Lipase inhibitory activity of constituents of Physalis glutinosa and Physalis latiphysa

The new labdane-type diterpenoid, physaglutinic acid (1), was isolated from the hexane extract of Physalis glutinosa. This compound is the first succinyl labdane isolated from a Physalis species. The known constituents of this plant were identified as the labdanes 12-O-acetylphysacoztomatin (3), physacoztomatin (4) and 12-epi-nicandrodiol (6). The flavonol retusin (8) was also isolated from this species. The investigation of the acetone extract of Physalis latiphysa resulted in the isolation of the known (+)- (Z)-labda-8(17),13-diene-15,16-diol (9), labdenediol (11), the epimeric mixture of physanicantriol (12) and 14 epi-physanicantriol (13), together with the sucrose esters, nicandroses B (15) and D (16). Also, a large amount of the flavonol glycoside rutin (17) was obtained from the methanol extract. The activity of eight of the isolated compounds and three of their derivatives as lipase inhibitors was determined. The mode of binding of active compounds 4, 10 and 16 was explored using molecular docking on the binding pocket of pancreatic lipase (PDB ID 1LPB).


Introduction
A revision of the literature regarding the constituents of Physalis species (family Solanaceae) reveals that the most frequently isolated metabolites from these plants are ergostane-type steroids with a δ or γ-lactone in the side chain, which are known as withanolides [1,2].However, in some species of this genus, such as P. coztomatl [3], P. sordida [4], and P. nicandroides var.attenuatta [5], labdane-type diterpenoids, as compounds 1 to 14, are some of their major constituents.Even more, despite a detailed study of P. nicandroides [6], withanolides were not detected and only a series of labdanes and sucrose esters were isolated.Now, as continuation of our investigation of Physalis species, we carried out the studies of P. glutinosa Schltdl.and P. latiphysa Waterf.that led to the isolation of labdanes, sucrose esters, and flavonoids, and in which the presence of withanolides was not detected either.It should be noted that among the compounds isolated from P. glutinosa we found the first succinyl-labdane reported from a Physalis species.This is interesting since succinyl-labdanes are present in the Araucariaceae, Leguminosae and Asteraceae families [7,8], but also in the polylabdanoid Class Ia amber or resinites [9], which were formed by polymerization of plants resins throughout millions of years, and in which the role of succinic acid is to cross-link their macromolecular structure [10].
On other hand, obesity is one of the major threats to worldwide health; it is estimated that more than one billion people in the planet are obese and this state is associated with several metabolic disorders such as diabetes mellitus, cardiovascular diseases, dyslipidemia, and cancer, among others [11].One of the therapeutical approaches to obesity consists of inhibiting pancreatic lipase, which hydrolyses triglycerides into fatty acids and glycerol, thus allowing intestinal cells to absorb dietary fats.Since orlistat is the only lipase inhibitor approved by the Food and Drug Administration (FDA), there is a need for novel drugs targeting this enzyme [12,13].As far as we know, P. glutinosa and P. latiphysa do not have a documented history of medicinal uses, but considering that several labdanes and their derivatives have shown potential to inhibit pancreatic lipase [14,15], it was decided to evaluate the in vitro lipase inhibitory activity of six of the isolated labdanes, as well as of three of their derivatives.To explore other possible types of drug candidates, a flavonoid and a sucrose ester were included in the bioassay (Fig. 1).

