Comparative study on inhibitory effects of ginsenosides on human pancreatic lipase and porcine pancreatic lipase: structure-activity relationships and inhibitory mechanism

Abstract The inhibitory effects of twenty-six ginsenosides on human pancreatic lipase (hPL) and porcine pancreatic lipase (pPL) were studied. Study reveals that nine ginsenosides have moderate inhibitory effects against hPL, and good selectivity over pPL. By contrast, (S)-Rh2 showed good inhibitory effects on pPL over hPL. SAR analysis indicated that introduction of the O-glycosyl group(s) at C-3/C-7 site is unbeneficial for hPL inhibition, ginsenosides with A-skeleton is more beneficial than ginsenosides with B-/C-skeleton. Inhibition kinetic analysis indicated that Rg3 and (S)-Rh2 inhibited hPL-catalyzed DDAO-ol hydrolysis in a mixed manner. Molecular docking studies have confirmed that Rg3 and (S)-Rh2 inhibit hPL via many Pi-hydrogen interactions and hydrogen bonds with catalytic residues of hPL. These results indicated that pPL as an enzyme source could not fully represent the inhibitory effect of the tested compounds on hPL, and hPL should be used as far as possible to evaluate the inhibitory effect of PL. Graphical Abstract


Introduction
For thousands of years, metabolic diseases including obesity, arteriosclerosis, and type II diabetes have been severe and global public health problems in several countries, which are inseparable from the lack of exercise of modern people and the increased intake of high-sugar and high-fat (Motamed et al. 2018).Obesity epidemic of the globe continues, affecting more than 2 billion people (Caballero 2019).Studies have shown that the significant risk factors for the frequent occurrence of metabolic diseases are the increase of fatty acid, cholesterol and other esters (Petrie et al. 2018).Accordingly, it is an urgent research direction to find effective drugs to regulate lipid metabolism around the critical target of lipid hydrolysis for treatment of metabolic diseases such as fatty liver disease, obesity and hyperlipidemia.
An essential part of the hydrolysis and absorption of dietary lipids is played by human pancreatic lipase.Studies have shown that inhibition of PL activity can lead to impaired absorption of fat hydrolysate in food and contribute to the adjuvant treatment of obesity, insulin resistance and hypertension (Liu et al. 2019;Jin et al. 2022).hPL has been identified as an essential enzyme targeted for the therapy of obesity (Bessesen and Van Gaal 2018).Since more than 20 years ago, orlistat is an irreversible and potent mammalian PLs (hPL and pPL) inhibitor that has been licensed for therapy of obesity.Nevertheless, the available PL inhibitor has been reported with various adverse effects (diarrhea, high carcinogenic risk, and pancreatic damage) after long-term medication (Bessesen and Van Gaal 2018).Various bioactive composites from Chinese medicinal herbs have been found to use as PLs inhibitors and thereby prevent excess dietary glycerides intake (Liu et al. 2018;Hou et al. 2020).Over the past years, porcine pancreatic lipase (pPL) has been broadly utilised as the enzyme source to screen for PL inhibitors and leading to the discovery of a large number of pPL inhibitors (Hou et al. 2020;Zhang et al. 2021;Zhang et al. 2022).However, due to the existence of species between human and porcine, the amino acid sequence identity of human pancreatic lipase (hPL) and porcine pancreatic lipase (pPL) is 86%, which may lead to the difference of inhibitor response between hPL and pPL (Point et al. 2012).
Radix ginseng Meyer, the root or rhizome of Panax ginseng, contains many chemical components, such as saponins, polysaccharides, peptides, fatty acids, amino acids, polyacetylenes, etc (Liu et al. 2022).As the main effective substance of ginseng (about 3%~6%), ginsenoside is a triterpene saponin including dammarane-type tetracyclic triterpenoid saponins and oleanane-type pentacyclic triterpenoid saponins (Chen et al. 2019).Studies have shown that ginsenosides could also lower blood lipids.Ginsenoside Rg1 can reduce body weight and fat storage, and effectively regulate the level of serum leptin, neuropeptide Y and cholecystokinin in the hyperlipidemia model rats (Gao et al. 2020).In addition, ginsenosides have been reported the inhibitory effect on PL (Li and Ji 2017).However, most of the reported inhibitory activities of ginsenosides are based on the screening method of pPL.Natural ginsenosides' inhibitory effects against hPL have not been systematically studied.The inhibitory effects of a series of natural ginsenosides against hPL were evaluated using fluorescence-based biochemical assays in this work.Furthermore, a comparative study on the inhibitory effect of ginsenosides on hPL and pPL was carried out.
7.80 µM, 2.89 µM, respectively (Table 1).Meanwhile, IC 50 values of CK and Rh3 were also determined as 8.74 µM and 12.72 µM, respectively.These results suggest that ginsenosides have different inhibitory effects on pPL from hPL.It was found that some of these natural ginsenosides have the moderate inhibitory effects on hPL, and good selectivity over pPL, including Rb1, Rb2, Rb3, Rc, Rd, Rg3, F2, Rh4 and F4.By contrast, (S)-Rh2 showed good inhibitory effects on pPL over hPL.These results indicated that pPL as an enzyme source could not fully represent the inhibitory effect of the tested compounds on human PL, and hPL should be used as far as possible to assess PL inhibition.

Inhibition kinetic analyses of ginsenosides on hPL
Rg3 and (S)-Rh2 were utilized to further study the inhibition kinetic and inhibition constants (K i ) on hPL in order to better understand the inhibitory mechanism of ginsenoside-type hPL inhibitors.The experiments for Rg3 and (S)-Rh2's time-dependent inhibition were then performed (Figure S4).Rg3 and (S)-Rh2, two ginsenosides which potently inhibit hPL, were not time-dependent inhibitors, which suggested that Rg3 and (S)-Rh2 were reversible hPL inhibitors.Subsequently, the inhibition kinetics of DDAO-ol hydrolysis catalyzed by hPL with Rg3 and (S)-Rh2 was investigated.The results showed that Rg3 and (S)-Rh2 effectively inhibited hPL in a mixed manner (Figure S5), with K i values of 1.37 µM and 1.50 µM, respectively (Figure 1) (Sun et al. 2019) (Table 2).

Docking simulations
Molecular docking simulations were used in order to get insight into the interaction mode between the inhibitors (Rg3 and (S)-Rh2)) and target (hPL) (Hou et al. 2022;Ma et al. 2022).The binding models between Rg3, (S)-Rh2 and hPL were investigated firstly.As shown in Figure S6, both Rg3 and (S)-Rh2 could dock well into the catalytic cavity of hPL.Notably, Phe215, a crucial residue above the catalytic pocket of hPL, may generate a strong and powerful hydrogen bonding contact with these ligands (Figure S6A).Such erroneous conformational alterations may be devastating to hPL's hydrolytic activity.Besides Pi-hydrogen interaction between Phe215 and Rg3(Figure S6B) and the sidechain acceptor bonding between Ser152 and (S)-Rh2 (Figure S6F), Rg3 formed Pi-hydrogen interaction with Phe215, as depicted in Figure S6B, while (S)-Rh2 formed backbone donor bonding interactions with Ala259, as depicted in Figure S6H.Moreover, in terms of scoring alone, Rg3 is also more favorable to combining with hPL than (S)-Rh2.As shown in Figure S6C and S6H, Rg3 is better encapsulated by enzyme activity pockets than (S)-Rh2.These results, which were compatible with the inhibition types findings that Rg3 and (S)-Rh2 potently suppress hPL activity in a mixed manner, suggested that Rg3 and (S)-Rh2 could bind on hPL in an allosteric approach firmly.

Experimental
The materials and methods adopted for the study have been described in the Supplementary Material.

Conclusion
As we all know, hPL is one of the most important enzymes involved in the digestion of dietary lipids, such as triacylglycerols, in the intestine.However, long-term treatment with the hPL inhibitor orlistat, which has been licensed for the treatment of obesity for over 20 years, has been associated with various adverse effects, including pancreatic damage, diarrhea, and a high risk of cancer (Bessesen and Van Gaal 2018).Over the past few thousand years, Panax ginseng has demonstrated its medicinal and health-promoting properties, including its potential for weight loss and fat reduction (Liu et al. 2021).However, little is known about the inhibitory potential of ginsenosides, the active components of Panax ginseng, against hPL.This study identified that among all the ginsenosides tested, ginsenoside Rg3 exhibited the most potent anti-hPL effect, with an IC 50 value of 2.47 µM.Six major ginsenosides, including Rh3, F2, CK, Rb3, Rg3, and (S)-Rh2, were identified as important constituents responsible for hPL inhibition.In vitro systems showed time-dependent and concentration-dependent features in the elimination of Rg3 and the generation of Rh2.This suggests that the in vitro metabolic system is effective for the metabolism of glycosides, as demonstrated by the considerable decrease in Rg3 and the generation of Rh2 (Zhao et al. 2014).Therefore, local exposure of Rg3 in the gastrointestinal tract can reach 12.7 µM, which is much higher than its IC 50 values (around 2.47 µM).Moreover, although Rg1 displays weak inhibitory effects against hPL, its content is the higher of these, reaching 3.15 mg/g (Li et al. 2018).Further studies revealed that ginsenosides have different inhibitory effects on pPL from hPL.SAR analysis of these ginsenosides suggested that introduction of the O-glycosyl group(s) at C-3 site or C-7 site is unbeneficial for hPL inhibition, ginsenosides with A-skeleton is more beneficial for hPL inhibition than ginsenosides with B-skeleton and C-skeleton.Collectively, our findings reveal that ginsenosides are one of the vital anti-obesity constituents in Panax ginseng and clarify the inhibitory mechanisms on hPL.These findings provide compelling proof for the anti-obesity and reducing lipids effects of ginsenosides and suggest that Panax ginseng could be developed as a dietary supplement to prevent and treat obesity.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was financially supported by the Shanghai Three-year Action Plan to Further Accelerate the Development of Traditional Chinese Medicine [ZY(2021[ZY( -2023))-0103] and the NSF of China [81973393].
5. By analyzing these compounds, e.g., CK, Rh4 and Panaxadiol containing hydroxyl group at C-3 and C-13 site and hydrogen at C-7 site, PPT, F1 and Panaxatriol with hydroxyl group at C-3, C-7 and C-13 site, we could conclude that ginsenosides with A-skeleton are more beneficial for hPL inhibition than ginsenosides with B-skeleton and C-skeleton, and ginsenosides with B-skeleton are usually better than C-skeleton.

Table 1 .
the inhibitory efects of natural ginsenosides against hPl and pPl.

Table 2 .
the inhibitory effects of (s)-rh2 and rg3 towards hPl and pPl.