyte binding protein 2 (aP2), fatty acid synthase (FASN), and sterol regulatory element-binding protein 1 (SREBF 1). They also enhanced the expression of cell cycle genes, i.e., cyclin D1 (CCND1) and C-Myc, and reduced cyclin A2 expression. When further investigated, it was also observed that these HPMFs accelerate lipid breakdown (lipolysis) and enhance lipolytic genes expression. Moreover, they also reduced the secretion of proteins (adipokines), including pro-inflammatory cytokines, from mature adipocytes. Taken together, this study concludes that these HPMFs have anti-obesity effects, which are worthy of further investigation.
Effects of Ilex paraguariensis beverages on in vivo triglyceride and starch absorption in mice
2022, Biocatalysis and Agricultural Biotechnology
Citation Excerpt :
Prominent among the inhibitors of the pancreatic lipase is orlistat, a lipstatin, isolated from the bacterium Streptomyces toxytricini, used for long-term treatment of obesity (McClendon et al., 2009). In spite of the effectiveness of orlistat and related compounds there has been continuous search for new inhibitors of fat absorption driven basically by the hope that natural compounds could eventually provide mild antiobesity action if continuously ingested along with regular meals (Akhlaghi and Najafpour-Darzi, 2021; Patil et al., 2015; Dlamini et al., 2022; Mateos et al., 2018; Souza et al., 2015). In the context of the trend that was detailed in the preceding paragraph, yerba mate (Ilex paraguariensis), a plant whose leaves are rich in polyphenolics and xanthines, has been considered as a possible adjuvant in the treatment of obesity and diabetes (Souza et al., 2015; Correa et al., 2018).
The purpose of this work was to characterize the inhibition of fat absorption by the four most popular beverages prepared with yerba mate (Ilex paraguariensis): (a) a hot water infusion of the dry green leaves (chimarrão); (b) a room temperature aqueous infusion of dry green leaves (tererê); (c) a hot water infusion of previously roasted leaves (mate tea); (d) a variant of the latter form, called instant or soluble mate tea. Triglyceride and starch tolerance were measured in vivo using mice and complemented by in vitro enzymatic assays and in silico simulations. All extracts diminished triglyceride absorption in vivo to similar extents at the doses of 250 mg/kg. No such effect was detected for starch absorption. The pancreatic lipase was strongly inhibited by the hot- and room temperature-water extracts of the green dried leaves but less strongly by mate tea and instant mate. Among the substances that can be found in the extracts, docking simulations revealed that the strongest inhibitors of the pancreatic lipase were isorhamnetin-3-O-rutinoside > kaempferol-3-O-rutinoside > 3,4-O-dicafeoylquinic acid > quercetin-3-O-rutinoside > 3,5-O-dicaffeoylquinic acid. Considering the amounts in the various extracts, however, the most important inhibitors were the mono- and di-caffeoylquinic acids. The inhibitory activity on triglyceride absorption is a property that fits well into the general and widespread notion that yerba mate can help to reduce weight gain and to stabilize the overall lipid metabolism at a health beneficial level.
Inhibitory kinetics and bioactivities of Nuciferine and Methyl Ganoderate on Mucor miehei lipase and 3T3-L1 preadipocytes
2020, International Journal of Biological Macromolecules
Citation Excerpt :
Studies have shown that flavonoids, alkaloids, and phenolic acids in lotus leaves can inhibit lipase activity and exhibit weight loss effects [5]. Ganoderma Lucidum is one of the sources of traditional Chinese herbal medicine, which has been used for centuries to improve blood pressure, reduce blood fat and resistance, and facilitate oxidation and immune regulation [6]. Methyl Ganoderate, as a kind of Ganoderma triterpenes, has an acetylcholinesterase inhibitory effect in previous reports from Lee et al. [7].
In this study, inhibitory kinetics of Nuciferine and Methyl Ganoderate extrated from Lotus Leaves and Ganoderma lucidum on Mucor miehei Lipase were studied first. The molecular structure of Nuciferine and Methyl Ganoderate were determined. The inhibitory effects of two extracts on lipase were reversible, with the IC₅₀ values of 0.194 and 0.332 mg/mL, respectively. The inhibition kinetic analysis by Lineweaver-Burk plots showed that they were a mixed-type inhibitor of lipase, with inhibition constants K_I of 0.16 and 0.29 mg/mL, and K_IS of 0.36 and 0.49 mg/mL, respectively. Results of spectral analysis showed that the UV absorption and the molecule fluorescence spectrum of the lipase hydrolyzate were significantly decreased after the inhibitor was added. The molecular docking further suggested that the interaction site between the active substance and inhibitor was located in an α-helix and a β-sheet of the lipase, and the lipase active site was interfered by the inhibitor near the cap structure. In addition, the proliferation and differentiation of 3 T3-L1 preadipocytes were inhibited by two extracts. Total triglycerides and cholesterol were significantly reduced in the cells. The results confirmed that Nuciferine and Methyl Ganoderate can be used as potential obesity treatment drugs.
Lipase Inhibitors for Obesity: A Review
2020, Biomedicine and Pharmacotherapy
Citation Excerpt :
Studies have found that flavonoids and saponins not only have lipase inhibition but also α-glucosidase inhibitory activity, which can prevent hyperglycaemia, alleviate hyperinsulinaemia, increase glucose tolerance, and prevent and treat obesity. Ercan [100] used a static in vitro digestion method to determine the total saponin content [101] and in vitro bio-affinity of the tribulus and chickpea. At the same time, the in vitro inhibitory effects of lipase, α-amylase and α-glucosidase on the selected food samples were evaluated.
With the rapid increase in the population of obese individuals, obesity has become a global problem. Many kinds of chronic metabolic diseases easily caused by obesity have received increasing attention from researchers. People are also striving to find various safe and effective treatment methods as well as anti-obesity medicines. Pancreatic lipase (PL) inhibitors have received substantial attention from researchers in recent years, and PL inhibitors from natural products have attracted much attention due to their structural diversity, low toxicity and wide range of sources. They have been used in the intestinal tract, blood, and the central nervous system with no side effects, and these advantages could lead to a new generation of diet pills or health care products with great development potential. This article is mainly aimed at discussing the research of obesity drug treatment with PL inhibitors and offers a brief review of related properties and the use of PL inhibitors in the field of weight loss.
Natural anti-obesity agents and their therapeutic role in management of obesity: A future trend perspective
2019, Biomedicine and Pharmacotherapy
In the present scenario, obesity is a challenging health problem and its prevalence along with comorbidities are on the rise around the world. According to world health organization and organisation for economic co-operation and development epidemiology reports, overweight and obesity are the fifth foremost causes of deaths globally. The increasing rate of obesity is becoming a mammoth problem which enormously affects an individual’s quality of life. The conventional therapy of obesity mainly involves synthetic moieties and surgical procedures, which has many harmful side effects and chances of recurrence with severity. Hence, the Present review is a metanalysis of all the available data on the use of the plants with their biological source, active phytochemical constituents and a probable mechanism of action as natural anti-obesity agents. The metanalysis of data during the period of 2000–2018 was performed with the help of scientific data search engine National Center for Biotechnology Information (NCBI/PubMed). This data reveals the need and scope of further research in the development of new natural phytoconstituents for the management of obesity.
Antilipase activity guided fractionation of Vinca major
2018, Journal of King Saud University - Science
Citation Excerpt :
Therefore, a safe pancreatic lipase inhibitor is still highly sought therapeutic molecule in the drug/pharmaceutical market, and plant derived natural products are suited option for the same. Several plants have been reported in literature for their lipase inhibitory effects, viz. Platycodon grandiflorus (Zhao et al., 2005), Cyclocarya paliurus (Kurihara et al., 2003), Dioscorea nipponica (Kwon et al., 2003), Scabiosa tschiliensis (Zheng and Koike, 2004), Panax japonicas (Han et al., 2005), Aesculus turbinate (Kimura et al., 2006), Acanthopanax sessiliflorus (Yoshizumi et al., 2008), Glycyrrhiza auralensis (Won et al., 2007), Peganum harmala, Inonotus hispidus (Benarous et al., 2015), Calatropis procera (Patil et al., 2015), Malus domestica, Vitaceae vitis (Danis et al., 2015) etc. Our laboratory is engaged in discovering the lipase inhibitory natural products from the Western Himalayan flora (Shimla, India).
Methanolic extract of flowers of Vinca major (VFE) reduced the activity of porcine pancreatic lipase (PPL) which was reported in our previous study. To identify the antilipase molecule(s) in the VFE, the antilipase activity-guided fractionation for the purification of lipase inhibitory molecule(s) by Silica Gel and Sephadex G-50 column chromatography was attempted, and the final anti-lipase fraction was examined by ESI-MS and FT-IR spectroscopy. Two molecules namely, Majoridine and Akuammine were elucidated to be present in the flowers of V. major which possessed high PPL inhibition activity. Further, both molecules were looked in silico for their interactions with PPL and other important proteins of lipid metabolism using molecular docking studies. Majoridine and Akuammine demonstrated significant binding interactions with active site residues of PPL.

View all citing articles on Scopus

View full text

In vitro lipase inhibitory effect and kinetic properties of di-terpenoid fraction from Calotropis procera (Aiton)

Abstract

Introduction

Section snippets

Reagents and chemicals

Plant material and extraction

Screening of lipase inhibitory activity using chromogenic polysorbate plate assay

Estimation of kinetic parameters

Discussion

Conclusion

Acknowledgment

Drug. Discov. Today

Drug Discov. Today

Bioorg. Med. Chem. Lett.

Tetrahedron

J. Ethnopharmacol.

Asian Pac. J. Trop. Biomed.

J. Pharm. Sci.

Endothelial lipase inhibitors for the treatment of atherosclerosis and cardiovascular disorders

ACS Med. Chem. Lett.

Inhibition of candida rugosa lipase by different extracts of five algerian plants and their antioxidant activities

Curr. Enzym. Inhib.

Pancreatic lipase inhibition activity of trilactone terpenes of Ginkgo biloba

J. Enzym. Inhib. Med. Chem.

Screening of some medicinal plants for their pancreatic lipase inhibitory potential

Jordan J. Pharma. Sci.