Results and discussion
Compound 1, isolated as a colorless viscous liquid, exhibited a broad IR band between 2800 and 3600 cm −1 , indicative of a carboxylic acid, and another band at 1731 cm −1 that was attributed to the carbonyls of the acid and/or a saturated ester.22.3; δ H 1.72).The HMBC spectrum showed the correlations of H-14 with the carbons of the oxymethine at C-12 (δ C 78.4; δ H 5.28) and of CH 3 -16.The HMBC correlation of H-12 with the carbonyl carbon of the acetyl group and that of H 2 -15 with C-1'of the succinyl group determined the position of these groups.The E-configuration of the C-13-double bond was based on the NOE effects between H 2 -15 and H 3 -16 in the NOESY spectrum (Fig. 2).Previously, it was observed that in 12-hydroxylabdanes with a vinyl C-8, the C-12-configuration affects the chemical shift of the H-9 signal, which in (12 R)-hydroxy compounds resonates at ∼δ H 2.05 and at ∼δ H 1.70 in their 12-O-acetyl derivatives, while in the (12 S)-hydroxy compounds, it appeared at ∼δ H 1.50 and at ∼δ H 1.40 in their 12-O-acetyl derivatives [5].In compound 1, this signal was observed at δ H 1.72, thus suggesting a (12 R)configuration.Supporting this assumption, a close similarity was observed between the NMR spectra of compounds 1 and 12,15-di-O-diacetylphysacoztomatin (5) [4].In fact, except for the signals of the different C-15-O-acyl groups, the NMR spectra of 1 and 5 were practically superimposable.Compound 5 was isolated from P. sordida and it was also obtained upon acetylation of physacoztomatin (4), whose structure was confirmed by X-ray analysis [3,4].In this way the structure of 1, named physaglutinic acid, was established as (12 R)-O-acetyl-15-O-succinyl-labda-7,13(E)-diene.
Regarding to the study of Physalis latiphysa Waterf., the examination of the acetone extract led to the isolation of the known compounds (+)-(Z)-labda-8(17)-13-diene-15,16diol (9) [17], labdenediol (11) [18,19], a mixture of the epimers physanicantriol (14 S) (12) and 14 epi-physanicantriol (14 R) (13) [6], and nicandroses B (15) and D (16) [20], while the flavonol glycoside rutin (17) [21] was obtained from the methanol extract (Fig. 1B).The chemical profile found in this species was very similar to the one found in P. nicandroides [6].The structural elucidation of all the known compounds isolated from both Physalis species was carried out by analysis of their physical and spectroscopic data, which were compared with those reported.In the case of compound 9, its NMR data recorded in CDCl 3 were used for comparison, however, since it is sensitive to acid, its spectra were acquired in acetone-d 6 (Table 2).In this solvent, some peculiarities such as duplicated signals for C-15 and C-16, and the integral for the proton signal (t, J = 5.5 Hz) of each of the hydroxyl groups bonded to CH 2 -15 and CH 2 -16 that corresponds to a half proton, were observed.An explanation for these facts is the existence of two different hydrogen bonds which are stabilized by acetone (Fig. 3).
Since another objective of this work was to discover new inhibitors of pancreatic lipase and considering that labdanes such as the dialdehyde (E)-labda-8(17),12-diene-15,16-dial have this activity (IC 50 14.63 μM) [14], six of the isolated labdanes were evaluated.Also, in order to explore a greater structural diversity, derivatives 7 [22], 10, and 14 [23] were prepared and included in the bioassay, together with the flavonoid 8 and the sucrose ester 16.
To gain further insight on the mode of binding of active compounds 4, 10 and 16, a molecular docking analysis was conducted.Figure 4A shows the lipase-colipase complex and the catalytic site, where dockings were simulated.As depicted in Fig. 4B, the active compounds fit in the catalytic site in a similar manner as the co-crystallized inhibitor.As expected, the docked compounds interact with at least two residues (Ser152, His263) of the catalytic triad (Ser152, Asp176, and His263).Some of these interactions are stabilized by one or two hydrogen bonds, conferring a better aliphatic ester which resembles the hydrophobic chain of the alkyl phosphonate (previously co-crystallized with lipase) [26], whose docking score was −6.79 kcal/mol.Thus, increasing the length of the chain seems to increase a Value expressed is the mean ± standard error of the mean of six independent determinations.NC Not calculated the binding affinity [27].This observation also agrees with our experimental results, as compounds 4 and 10 are lacking this moiety and are less effective lipase inhibitors.
Table 4 shows the resulting protein-ligand interactions of the docking simulations, and Fig. 5 depicts the docking poses of analyzed lipase inhibitors.Orlistat acts by binding covalently to the serine residues on the active sites of both gastric and pancreatic lipase, thus preventing them from interacting with its substrate [26].The amino acids involved in orlistat's inhibition are Ser152, Asp176, and His263.
Several sucrose esters isolated from other Solanaceae species have demonstrated a variety of biological such as antibacterial and antifungal, and others associated to ecological interactions (antifeedant, antiovipositional, insecticidal, molluscicidal, aphicidal).Noteworthy, the hypoglycemic activity of peruviosides A-F, sucrose esters isolated from Physalis peruviana, has been related to their ability to inhibit intestinal α-amylase, an enzyme responsible for the hydrolysis of carbohydrates [27,28].Furthermore, structure-activity relationships showed that the length of the C-2 fatty acid of sucrose affects the inhibitory activity.This relationship for lipase inhibitory effect needs to be evaluated for other sucrose esters, but our results suggest the potential of these natural products to act as a novel class of lipase inhibitors.
Fig. 4 Crystal structure of the lipase-colipase complex (PDB ID: 1LPB).A Lipase-colipase complex (cyan ribbons represents the lipase, magenta ribbons represent colipase moiety) with the docked molecules (yellow sticks for query molecules, blue sticks for co-crystallized inhibitor).B Detailed image of the site of interaction, showing the docked molecules into the catalytic pocket, depicted a line-surface representation (green surface indicates hydrophobic interactions, blue surface indicates polar interactions and red surface specifies exposed residues)

