Pharmaceutical Food Science: Search for Bio-Functional Molecules Obtained from Natural Resources to Prevent and Ameliorate Lifestyle Diseases

Abstract

In this review, our resent pharmaceutical food science research for bio-functional molecules obtained from natural resources that contribute to i) suppression of postprandial blood glucose elevation and/or improvement of glucose tolerance and ii) reduction of visceral fat accumulation and improvement of lipid metabolism were summarized. Based on studies using MONOTORI science, salacinol (1), neokotalanol (4), and trans-tiliroside (20) have been approved or notified by the Consumer Affairs Agency in Japan as functional substances in food with health claims, Food for Specified Health Use and Food with Functional Claims.

1. Introduction

Among the natural resources, various products are used for medicinal purposes, including crude drugs, traditional medicines, and medicinal plants. Some of them are used for both medicinal and edible purposes and are used as a health food (functional food) that are expected to have the tertiary function of foods, the body-modulating function, with the goal of contributing to the maintenance and improvement of people’s health through food.^1–3) Health food (functional food) includes the labels “food with health claims” or “so-called health food”; the former is allowed as a label of their functionality (function claim) in the food category by the Consumer Affairs Agency in Japan, whereas the latter is not. According to the Ministry of Health, Labour and Welfare, Japan, the foods labeled “so-called health food” are not defined by law but refer to food that is widely sold or used as food contributing to health conservation and enhancement.⁴⁾ Among the “food with health claims,” Food for Specified Health Use (FOSHU) and Food with Function Claims (FFC) are widely used for self-care/prevention and health maintenance/enhancement practices, which may contribute to reducing the burden on limited medical resources^5–12) (Fig. 1). Based on this background, we have been conducting pharmaceutical food science research^13–15) to search for bio-functional products from natural resources that contribute to the prevention of lifestyle diseases, or the improvement of their initial symptoms. This review summarizes the author’s efforts to investigate bio-functional natural products with preventive and remedial effects in the early phase of diabetes and dyslipidemia using MONOTORI science from several natural resources.

Fig. 1. Pharmaceutical Food Science: Characterization of Bio-Functional Products from Natural Resources Based on MONOTORI Science

2. Search for Bio-Functional Natural Products That Suppress Postprandial Blood Glucose Elevation and/or Improve Glucose Tolerance

Diabetes mellitus is a chronic disease that occurs when the pancreas does not produce enough insulin or when the body cannot effectively use the insulin it produces. Insulin is a hormone that regulates blood glucose levels. Hyperglycemia, also called raised blood glucose or raised blood sugar, is a common effect of uncontrolled diabetes mellitus that over time leads to serious damage to many of the body’s systems, especially the nerves and blood vessels.^16–19) According to the International Diabetes Federation Atlas Tenth Edition 2021, an estimated 537 million adults have diabetes, and this number is predicted to rise to 643 million by 2030 and 783 million by 2045. In addition, 541 million adults have impaired glucose tolerance (IGT), which places them at high risk of type 2 diabetes mellitus (T2DM).²⁰⁾ Currently available glucose-lowering agents are classified into the following categories: insulin secretagogue [sulfonylureas, and rapid-acting insulin secretagogues (i.e., glinides)]; dipeptidyl-peptidase-4 (DPP-4) inhibitors; insulin sensitizers (biguanides and thiazolidinediones); α-glucosidase inhibitors; sodium-glucose cotransporter (SGLT) 2 inhibitors; and non-insulin injectables glucagon-like peptide 1 (GLP-1) receptor agonists.²¹⁾ In addition, aldose reductase inhibitors are a class of drugs to prevent eye and nerve damage in people with diabetes.²²⁾ Among these anti-diabetic targets, this review describes our exploratory study of bio-functional molecules with α-glucosidase, DPP-4, and aldose reductase inhibitory activities.

2.1. α-Glucosidase Inhibitory Activity

α-Glucosidase is an exo-type carbohydrase widely distributed in microorganisms, plants, and animal tissues that catalyzes the release of α-glucose from the non-reducing end of substrates. The inhibition of this enzyme slows the elevation of blood sugar levels following a carbohydrate meal. It is a membrane-bound enzyme present in the epithelium of the small intestine that facilitates glucose absorption by catalyzing the hydrolytic cleavage of oligosaccharides into absorbable monosaccharides. By the inhibition of α-glucosidase in the intestine, the rate of hydrolytic cleavage of oligosaccharides is decreased and the process of carbohydrate digestion spreads to the lower part of the small intestine. The spread of the digestive process delays the overall absorption rate of glucose into the blood. This is one of the best strategies to decrease the postprandial rise in blood glucose and, in turn, helps avoid the onset of late diabetic complications.²³⁾ Acarbose, voglibose, and miglitol are the currently available α-glucosidase inhibitors approved for the prevention and/or management of T2DM.²⁴⁾

