Heat stability of the in vitro inhibitory effect of spices on lipase, amylase, and glucosidase enzymes

Abstract This study investigated the effect of boiling on the inhibitory action of spices on digestive enzymes. Unboiled extracts of Trigonella foenum‐graecum (seed) (25.42%), Myristica fragrans (seed) (22.70%), and Cuminum cyminum (seed) (19.17%) showed significantly (p < 0.05) a higher lipase inhibitory activity than their respective boiled extracts (20.23%, 15.74%, and 12.57%). Unboiled extracts of Cinnamomum zeylanicum (stem bark) (−16.98%) and Foeniculum officinale (seed) (−16.05%) showed an activation of lipase enzyme, and boiling significantly (p < 0.05) changed the activity into lipase inhibition as 8.47% and 9.54%, respectively. Unboiled extracts of Coriandrm sativum (seed), C. cyminum, and Elettaria cardamomum (seed) showed an activation of amylase enzyme, and boiling these extracts significantly reduced the enzyme activation. The other unboiled extracts showed a higher amylase inhibition than the boiled extracts, whereas the boiled extracts of C.longa (rhizome) and Syzygium aromaticum (flower) exhibited significantly (p < 0.05) lower values. Unboiled extracts of C. zeylanicum, M. fragrans, and S. aromaticum showed an insignificantly higher glucosidase inhibitory activity than the boiled extracts. Inhibition of digestive enzymes by nutritional intervention is one avenue to be considered in treating diet‐induced obesity and in the management of postprandial hyperglycemia. Spices, used as food additives, could be a potential source of digestive enzyme inhibitors. The current study revealed that unboiled extracts of T. foenum‐graecum (seed), C. cyminum (seed), and M. fragrans (seed) are more effective than the boiled extracts as an antiobesity therapy. Moreover, it endorses the use of infusion of T. foenum‐graecum seeds as an antiobesity therapy.

garlic, and ginger have been used in Asia and the Middle East for many centuries. Sri Lanka is well known for a variety of spices such as cinnamon, pepper, cloves, cardamoms, nutmeg, mace, and vanilla that grow in abundance all over the island in different types of soil and climatic conditions. These spices are an important part of the agricultural exports of Sri Lanka.
Pancreatic lipase, which hydrolyzes triglycerides into glycerol and fatty acids, is the key enzyme for dietary fat digestion in the small intestine (Mukherjee & Sengupta, 2013). An inhibitor of pancreatic lipase can reduce fat absorption and can be used as a therapeutic agent for treating diet-induced obesity in humans. Orlistat is the only clinically approved pharmacological agent used as a pancreatic lipase inhibitor (Weibel, Hadvary, Houchuli, Kupfer, & Lengsfeld, 1987).
Inhibition of carbohydrate hydrolyzing enzymes such as αglucosidase and pancreatic α-amylase is one of the therapeutic approaches for delaying carbohydrate digestion, resulting in reduced postprandial hyperglycemia which is critical in the management of diabetes mellitus (Sudhir & Mohan, 2002). α-Amylase catalyzes the hydrolysis of α-1,4-glucosidic linkages in starch and related polysaccharides.
α-Glucosidase secreted from intestinal epithelium is responsible for the degradation of oligosaccharides, trisaccharides, and disaccharides into monosaccharides. Inhibition of amylase and glucosidase enzymes would delay carbohydrate digestion and glucose absorption and thereby reduce postprandial hyperglycemia. Drugs that target carbohydrate hydrolyzing enzymes include acarbose, miglitol, voglibose, nojirimycin, and 1-deoxynojirimycin, which reduce postprandial hyperglycemia by delaying glucose absorption (Bischoff, 1994).
The synthetic inhibitors of digestive enzymes lead to several adverse events in the gastrointestinal tract (Chiasson et al., 2002;Kaila & Raman, 2008). In this context, spices are an attractive source for the identification of newer digestive enzyme inhibitors that lack some of the adverse reactions of these synthetic enzyme inhibitors.
Spices are usually used as food additives and in many recipes worldwide "cooking, baking, and roasting were applied to the spices." Digestive enzyme inhibitors belonging to a variety of secondary metabolite groups such as polyphenols (flavonoids, phenolic acids, and proanthocyanidins) (Karamać & Amarowicz, 1996;Kim, Kwon, & Son, 2000;Shimura et al., 1994;You, Chen, Wang, Luo, & Jiang, 2011) and saponins (Zhao & Kim, 2004) have been identified, and these metabolites have been isolated in spices as well (Villupanoor, Chempakam, & Zachariah, 2008). Therefore, the heat stability of inhibitors is vital for the inhibitory action to persist after cooking (Adefegha, Oboh, Oyeleye, & Osumo, 2016). The heat stability of the lipase, amylase, and glucosidase inhibitors of spices after processing (cooking at 100°C) has not been clearly investigated. Therefore, the investigation of the effects of boiling of spices on the lipase, amylase, and glucosidase inhibitory activities is important.
Hence, this study was designed to determine the lipase inhibitory activity, amylase inhibitory activity, and glucosidase inhibitory activity of unboiled and boiled crude methanol extracts of ten popular spices in Sri Lanka. Moreover, the heat stability of antioxidants of these spices was determined. The real-time lipase inhibition assay used in this study was also optimized using the lipase assay developed by Choi et al. in 2003.

| Lipase inhibition assay
Lipase inhibition assay used during the current study was adapted from the lipase assay reported by Choi, Hwang, and Kim (2003).
In this assay, free thiol groups that are generated by the lipase hydrolysis of DMPTB reduce DTNB to create a yellow color, which is spectrophotometrically quantified. The substrate mixture con-

| Amylase inhibition assay
The amylase inhibition assay was performed using the preincubation chromogenic method adapted from Geethalakshmi, Sarada, Marimuthu, and Ramasamy (2010). The plant extract (40 μl, 20 mg/ ml in dimethylsulfoxide, DMSO), 160 μl of distilled water, was preincubated with the addition of 200 μl of the enzyme solution for 5 min at 37°C before reacting with the starch solution (400 μl) for 15 min.
The mixture (200 μL) was removed and added into a separate tube containing 100 μL 3,5-dinitrosalicylic acid (DNS) color reagent solution and placed in a 85°C water bath. After 15 min, this mixture was diluted with 900 μl distilled water and removed from the water bath.
α-Amylase activity was determined by measuring the absorbance of the mixture at 540 nm. Acarbose was used as the control/standard inhibitor.
Percentage maltose generated was calculated from the equation obtained from the maltose standard calibration curve (0%-0.2%, w/v maltose) % reaction = (mean maltose in test/mean maltose in control) × 100.

| Glucosidase inhibition assay
The inhibition of α-glucosidase activity was determined using the modified published method (Elya et al., 2012). 10 mM pNPG solution (400 μl), 400 μl PBS buffer, and 100 μl plant extract (10 mg/ml in dimethylsulfoxide, DMSO) were preincubated for 3 min at 37°C. After preincubation, 100 μl glucosidase enzyme (0.15 U/ml) was added to all and incubated for 15 min. The reaction was terminated by the addition of 2000 μl Na 2 CO 3 (200 mM). The absorbance of the mixture at 405 nm was measured. Acarbose was used as the control/standard inhibitor The percentage lipase inhibition was calculated by the following formula;

| Determination of the heat stability of the inhibitory effect
The extracts were dissolved in the corresponding buffer, and the solutions were boiled for 20 min in a boiling water bath at 100°C.
Then, the extract solutions were cooled, and volumes were adjusted to original volumes with buffer and assayed for lipase or amylase or glucosidase inhibitory activities.

| 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay
DPPH solution (1.8 ml) of 0.004% was mixed with 200 μl extract solution (10 mg/ml in methanol) to get a final concentration of 1 mg/ ml. These solution mixtures were kept in the dark for 30 min, and absorbance was measured at 517 nm. The absorbance was recorded and % scavenging activity was calculated (Tepe, Eminagaoglu, Akpulat, & Aydin, 2005). BHA (1 mg/ml) was used as the control/ standard for the assay. TA B L E 1 Percentage yield (%) of crude methanol extracts of spices The extracts were boiled in a water bath at 100°C for 20 min.
Then, the extract solutions were cooled, and volumes were adjusted to original volumes with buffer and assayed for the antioxidant activity by conducting the DPPH radical scavenging assay.

| Statistical analysis
All experiments were performed in three different sets, with each set in triplicates. Results were expressed as the mean ± SD of n experiments. The data were analyzed by repeated measurements of one-way analysis of variance (ANOVA) using Minitab version 17.
Differences between means were considered significant if p < 0.05 as denoted in applicable tables, figures, and the text.

| Yield of plant extracts
The yields of crude methanol extracts obtained from 100 g of each plant material from the spices studied are tabulated in

| Optimization of lipase inhibition assay
The lipase concentration employed in the assay was restricted to a maximum of 8U/ml due to the lack of solubility of lyophilized powder of lipase at higher concentrations. As shown in Figure

| Inhibition of lipase activity
Among the unboiled extracts tested, the crude methanol extract of Trigonella foenum-graecum was the most effective inhibitor of pancreatic lipase activity, while M. fragrans, C. cyminum, E. cardamomum, and C. sativum also showed an inhibitory activity ( Table 2). The extract from S. aromaticum did not inhibit pancreatic lipase, whereas C. longa, F. officinalis, and C. zeylanicum crude methanol extracts showed an activation of the lipase enzyme (Table 2).
All the boiled extracts exhibited a pancreatic lipase inhibitory activity (Table 2)

| Inhibition of amylase activity
All ten unboiled crude methanol extracts were subjected to the amylase inhibition assay, using the preincubation method and only six crude extracts exhibited inhibition activity (Table 3). In those extracts, the pancreatic amylase inhibitory activity was in the following order, from the highest to the lowest: S. aromaticum > C. longa > C. zeylanicum > F. officinale > B. juncea > T. foenum-graecum (Table 3). Other four unboiled extracts showed an activation of the pancreatic amylase enzyme (Table 3).
All the unboiled crude extracts which showed an amylase inhibitory activity exhibited a lower inhibitory activity in the boiled form (Table 3). Among them, the boiled extracts of S. aromaticum and C. longa showed a significantly (p < 0.05) lower inhibitory activity (Table 3). Further, the unboiled extract of C.cyminum, which exhibited an activation of amylase enzyme, inhibited the enzyme in the boiled form (Table 3). The other three boiled extracts were showing activation of the pancreatic amylase enzyme (Table 3), whereas C. sativum and E. cardamomum extracts showed a significant (p < 0.05) reduction in the enzyme activation in the boiled form.
Boiled extracts of six spices exhibited a glucosidase inhibitory activity, and those activities were not significantly (p < 0.05) lower than the unboiled extracts (Table 4). Meanwhile, the boiled extracts of B. juncea, C. sativum, C. cyminum, and F. officinale showed neither activation nor inhibition of the α-glucosidase enzyme.

| Antioxidant activity
All the ten unboiled and boiled extracts of spices had pronounced radical scavenging antioxidant activity (

| D ISCUSS I ON
Cooking represents an indispensable prerequisite in obtaining quality, palatability, and digestibility of food (Adefegha & Oboh, 2011). It is well known that thermal processing such as boiling, frying, and microwave cooking might be able to modulate the secondary metabolites present in plant materials (Kaur & Kapoor, 2001;Rohn, Buchner, Driemel, Rauser, & Kroh, 2007) and also may modulate the biological activity of plant secondary metabolites (Estbeyoglu, Ulbrich, Rehberg, Rogn, & Rambach, 2015). Studies conducted to investigate the thermal stability of secondary metabolites of spices are very scarce. In such studies, thermal processing was reported to cause reduction in flavonoid and polyphenol contents of spices based on the magnitude and extent of heat and duration of heating (Adefegha et al., 2016;Settharaksa, Jongjareonrak, Hmadhlu, Chansuwan, & Siripongvutikorn, 2012).
Despite the large number of research studies conducted to investigate inhibitory potential of spice crude extracts on lipase, amylase, and glucosidase enzymes, the current study is the first reported study revealing the heat stability of the lipase, amylase, and glucosidase inhibitors in crude spice extracts. The results of the current study revealed that unboiled spice extracts had a higher lipase and amylase inhibitory activities than boiled extracts. Major compounds present in spices such as apegenins in T. foenum-graecum (seed) (Fernando, 2016), eugenol in M. fragrans and S. aromaticum (Mnafgui et al., 2013), and cucuminoids in C. longa (Lekshmi, Arimboor, Raghu, & Menon, 2012)  Notes. 2,3-Dimercapto-1-propanol tributyrate (DMPTB) was used as the substrate, and the final concentration of the crude extracts was at 1 mg/ ml. The amount of thiol released was measured after the incubation for 6 mins at 37°C with 412 nm. Orlistat is taken as standard inhibitor. Results were presented as mean ± standard deviation, and mean was taken as the average of three readings of three different experiments. "-" indicates a promotion of pancreatic lipase activity. NA, not applicable.
a The inhibitory activity in boiled extract is significantly (p < 0.05) different to the corresponding unboiled extract.
TA B L E 3 Percentage amylase inhibitory activity of unboiled and boiled crude methanol extracts of spices  Geethalakshmi et al. (2010) was adapted, and the final concentrations of the crude extracts were 1 mg/ml. The amylase inhibition was analyzed by amount of maltose production from starch at 517 nm after incubation at 37°C. Acarbose was used as the standard inhibitor. Results were presented as mean ± standard deviation, and mean was taken as the average of three readings of three different experiments. "-" indicates a promotion of pancreatic amylase activity. NA, not applicable.
a The inhibitory activity in boiled extract is significantly (p < 0.05) different to the corresponding unboiled extract.
changes in the concentration of these secondary metabolites due to thermal processing such as boiling (Baker, Chogan, & Opara, 2013;Tomaino et al., 2005), and this might be the reason for the change in the inhibitory activities between the boiled and the unboiled extracts observed in this study. Further, flavonoids in spices exist in free and conjugated forms and may breakdown by enzyme, acid, or heat treatment (Cartea, Francisco, Soengas, & Velasco, 2011), and change in the inhibitory activities of boiled and unboiled extracts may be due to the variation of the inhibitory activities in these two forms. However, recent literature data did not show a consistent trend for the effects of thermal processing on secondary metabolite contents in spices (Baker et al., 2013;Khatun, Eguchi, Yamaguchi, Takamura, & Matoba, 2006).
This suggests that the effect of thermal processing on secondary metabolites varies in different spices and deserves further research.
Therefore, the reason for the modulation in the inhibition of lipase, amylase, and glucosidase activities as a result of boiling of the spices could not be categorically stated.
Among crude extracts, T. foenum-graecum (seed), C. cyminum (seed), and M. fragrans (seed) extracts showed lipase inhibitory activity in unboiled and boiled forms. Further, the unboiled form of these extracts showed a significantly higher inhibitory activity than the boiled form, and this suggested the extraction procedures which do not involve heating to be more effective than the extraction procedures which involve heating in extracting lipase inhibitors from these spices. Unboiled extracts of T. foenum-graecum (seed), C. cyminum (seed), and M. fragrans (seed) had more potential in inhibiting the major lipid digesting enzyme pancreatic lipase. Therefore, these extracts may have more potential as dietary therapy in controlling obesity and dyslipidemias.
In Asian countries, a drink prepared from T. foenum-graecum (seed) is consumed as an infusion or as a decoction. Therefore, considering results from the current study, the use of T. foenum-graecum (seed) extract in the form of infusion could be recommended to be more beneficial than the decoction in controlling obesity and dyslipidemias. In the past, seeds of M. fragrans were added to puddings as a flavoring agent but, at present, this practice has been discontinued in Sri Lanka. This study shows the hypolipidemic potential of M. fragrans seeds in the boiled form and endorses the need of resuscitation of the old culinary practice.
Research has revealed that stronger inhibition of α-glucosidase activity and mild inhibition of α-amylase activity of drugs/extracts could address the major drawbacks of currently used synthetic αglucosidase and α-amylase inhibitors (Bischoff, 1994 The inhibitory activity in boiled extract is significantly (p < 0.05) different to the corresponding unboiled extract.
Boiling the spices modulated the inhibitory potentials for lipase, amylase, and glucosidase enzymes that are already present in the spices. Conducting in vivo studies using unboiled and boiled extracts to prove the hypothesis developed by the in vitro studies is beneficial. Overall, the properties investigated in this study will be of value in the use of spices as antiobesity and antidiabetic agents. Shyamali Mapa Senanayake of English Teaching Unit, Faculty of Medicine, University of Peradeniya, Sri Lanka, are highly appreciated.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

E TH I C A L A PPROVA L A N D CO N S E NT TO PA RTI CI PATE
Ethics approval and consent to participate is not applicable to this manuscript, since it does not report on or involve the use of any animal or human data or tissues.

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