Conclusions
Physaglutinic acid (1) was isolated from Physalis glutinosa.This new compound is the first succinyl labdane isolated from a Physalis species.Labdane diterpenoids were the main constituents of P. glutinosa and P. latiphysa; flavonoids were also present in both species, while sucrose esters only were found in P. latiphysa.Withanolides were not detected in either of the two species, increasing to three the number of Physalis species that produce labdanes, but not withanolides.In bioassays that evaluated the ability of nine labdanes, one flavonoid, and one sucrose ester to inhibit pancreatic lipase activity, only labdanes 4 and 10 and sucrose ester 16 were active.The highest activity corresponded to 16. Docking analysis seems to support the hypothesis that the length of the ester chain in C-2 of glucose is important for the lipase inhibitory activity.C at 125 MHz) using CDCl 3 as solvent and TMS as internal standard.EIMS were recorded on a JEOL JMS-SX102 A mass spectrometer.FABMS spectra were determined on a JEOL Mstation JMS-700 mass spectrometer, using dinitrobenzyl alcohol as matrix.DARTMS and HRDARTMS were measured on a JEOL AccuTOF JMS-T100LC.Analytical thin layer chromatography (TLC) was performed on precoated Alugram® Sil G/UV 254 plates, using UV and Ce(SO 4 ) 2 /H 2 SO 4 for spot detection.Preparative TLC was carried on Macherey-Nagel Sil G-200 UV UV 254 plates (2.0 mm thickness).Vacuum-assisted column chromatography (VCC) was performed on silica gel 60 G (Macherey-Nagel), unless another stationary phase, such as Tonsil Clay Acticil 220-FF (Química Rique S. A.), is specified.
Fig. 5 Relevant interactions of compounds (cyan sticks, surface) 4 (A), 10 (B), 16 (C), and orlistat (D) with lipase protein (PDB ID 1LPB).In all cases, the interaction pocket is represented as white sticks, highlighting catalytic triad residues (Ser152, Asp176 and His263) as orange sticks.Hydrogen bonds are shown as green dotted lines