In our search for new bio-functional natural products with α-glucosidase inhibitory activity, we found that extracts from the roots and stems of Salacia reticulata Wight and related genus Salacia plants, such as S. oblonga Wall. and S. chinensis L., exhibit activity.^25–29) To characterize the active constituents, an isolation study was conducted using bioassay-guided separation to monitor α-glucosidase inhibitory activity. As a result, a new class of naturally occurring pseudo-sugars, salacinol (1) and kotalanol (2) etc., bearing a thiosugar sulfonium sulfate inner salt comprised of 1-deoxy-4-thio-D-arabinofuranosyl cation and 1-deoxy-aldosyl-3-sulfate anion, and their de-O-sulfonated analogs, neosalacinol (3) and neokotalanol (4) etc., were isolated as active constituents^25–34) (Fig. 2). The inhibitory activities against human small intestinal maltase of their sulfonium constituents (1: IC₅₀ = 4.9, Ki = 0.44 µM and 4: IC₅₀ = 3.9, Ki = 0.33 µM) are as high as those of acarbose (IC₅₀ = 15.2, Ki = 2.6 µM), voglibose (IC₅₀ = 1.3, Ki = 0.17 µM), and miglitol (IC₅₀ = 3.7, Ki = 0.57 µM), which are used clinically.^35,36) The total and high potency analog synthesis and docking studies are also being conducted to elucidate the structure–activity relationships of these candidates.^37–43) Indeed, our research group has been studying the synthesis of highly active analogs based on the structure–activity relationship studies, which will be summarized in a future issue.^34,37–43)

Fig. 2. New Class of α-Glucosidase Inhibitors, Salacinol (1), Kotalanol (2), Neosalacinol (3), and Neokotalanol (4), Obtained from Genus Salacia Plants

2.2. DPP-4 Inhibitory Activity

The serine protease DPP-4 is widely expressed in endothelial cells throughout the body and is found in a circulating soluble form.⁴⁴⁾ The incretin hormone GLP-1 is released from intestinal L-cells into the circulation in response to the ingestion of food and stimulates both insulin biosynthesis and secretion.^45,46) Apart from several other beneficial effects, GLP-1 regulates insulin in a strictly glucose-dependent manner, and inhibition of the enzyme DPP-4, which rapidly inactivates GLP-1, increases the half-life of GLP-1 and prolongs its effects. Gliptins, such as sitagliptin, vildagliptin, saxagliptin, and alogliptin, are the currently available DPP-4 inhibitors approved for T2DM.^47–50)

We found that a methanol extract of “Everlasting Flower,” the flowers of Helichrysum arenarium (L.) Moench (common names; dwarf everlasting and immortelle) was found to inhibit blood glucose elevation in sucrose-loaded mice as well as the enzymatic activity against DPP-4 (IC₅₀ = 41.2 µg/mL).^51,52) During the MONOTORI study of the extract,^53–55) we obtained new compounds having unusual structures for natural products, e.g., everlastosides A (5) and B (6), megastigman glycosides having a γ-lactone linkage between C-5 and C-12,⁵⁴⁾ and arenariumoside VI (7) and related analogs (arenariumosides V and VII), dimeric dihydrocalcone glycosides⁵¹⁾ (Fig. 3). Our detailed chemical study of the flowers of H. arenarium has been summarized in a previous review.⁵²⁾ As for the active constituents with DPP-4 inhibitory activity, a chalcone glycoside, chalconaringenin 2′-O-β-D-glucopyranoside (8, IC₅₀ = 23.1 µM), was found to be the most potent isolate.⁵¹⁾

Fig. 3. Isolates from Helichrysum arenarium (L.) Moench

2.3. Aldose Reductase Inhibitory Activity

Aldose reductase is the first enzyme in the polyol pathway that catalyzes the reduction of the aldehyde form of D-glucose to D-sorbitol with the concomitant conversion of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide phosphate (NADP⁺). The polyol pathway plays a significant role in the development of degenerative complications in diabetes such as neuropathy, nephropathy, retinopathy, cataract formation, and cardiovascular diseases. In diabetes mellitus, caused by abnormal glucose metabolism and elevated blood glucose levels, a significant flux of glucose through the polyol pathway is induced in tissues such as the nerves, retina, lens, and kidneys. Because sorbitol does not readily diffuse across cell membranes and accumulates intracellularly, it has been implicated in the chronic complications of diabetes. These findings suggest that aldose reductase inhibitors prevent the conversion of glucose to sorbitol and may prevent and/or treat diabetic complications.^56–60)

During our investigation of aldose reductase inhibitors obtained from natural resources, we identified several flavonoids,^{13,28,61–67)} quinic acid derivatives,⁶⁶⁾ terpenoids,⁶⁸⁾ stilbenoids,⁶⁹⁾ and phenylethanoids^70,71) as active compounds. The structural requirements of flavonoids and related compounds have been suggested and summarized in our previous reports.^13,63) The active phenylethanoids isolated from the stems of Cistanche tubulosa (Schenk) Wight have as major constituents acteoside (9, IC₅₀ = 1.2 µM) and echinacoside (10, 3.1 µM), which show relatively potent inhibitory activity (Fig. 4). Among the isolates, 2′-acetylacteoside (11, 0.071 µM) was the most potent inhibitor, and its inhibitory activity is equivalent to that of a clinically used inhibitor, epalrestat (0.072 µM).^70,71)

Fig. 4. Phenylethanoids with Aldose Reductase Inhibitory Activity Obtained from Cistanche tubulosa (Schenk) Wight

2.4. Suppression of Postprandial Blood Glucose Level Elevation and Amelioration of IGT

In diabetes, the postprandial phase is characterized by a rapid and substantial increase in blood glucose levels, and the possibility that these postprandial hyperglycemic spikes may be relevant to the pathophysiology of late diabetic complications has recently received much attention. Therefore, improving postprandial hyperglycemia may form part of the strategy for the prevention and management of cardiovascular disease in diabetes.⁷²⁾ In addition, the development of T2DM can be prevented or delayed in individuals with IGT by implementing lifestyle changes or through the use of therapeutic agents.¹⁷⁾ Indeed, α-glucosidase inhibitor acarbose can be used either as an alternative or in addition to lifestyle changes to delay the development of T2DM in people with IGT,⁷³⁾ and voglibose has been approved in Japan.⁷⁴⁾ As described above, thiosugar sulfoniums with potent α-glucosidase inhibitor activity obtained from Salacia plants, suppressive effects of salacinol (1) and neokotalanol (4) as well as the related isolates, neosalacinol (2) and kotalanol (3), on postprandial blood glucose level elevation using in vivo maltose-loaded mice model were found⁴¹⁾ (Table 1). These thiosugar sulfoniums have unique structures with remarkable α-glucosidase inhibitory activities and are promising candidates for a new class of antidiabetic agents in the future.

Table 1. Suppressive Effects of the Sulfonium Constituents Obtained from Genus Salacia Plants (1–4) on Blood Glucose Level Elevation in Maltose-loaded Mice

Treatment	Dose (mg/kg, p.o.)	N	Blood glucose (mg/dL)a)
			0 min	15 min	30 min	60 min	120 min	180 min
Normal	—	4	104.6 ± 3.5	98.3 ± 6.2##	108.5 ± 5.3##	103.3 ± 5.1##	99.8 ± 6.3	89.3 ± 6.4
Control	—	6	96.3 ± 5.4	170.2 ± 6.9	198.2 ± 8.5	162.7 ± 9.9	115.8 ± 7.5	109.0 ± 8.1
Salacinol (1)	0.3	6	95.0 ± 2.5	150.2 ± 9.3	177.7 ± 5.7	152.3 ± 6.8	126.7 ± 4.5	100.7 ± 6.6
	1	6	82.8 ± 6.1	127.5 ± 9.1**	145.0 ± 10.3**	143.5 ± 7.5	124.8 ± 11.4	107.7 ± 7.0
	3	6	88.2 ± 3.6	125.8 ± 8.6**	136.0 ± 10.0**	138.2 ± 5.0	111.6 ± 6.1	96.7 ± 2.9
Neosalacinol (3)	0.3	6	93.7 ± 3.6	171.4 ± 12.1	183.1 ± 13.2	166.2 ± 14.1	112.3 ± 8.9	89.8 ± 6.3
	1	6	93.2 ± 6.3	142.7 ± 7.5	156.0 ± 7.9*	143.5 ± 5.1	110.2 ± 4.0	102.8 ± 4.1
	3	6	84.8 ± 5.9	132.2 ± 9.1*	141.3 ± 14.2**	147.9 ± 8.2	116.7 ± 3.1	103.7 ± 7.9
Normal	—	4	82.0 ± 4.5	104.6 ± 10.5##	111.3 ± 11.0##	105.5 ± 10.0	90.5 ± 7.5	88.3 ± 4.4
Control	—	8	79.6 ± 3.5	179.5 ± 8.1	195.5 ± 7.8	137.9 ± 11.6	103.1 ± 4.2	91.1 ± 2.7
Kotalanol (2)	0.3	6	73.2 ± 2.7	150.7 ± 13.0	147.2 ± 6.7**	125.8 ± 9.4	106.5 ± 3.1	93.5 ± 5.6
	1	6	83.8 ± 3.8	141.7 ± 6.4*	135.2 ± 8.0**	121.7 ± 8.6	93.1 ± 7.7	85.4 ± 9.1
	3	6	83.7 ± 3.9	124.2 ± 2.7**	127.8 ± 3.8**	102.0 ± 6.9	101.2 ± 3.7	78.0 ± 6.6
Neokotalanol (4)	0.3	6	81.8 ± 6.8	152.9 ± 10.1	160.3 ± 6.4**	145.0 ± 8.7	101.3 ± 4.4	84.7 ± 8.9
	1	6	75.7 ± 7.8	116.0 ± 7.3**	123.0 ± 9.6**	101.7 ± 14.1	97.4 ± 6.9	74.8 ± 9.3
	3	6	80.7 ± 4.8	111.7 ± 6.3**	113.5 ± 5.0**	101.3 ± 5.7*	91.7 ± 3.6	77.0 ± 6.1
Normal	—	4	92.5 ± 11.2	109.0 ± 18.8##	126.3 ± 20.2##	115.3 ± 10.6##	99.3 ± 11.7	88.3 ± 8.4
Control	—	8	87.5 ± 2.3	193.3 ± 9.1**	209.9 ± 18.9	157.3 ± 11.7	107.4 ± 2.7	96.6 ± 2.5
Acarbose	3	6	83.2 ± 4.2	159.7 ± 6.3*	187.7 ± 11.3	163.5 ± 8.1	113.2 ± 7.1	99.2 ± 5.2
	10	6	90.7 ± 6.5	129.3 ± 4.1**	165.2 ± 5.1*	150.5 ± 4.8	113.3 ± 4.7	98.8 ± 5.6
	30	6	85.5 ± 3.2	116.2 ± 7.8**	138.8 ± 6.5**	145.2 ± 6.3	109.0 ± 6.7	96.7 ± 4.0
Normal	—	4	78.1 ± 11.8	101.3 ± 7.1##	94.3 ± 9.4##	94.5 ± 8.6##	86.3 ± 8.2	85.8 ± 3.6
Control	—	8	75.6 ± 7.5	187.3 ± 5.1	178.6 ± 9.7	150.4 ± 8.2	98.9 ± 4.4	91.8 ± 6.3
Voglibose	0.03	6	77.0 ± 6.2	174.0 ± 9.1	183.0 ± 9.4	152.2 ± 7.1	101.2 ± 3.9	94.5 ± 6.2
	0.1	6	73.3 ± 5.6	153.8 ± 7.7*	161.0 ± 3.9	144.2 ± 6.1	97.3 ± 4.6	86.2 ± 2.5
	0.3	6	81.7 ± 7.8	129.7 ± 9.7**	126.8 ± 9.3**	128.7 ± 9.3	101.0 ± 4.3	87.8 ± 6.7

Animals. Male ddY mice (6-weeks-old) were purchased from Kiwa Laboratory Animal Co., Ltd., Wakayama, Japan. The animals were housed at a constant temperature of 23 ± 2 °C, and were then fed a standard laboratory chow (MF, Oriental Yeast Co., Ltd., Tokyo, Japan). The animals were fasted for 20–24 h prior to the beginning of the experiment, but were allowed free access to tap water. All of the experiments were performed on conscious mice unless otherwise noted. The experimental protocol was approved by the Experimental Animal Research Committee at Kindai University. Effects on blood glucose levels in maltose-loaded mice. The experiments were performed according to the method as described in our previous reports with a slight modification.⁴¹⁾ After fasting, the mice were orally administrated a 10% (w/v) maltose solution (1 g/kg) with or without a test sample. At 0, 15, 30, 60, 120, and 180 min after the administration, blood samples were taken from the tail vein and immediately subjected to the measurement of blood glucose using the glucose oxidase method. As a baseline, distilled water was administrated to rats as a normal group. An intestinal α-glucosidase inhibitors, acarbose and voglibose, were used as reference compounds. a) Each value represents the mean ± standard error of the mean (S.E.M.). Asterisks (sharps) denote significant differences from control at * p < 0.05, **^(##) p < 0.01.

To develop Salacia-containing products, which contribute to the regulation of postprandial blood glucose elevation, to be approved as FOSHU or notified as FFC, we evaluated their quality,^{32,33,36,75–78)} efficacy, and safety, including clinical studies of Salacia extracts containing these potent α-glucosidase inhibitors (1 and/or 4), as functional substances.^{35,36,79–82)} To date, several Salacia-containing products are marketed as FOSHU and FFC in Japan; the findings mentioned above are summarized in another review article.³⁶⁾

The major phenylethanoids obtained from C. tubulosa (vide ante), acteoside (9), and echinacoside (10), also significantly suppress postprandial blood glucose level elevation in starch (1 g/kg)-loaded mice at doses of 250–500 mg/kg, p.o. The effects on glucose tolerance of 9 and 10 in the same starch-loaded mice are significantly improved without any changes in body weight gain or food intake after 2 weeks of continuous administration at doses of 125 or 250 mg/kg/d p.o.^71,72) (Fig. 5). We further focused on the regulatory mechanism of dietary glucose absorption of 9 and 10, and the effects on sodium-dependent glucose cotransporter (SGLT) 1-mediated gastrointestinal glucose absorption were examined. Using human intestinal Caco-2 cells and 2-deoxy-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG), the effects of 9 (IC₅₀ = 7.0 ± 1.5 µM) and 10 (7.8 ± 1.2 µM) on sodium-dependent 2-NBDG uptake via SGLT1 were found to inhibit than that of positive agent phlorizin (163.0 ± 1.7 µM).^72,83)

Fig. 5. Effects of Two-Week Administration of Phenylethanoids (9 and 10) on Blood Glucose Levels in Starch-Loaded Mice

Each value represents the mean ± S.E.M. (n = 5–9). Significantly different from the control: * p < 0.05, ** p < 0.01. Animals. Male ddY mice (10 w) were purchased from Kiwa Laboratory Animal Co., Ltd., Wakayama, Japan. The animals were housed at a constant temperature of 23 ± 2 °C, and were then fed a standard laboratory chow (MF, Oriental Yeast Co., Ltd., Tokyo, Japan). The animals were fasted for 20–24 h prior to the beginning of the experiment, but were allowed free access to tap water. All of the experiments were performed with conscious mice unless otherwise noted. The experimental protocol was approved by the Experimental Animal Research Committee at Kindai University. Effects on glucose tolerance after 2 weeks administration in starch-loaded mice. Each test sample was administered to mice once a day (10:00–12:00) for 2 weeks. Body weight was measured every day before administration of the test sample. After fasting for 20 h, α-starch (1 g/kg) solution was orally administered to the mice at 20 mL/kg. Through the same procedure, blood samples (approx. 0.1 mL) were collected from the infraorbital venous plexus under ether anesthesia 0.5, 1, and 2 h after the oral administration, and blood samples were taken from the tail vein and immediately subjected to the measurement of blood glucose using the glucose oxidase method. Reproduced with permission from J. Nat. Med., 68, 561–566. Copyright [2014]. Springer.

As further exploratory research on bio-functional natural products with suppressive effects on postprandial blood glucose level elevation, we investigated the following compounds: triterpenoid saponins obtained from Kochia scoparia (L.) Schrad. [syn. Bassia scoparia (L.) A. J. Scott] [e.g., momordin Ic (12)],^84,85) steroid saponins from Borassus flabellifer L. [e.g., dioscin (13)],⁸⁶⁾ steroidal alkaloid saponins from Solanum lycocarpum A. St.-Hil. [e.g., solamargine (14) and solasonine (15)],⁸⁷⁾ flavonoids from Sinocrassula indica A. Berger,⁸⁸⁾ oligostilbenoids from Shorea roxburghii G. Don [e.g., (−)-hopeaphenol (16), hemsleyanol D (17), (+)-α-viniferin (18), and (−)-balanocarpol (19)],⁶⁹⁾ and azasugars from Bombycis Feces and Bombyx Batryticatus [e.g., 1-deoxynojirimycin]⁸⁹⁾ (Fig. 6). The mode of action underlying the blood glucose level elevation inhibitory activity of the triterpenoid saponins in sugar-loaded animal models, including their gastric emptying inhibitory and gastrointestinal transit accelerative effects, have been summarized in a recent review.⁸⁵⁾

Fig. 6. Bio-Functional Natural Products with Suppressive Effects on Postprandial Blood Glucose Level Elevation

3. Search for Bio-Functional Natural Products That Reduce Visceral Fat Accumulation and Improve Lipid Metabolism

The global prevalence of obesity and its associated metabolic complications has increased significantly in recent years. It increases the risk of developing various pathological conditions, such as insulin resistance, T2DM, dyslipidemia, hypertension, and non-alcoholic fatty liver disease. Abdominal obesity is a major potential risk factor for metabolic syndrome.^90,91) Hyperlipidemia is defined as elevated blood lipid levels, including triglycerides and cholesterol. It significantly contributes to the manifestation and development of cardiovascular diseases, including atherosclerosis, which is one of the most common causes of mortality and morbidity worldwide. Various classes of lipid-lowering agents, including statins, fibrates, and bile acid sequestrants, are currently used to treat hyperlipidemia. Although these drugs have beneficial therapeutic effects, they are often associated with serious side effects, such as rhabdomyolysis, myopathy, and cholelithiasis. Therefore, new lipid-lowering agents with high therapeutic value and minimum tolerable side effects are needed.^92–94) This chapter describes exploratory research on bio-functional natural products that contribute to (1) reduction of visceral fat accumulation, (2) inhibition of lipid accumulation and lipid metabolism, and (3) anti-hyperlipidemic effects.

3.1. Reduction of Visceral Fat Accumulation

Acylated flavonol glycosides, such as trans-tiliroside (20), obtained from the rose hip (the seeds of Rosa canina L.), were found to significantly inhibit body weight gain owing to reduced visceral fat accumulation without affecting food intake in mice for two weeks at doses of 0.1–10 mg/kg/d.⁹⁵⁾ To clarify the structural requirement for the anti-obese effect through the reduction of visceral fat accumulation, the following related compounds were also evaluated. The aglycone component, kaempferol, and the acyl moiety, p-coumaric acid, did not show any activity. Its desacyl derivative, kaempferol 3-O-β-D-glucopyranoside, and its related analog, helichrysoside (21), which was isolated from Helichrysum kraussii Sch. Bip.⁹⁶⁾ and H. stoechas (L.) Moench,⁹⁷⁾ tended to reduce the body weight gain through reduction of visceral fat accumulation at a dose of 10 mg/kg/d (Fig. 7). In glucose tolerance tests in mice after two weeks of continuous administration, these acylated flavonol glycosides (20 and 21) were found to significantly suppress the increase in blood glucose levels at 30 and/or 60 min post-glucose loading by intraperitoneally administering 10 mL/kg of 10% (w/v) glucose solution.^95,98)

Fig. 7. Bio-Functional Natural Products with Visceral Fat Accumulation Inhibitory and Lipid Metabolism Improving Activities

3.2. Lipid Accumulation Inhibitory and Lipid Metabolism-Promoting Activities

Fatty liver is recognized as a risk factor for liver disease,^99,100) and a strong causal relationship between fatty liver and hyperinsulinemia owing to insulin resistance has been identified.^101,102) Therefore, fatty liver is closely related to obesity and T2DM.¹⁰²⁾ To search for bio-functional natural products that inhibit intracellular triglyceride (TG) accumulation, the effects of oleic acid-albumin-induced TG accumulation in human hepatoblastoma-derived HepG2 cells were examined. As a result, several flavonoids obtained from Sedum sarmentosum Bunge [e.g., sarmenosides II (22), V (23), and VII (24)],^103,104) and Camellia sinensis (L.) Kuntze,¹⁰⁵⁾ megastigmanes from S. sarmantosum,¹⁰⁶⁾ and triterpenoid saponins from Ilex paraguariensis A. St.-Hil.¹⁰⁷⁾ were identified as active molecules (Fig. 7). Intracellular TG accumulation in HepG2 cells increases the expression of lipogenesis-related proteins, such as sterol regulatory element-binding protein 1c and fatty acid synthase, when cultured in a high-glucose-containing medium.^108,109) Thus, we examined the inhibition of TG accumulation in HepG2 cells induced by high glucose culture conditions, as well as the lipid metabolism-promoting activity in high glucose-pretreated HepG2 cells. As shown in Fig. 7, and acylated flavonol glycosides (20 and 21),⁹⁸⁾ inhibited TG accumulation in high glucose-induced HepG2 cells. In addition, acylated flavonol glycosides (20 and 21),⁹⁸⁾ megastigmanes from S. sarmantosum,¹⁰⁶⁾ limonoids from C. guianensis [e.g., 7-deacetoxy-7-oxogedunin (25)],^110,111) and diterpenes obtained from Nigella sativa [e.g., nigellamine A₃ (26)],^112–114) showed lipid metabolism-promoting activity in high glucose-pretreated HepG2 cells (Fig. 7).

Among the aforementioned natural products, acylated flavonol glycosides (20 and 21) were also found to accelerate glucose consumption in HepG2 cells, suggesting that they might be considered possible candidates for the prevention of lipid and glucose metabolism-related disorders.⁹⁸⁾

As shown in Fig. 8, limonoid (25) activates AMP-activated protein kinase (AMPK) as part of its mechanism of action. Thus, the TG-reducing effects of 25 in high glucose-induced TG accumulation inhibitory activity are attenuated by the concomitant use of an AMPK inhibitor, compound C (dorsomorphin), which shows activity similar to that of berberine.¹¹⁵⁾ However, the effects of AMPK activation by 25 are less prominent than those of berberine, and it does not affect acetyl-CoA carboxylase phosphorylation associated with AMPK activation (data not shown). Further detailed mechanisms of action demonstrated that 25 improved intracellular lipid metabolism through autophagy.¹¹¹⁾

Fig. 8. Mechanisms of Action of Limonoid (25) on Intracellular Lipid Metabolism through Autophagy in HepG2 Cells Pretreated with High Glucose and Oleic Acid

(A) Effect of 25 on TG accumulation; (B) Oil red O staining of lipid droplets treated with or without 25 (20 µM) for 24 h; (C) Effects of 25 and berberine on TG accumulation in the presence of an AMPK inhibitor (compound C, 20 µM); (D, E) Effects of 25 (20 µM) and berberine (30 µM) on phosphorylation levels of AMPK determined by Western blotting analysis; (F) Time-dependent expression of microtubule-associated protein 1 light chain 3 (LC3), sequestosome 1 (SQSTM1/p62), and Run domain Beclin-1 interaction and cysteine-rich containing protein (Rubicon) after application of 25 (20 µM) and berberine (30 µM) determined by Western blotting analysis; (G) Mechanisms of action of 25 on intracellular lipid metabolism through autophagy. Each value represents the mean ± S.E.M. (n = 4). * p < 0.05, ** p < 0.01 vs. control cells treated with vehicle (Western Dunnett); ^# p < 0.05, ^## p < 0.01 vs. compound C-nontreated cells (Student’s t). Reproduced in part with permission from Int. J. Mol. Sci., 23, 13141. Copyright [2022]. MDPI.

3.3. Anti-hyperlipidemic Effects

We searched for bio-functional molecules with anti-hyperlipidemic effects obtained from natural resources and evaluated their suppressive effects on elevated blood TG levels in olive oil-loaded mice. To date, we identified triterpenoid saponins obtained from C. sinensis [e.g., chakasaponins and floratheasaponins],^116,117)Bellis perennis L. [e.g., perenissosides I (27) and II (28) and bellisoside E (29)],^14,118) (Table 2) Sapindus rarak D.C. [e.g., hederagenin 3-O-α-L-arabinopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-Larabinopyranoside (30)],¹¹⁹⁾ and I. paraguariensis [e.g., matesaponins 1 (31) and 2 (32)],¹⁰⁷⁾ sesquiterpenes from Cynara scolymus L. [e.g., cynaropicrin (33)],¹²⁰⁾ and oligostilbenoids from S. roxburghii [e.g., (−)-hopeaphenol (16), (+)-isohopeaphenol, hemsleyanol D (17), (+)-α-viniferin (18), and (−)-balanocarpol (19)],¹²¹⁾ as active molecules (25–200 mg/kg, p.o.) (Fig. 9).

Fig. 9. Bio-Functional Natural Products with Anti-hyperlipidemic Activities

Table 2. Inhibitory Effects of Perennisosides I (27) and II (28) and Bellisoside E (29) on Serum Triglyceride Elevation in Olive Oil-Treated Mice

Treatment	Dose (mg/kg, p.o.)	N	Serum triglyceride (mg/dL)a)
			2.0 h	4.0 h	6.0 h
Normal	—	6	141.4 ± 9.1**	101.6 ± 10.7	81.7 ± 9.3
Control	—	6	501.5 ± 64.0	239.0 ± 58.6	167.2 ± 26.6
Perennisoside I (27)	25	6	337.0 ± 47.7	357.7 ± 65.6	222.7 ± 41.3
	50	6	326.9 ± 50.5*	355.8 ± 67.5	203.7 ± 30.7
	100	6	135.7 ± 33.5**	278.7 ± 78.2	208.2 ± 31.4
Control	—	6	338.5 ± 61.9	207.0 ± 26.3	142.1 ± 18.4
Perennisoside II (28)	25	6	204.6 ± 25.5*	147.0 ± 31.2	87.0 ± 9.5**
	50	6	232.5 ± 31.8	180.4 ± 33.4	104.6 ± 7.5
	100	6	179.4 ± 15.3**	155.6 ± 24.5	80.6 ± 7.5**
Control	—	6	425.2 ± 28.7	336.7 ± 31.4	243.7 ± 32.0
Bellisoside E (29)	25	6	365.8 ± 67.5	382.5 ± 35.5	249.2 ± 40.6
	50	6	368.1 ± 51.6	389.5 ± 80.4	274.0 ± 55.5
	100	6	127.3 ± 10.4**	294.6 ± 72.8	276.1 ± 54.2
Normal	—	10	154.3 ± 9.3**	138.0 ± 9.8**	138.1 ± 12.3**
Control	—	10	387.1 ± 39.2	320.4 ± 61.3	276.5 ± 35.1
Orlistat	6.25	10	266.4 ± 31.1*	179.3 ± 17.2*	155.6 ± 13.2**
	12.5	10	187.9 ± 25.5**	176.0 ± 29.5**	189.7 ± 28.8*
	25	10	158.9 ± 28.7**	132.2 ± 10.5**	140.1 ± 13.7**

Animals. Male ddY mice (6-weeks-old) were purchased from Kiwa Laboratory Animal Co., Ltd., Wakayama, Japan. The animals were housed at a constant temperature of 23 ± 2 °C, and were then fed a standard laboratory chow (MF, Oriental Yeast Co., Ltd., Tokyo, Japan). The animals were fasted for 20– 24 h prior to the beginning of the experiment, but were allowed free access to tap water. All of the experiments were performed on conscious mice unless otherwise noted. The experimental protocol was approved by the Experimental Animal Research Committee at Kindai University. Inhibitory effects on serum TG elevation in olive oil-treated mice. Each test sample was administered orally to fasted mice and olive oil (5 mL/kg) was administered p.o. 30 min thereafter. Blood was collected from the infraorbital venosus plexus, 2, 4, and 6 h after olive oil treatment. Serum TG was determined by enzymatic method using a triglyceride E test Wako (Wako Pure Chemical Corporation, Osaka, Japan). Orlistat was used as reference compounds. a) Each value represents the mean ± S.E.M. Asterisks denote significant differences from control at * p < 0.05, ** p < 0.01. Reproduced in part with permission from J. Nat. Prod., 71, 828–835. Copyright [2008]. ACS.

The mode of action of these active molecules for the blood TG level elevation inhibitory activity was evaluated in olive oil-loaded mice, including pancreatic lipase inhibitory activity and the inhibitory effects of gastric emptying. Saponins (27–32) and oligostilbenoids (16–19) inhibit pancreatic lipase, which plays an important role in lipid digestion.^{117,121–123)} On the other hand, sesquiterpene (33) significantly inhibits gastric emptying in an olive oil-loaded model at the effective dose for anti-hyprelipidemic activity.¹²⁰⁾ Because the saponin-rich fraction of B. perennis containing 27–29 also showed gastric emptying inhibitory activity, these saponin constituents were suggested to have the same mechanisms of action.¹²⁴⁾ Consequently, the above-mentioned evidence indicates that these edible natural resources could be applied in health foods (functional foods) and beverages to reduce the absorption of excess dietary fat.

4. Conclusion

In this review, we discuss our recent pharmaceutical food science research on bio-functional products from natural resources that contribute to the prevention of lifestyle diseases or the improvement of their initial symptoms. Indeed, some food materials, such as genus Salacia plants and rose hip, have been used as FOSHU and/or FFC. Although evaluation in human trials is essential for practical research, it is necessary to obtain bio-functional products from natural resources using “MONOTORI science” as exploratory research and to evaluate “quality management science” by the obtained functional substances. Both approaches are fundamental to pharmaceutical food science as well as a basis for pharmaceutical sciences. Even in recent years, MONOTORI science has been vigorously conducted by incorporating various ideas and methods in the field of natural product chemistry in the pharmaceutical and other peripheral areas.¹²⁵⁾ In addition, the importance of quality control and quality assurance of health foods has been emphasized.^126,127) I hope that further progress in research on pharmaceutical food science will lead to the extension of people’s healthy life expectancy.¹²⁸⁾

Acknowledgments

These studies were performed at the KPU and the Pharmaceutical Research and Technology Institute, Kindai University. The authors express their sincere appreciation to Emeritus Professors Masayuki Yoshikawa and Hisashi Matsuda of KPU and Osamu Muraoka of Kindai University for their fruitful discussions and encouragement. The authors would like to thank Professor Kiyofumi Ninomiya (Shujitsu University) and all the current and previous members of our laboratory. This work was financially supported, in part, by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS KAKENHI Grant Nos. 20790022, 22790026, 24590037, 15K08008, 18K06726, and 22K06688).

Conflict of Interest

The author declares no conflict of interest.

Notes

This review of the author’s work was written by the author upon receiving the 2022 Pharmaceutical Society of Japan Award for Divisional Scientific Promotion.

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