Lipase inhibition assay
A previously reported colorimetric assay based on the hydrolysis of p-nitrophenylpalmitate was conducted with modifications [24].Compounds were dissolved in a mixture of acetonitrile-ethanol (33.3:66.7) to achieve a 5 mM concentration and were kept in refrigeration (4 °C) upon use.Then, the following solutions were put in a 96-well microplate (added in order): 75 mM Tris-HCl buffer (pH 8.5), in a volume ranging from 50 to 80 μL, 10 μL of p-nitrophenylpalmitate (3 mM), test compound in a volume ranging from 1 to 30 μL, and 10 μL of type II porcine pancreatic lipase (EC 3.1.1.3;prepared at 10 mg/mL in 75 mM Tris-HCl buffer, pH 8.5).Final volume of each well was 100 μL.This mixture was incubated at room temperature for 5 min and afterwards, at 37 °C for 25 min.Then, absorbance was determined at 405 nm using a Synergy HT Multi-Mode Microplate Reader (Biotek, Winooski, VA, USA).Negative control consisted of 80 μL of buffer, 10 μL of substrate, and 10 μL of enzyme.Orlistat was used as a positive control (200 μM in ethanol).Final concentrations of evaluated compounds ranged from 0.3 to 300 μM, and then from 50 to 500 μM for compound 16.Six independent curves were constructed to obtain six replicates of each concentration.Orlistat concentrations used ranged from 0 to 200 μM.Percentage of inhibition was calculated using the following equation: Where A negative control is the mean absorbance of the wells used as negative control and A sample is the absorbance of each well.With the percentage of inhibition, the IC 50 values were calculated using a non-linear regression using GraphPad Prism 9.0 software (GraphPad Inc., US).

Molecular docking
Crystal structure of the pancreatic lipase in complex with colipase and inhibited by an alkyl phosphonate (methoxyundecylphosphinic acid) was retrieved from the Protein Data Bank under the PDB ID 1LPB (https://www.rcsb.org/structure/1LPB).Chain B was used for docking simulation since it holds the catalytic site.The chemical models of the ligands (4, 10 and 16) were built with MOE (Molecular Operating Environment, v. 2022.02) and then subjected to energy minimization with a single point procedure using the MMFF94x force field implemented in the same software.In addition, the positive control orlistat and the co-crystallized inhibitor methoxyundecylphosphinic acid were also docked as reference.The docking involves a two-step protocol; first a placement algorithm where the ligands are placed in the site of interaction (conformation and fit are evaluated) and second, a refinement method to rank the final poses.The placement method was carried out using the triangle matcher algorithm, and the refinement was achieved using an induced fit method.

Table 1
Its molecular formula was determined as C 26 H 40 O 6 by the [M + NH 4 ] + ion peak at m/z 466.31965, and by the 26 carbon signals in the 13 C NMR spectrum ( (2) presence of the free carboxylic acid was probed by the obtention of the methyl ester(2)whose NMR spectra showed the signals for the OMe group (δ C 51.8; δ H 3.69) and the high field shift of the new ester carbonyl (δ C 172.7, C-4").The remaining NMR signals of compound 1 were attributed to a labda-7,13(E)-diene with oxygenated functions at C-12 and C-15.Thus, the presence of the double bond at C-7 was deduced from the signals for CH-7 (δ C 123.0; δ H 5.41), C-8 (δ C 134.3), and CH 3 -17 (δ C 22.3; δ H 1.72), while that at C-13 was consistent with the signals for C-13 (δ C 140.4), CH-14 (δ C 125.1; δ H 5.57), CH 2 O-15 (δ C 60.9; δ H 4.65), and CH 3 -16 (δ C

Table 1 1
H and13C NMR Data of Compounds 1 and 2 (500 MHz, CDCl 3 ) Fig. 2 Key HMBC and NOESY correlations for compound 1docking score.In this study, the positive control orlistat ranks as the ligand with the highest docking score (−8.50 kcal/mol), followed by 16 (−8.35kcal/mol), 10 (−7.16 kcal/mol), and 4 (−7.02kcal/mol), in agreement with our in vitro data.A possible explanation of these results is that unlike 4 and 10, nicandrose D (16) holds a C 10

Table 2 .
Oxidation of compound 9 Pyridinium chlorochromate (99.6 mg) was added to a solution of 9 (42.5 mg) in CH 2 Cl 2 (10 mL).The suspension was stirred at rt by 30 min, then filtered through a Si gel G column with CH 2 Cl 2 as eluent to afford 34.0 mg of a residue which was purified by a VCC eluted with hexane to obtain 27.1 mg of compound 14: