molecules

Review The Biological Effects of Leaves Containing the Cyclic AMP Phosphodiesterase 4 Inhibitor Phillyrin

Sansei Nishibe 1,*, Kumiko Mitsui-Saitoh 2, Junichi Sakai 2 and Takahiko Fujikawa 3,*

1 Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan 2 Faculty of Health and Sport, Nagoya Gakuin University, 1350 Kamishinano, Seto, Aichi 480-1298, Japan; [email protected] (K.M.-S.); [email protected] (J.S.) 3 Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, 3500-3 Minamitamagaki-cho, Suzuka-City, Mie 513-8670, Japan * Correspondence: [email protected] (S.N.); [email protected] (T.F.); Fax: +81-11-812-5460 (S.N.); +81-59-340-0595 (T.F.)

Abstract: Forsythia fruit ( Vahl ()) is a common component of Kampo medicines for treating the common cold, influenza, and allergies. The main polyphenolic compounds in the leaves of F. suspensa are pinoresinol β-D-glucoside, phillyrin and forsythiaside, and their levels are higher in the leaves of the than in the fruit. It is known that polyphenolic compounds stimulate lipid catabolism in the liver and suppress dyslipidemia, thereby attenuating diet-induced obesity and polyphenolic anti-oxidants might attenuate obesity in animals consuming high-fat diets.   Recently, phillyrin was reported as a novel cyclic AMP phosphodiesterase 4 (PDE4) inhibitor derived from forsythia fruit. It was expected that the leaves of F. suspensa might display anti-obesity effects and Citation: Nishibe, S.; Mitsui-Saitoh, K.; Sakai, J.; Fujikawa, T. The serve as a health food material. In this review, we summarized our studies on the biological effects of Biological Effects of Forsythia Leaves forsythia leaves containing phillyrin and other polyphenolic compounds, particularly against obesity, Containing the Cyclic AMP atopic dermatitis, and influenza A virus infection, and its potential as a phytoestrogen. Phosphodiesterase 4 Inhibitor Phillyrin. Molecules 2021, 26, 2362. Keywords: forsythia leaves; polyphenolic compound; forsythiaside; phillyrin; PDE4 inhibitor; https://doi.org/10.3390/ anti-obesity; atopic dermatitis; influenza A virus infection; phytoestrogen molecules26082362

Academic Editors: Ihsan˙ Çalı¸s, Horváth Györgyi and 1. Introduction Agnieszka Ludwiczuk Obesity has become an urgent worldwide public health problem in recent decades. Diet-induced obesity is closely associated with lifestyle-related diseases (.g., diabetes, Received: 23 March 2021 hyperlipidemia, hypertension), which are primary risk factors for cardiovascular dis- Accepted: 14 April 2021 Published: 19 April 2021 ease [1]. Therefore, preventing obesity is strongly encouraged to alleviate various lifestyle- related diseases. Forsythia suspensa Publisher’s Note: MDPI stays neutral Vahl (Oleaceae) is listed in Japanese Pharmacopoeia as the original with regard to jurisdictional claims in plant of the crude drug “forsythia fruit” [2], a common component in Kampo medicines published maps and institutional affil- for treating the common cold, influenza, and allergies. With at least 3000 years of con- iations. tinuous use, forsythia fruit is traditionally considered a detoxicant for treating so-called toxic and hot conditions. In addition, forsythia fruit is one of the main components of “Bofutsushosan” (), an extremely popular Kampo medicine in Japan with anti-obesity effects [3]. Wang et al. reported review with the title of “Phytochemistry, pharmacology, quality control, and future research of Forsythia suspensa (Thunb.) Vahl: A review” [4]. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. However, we could not find a description of the anti-obesity effects of the forsythia fruit or This article is an open access article leaves alone in the review. distributed under the terms and We found that the main polyphenolic compounds in leaves of F. suspensa are pinoresinol conditions of the Creative Commons β-D-glucoside, phillyrin and forsythiaside and their levels are higher in the leaves of the Attribution (CC BY) license (https:// plant than in its fruit [5,6]. Polyphenolic compounds stimulate lipid catabolism in the creativecommons.org/licenses/by/ liver and suppress dyslipidemia, thereby attenuating diet-induced obesity, and polyphe- 4.0/). nolic anti-oxidants can attenuate obesity in animals fed a high-fat diets (HFD) [7,8]. We

Molecules 2021, 26, 2362. https://doi.org/10.3390/molecules26082362 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 20

We found that the main polyphenolic compounds in leaves of F. suspensa are - noresinol β-D-glucoside, phillyrin and forsythiaside and their levels are higher in the leaves of the plant than in its fruit [5,6]. Polyphenolic compounds stimulate lipid catabo- Molecules 2021, 26, 2362 2 of 21 lism in the liver and suppress dyslipidemia, thereby attenuating diet-induced obesity, and polyphenolic anti-oxidants can attenuate obesity in animals fed a high-fat diets (HFD) [7,8]. We found that pinoresinol β-D-glucoside is a strong cyclic AMP phosphodiesterase (PDE)found inhibit that pinoresinolor in vitro β[9].-D -glucosideHowever, it is was a strong unclear cyclic whether AMP pinoresinol phosphodiesterase β-D-glucoside (PDE) isinhibitor a selectivein vitro PDE4[9 ].inhibitor However, or not. it was Phillyrin unclear also whether showed pinoresinol a moderateβ -inhibitionD-glucoside in isour a analysis.selective PDE4Phillyrin inhibitor was recently or not. Phillyrinreported alsoas a showednovel selective a moderate PDE4 inhibition inhibitor in [10 our]. As analysis. PDE4 inhibitionPhillyrin was is a recentlytherapeutic reported strategy as a for novel metabolic selective disorders PDE4 inhibitor [11], it is [10 expected]. As PDE4 that inhibition the fruit andis a therapeuticleaves of F. strategysuspensa formight metabolic display disorders anti-obesity [11], effect it is expecteds. that the fruit and leaves of F.However, suspensa might the use display of forsythia anti-obesity fruit effects.as a health food is currently prohibited in Japan by theHowever, Regulatory the use law of “Distinction forsythia fruit between as a health drugs food and is food currently stuffs” prohibited from Ministry in Japan of Health,by the RegulatoryLabor, and lawWelfare “Distinction (Japan). Therefore, between drugs we studied and food the stuffs”biological from effects Ministry of for- of sythiaHealth, leaves Labor, for and use Welfare as a health (Japan). food Therefore, material we as studied an alternative the biological to pharmaceuticals. effects of forsythia In addition,leaves for studies use as aof health F. suspensa food materialregarding as its an effects alternative on atopic to pharmaceuticals. dermatitis [12] and In addition, the pre- ventionstudies ofof F.influenza suspensa [13,14]regarding have its been effects reported. on atopic dermatitis [12] and the prevention of influenzaThis review [13,14] summarizes have been reported. our studies on the biological effects of forsythia leaves contain- ing phillyrinThis review and other summarizes polyphenolic our compounds studies on against the biological obesity, effectsatopic dermatitis, of forsythia and leaves influ- enzacontaining A virus phillyrin infection, and as well other as polyphenolic its potential as compounds a phytoestrogen. against obesity, atopic dermatitis, and influenza A virus infection, as well as its potential as a phytoestrogen. 2. Structures of Phillyrin and Forsythiaside 2. Structures of Phillyrin and Forsythiaside Pinoresinol β-D-glucoside, phillyrin and forsythiaside (caffeoyl glycoside of 3,4-di- Pinoresinol β-D-glucoside, phillyrin and forsythiaside (caffeoyl glycoside of 3,4- hydroxy-β-phenethyl alcohol) were isolated as the main polyphenolic compounds from dihydroxy-β-phenethyl alcohol) were isolated as the main polyphenolic compounds from thethe fruit fruit and and leaves leaves of of F. F.suspens suspensaa, respectively, respectively [5,6]. [5 ,The6]. contents The contents of the of compound the compoundss in the leaves are as follows; pinoresinol β-D-glucoside, 0.3–2%, phillyrin, 0.4–3% and forsythi- in the leaves are as follows; pinoresinol β-D-glucoside, 0.3–2%, phillyrin, 0.4–3% and asideforsythiaside,, 0.4–5% [ 0.4–5%6]. [6].

2.1. Phillyrin Phillyrin PhillyrinPhillyrin,, the glucoside of (+) (+)-epipinoresinol-epipinoresinol monomethyl monomethyl ether ether,, is widely distributed inin the leaves of Forsythia [[1155]. T Thehe position (C (C-4-4′0 or C C-4-4”)00) of the glucose linkage with the aglycone was previously undetermined. We We establishedestablished the position using the 13CC-NMR-NMR spectra spectra of of aglycone aglycone derivatives derivatives as as shown in Figure 11[ [1166].]. The aglycone was obtained via the hydrolysis of phillyrinphillyrin by β-glucosidase.-glucosidase. Pelter et al. reported that the 11′0 and 1” 100 carboncarbon atoms atoms of the the axial and equatorial aryl groups of 2,6 2,6-diaryl-3,7-dioxabicyclo-diaryl-3,7-dioxabicyclo [3.3.0][3.3.0] octane octane lignans lignans are clearly distinct from each other in thethe 1313CC-NMR-NMR spectra, spectra, that that is, is, thethe signals ofof formerformer areare assigned assigned to to approximately approximately 131 131 ppm, ppm, whereas whereas the the latter latter signals signals are areassigned assigned to approximately to approximately 134 134 ppm ppm for thefor the veratryl veratryl group group [17]. [17].

1’

RO 1”

Phillyrin : R=-D-glucose Phillygenin methyl ether : R=CH3 Phillygenin : R=H Phillygenin acetate : R=CH3CO Figure 1. ChemicalChemical structures structures of phillyrin and aglycone derivatives.

Comparing the spectral data of these three derivatives, appreciable differences of Comparing the spectral data00 of these three derivatives, appreciable differences of chem- icalchemical shift values shift values for C- for1” (equatorial C-1 (equatorial aryl group) aryl group) at 133.8 at ppm 133.8 for ppm phillygenin for phillygenin methyl methyl ether, ether, 133.1 ppm for phillygenin and 140.3 ppm for phillygenin acetate were observed via aryl carbon shielding, because of the effect of the substituent at the para position, whereas differences for C-10 (axial aryl group) at approximately 131 ppm were not observed. These

results indicated that the glucose linkage in phillyrin is at the C-400 position as shown in Table1. Molecules 2021, 26, x FOR PEER REVIEW 3 of 20

133.1 ppm for phillygenin and 140.3 ppm for phillygenin acetate were observed via aryl car- bon shielding, because of the effect of the substituent at the para position, whereas differences Molecules 2021, 26, 2362 for C-1′ (axial aryl group) at approximately 131 ppm were not observed. These results 3indi- of 21 cated that the glucose linkage in phillyrin is at the C-4” position as shown in Table 1.

Table 1. Chemical shifts for C-1′ and C-1” of phillyrin and phillygenin derivatives. Table 1. Chemical shifts for C-10 and C-100 of phillyrin and phillygenin derivatives. C-1’ C-1” C-10 C-100 Phillyrin 131.0 135.4 Phillyrin 131.0 135.4 Phillygenin methyl ether 131.0 133.8 PhillygeninPhillygenin methyl ether131.0 131.0 133.1 133.8 PhillygeninPhillygenin acetate 131.0 131.0 140.3 133.1 Phillygenin acetate 131.0 140.3 2.2. Forsythiaside 2.2. ForsythiasideWe isolated a novel caffeoyl glycoside of 3,4-dihydroxy-β-phenethyl alcohol for the first timeWe isolatedand named a novel the compound caffeoyl glycoside forsythiaside of 3,4-dihydroxy- [18]. The 1Hβ-NMR-phenethyl spectrum alcohol of acetate for the offirst time andide named revealed the the compound presence forsythiasideof five alcoholic [18 acetoxyl]. The 1H-NMR and four spectrum phenolic of acetoxyl acetate groups.of forsythiaside Alkalinerevealed treatment the of presence forsythiaside of five followed alcoholic by acetoxyl acid hydrolysis and four phenolicgave caffeic acetoxyl acid andgroups. 3,4-dihydroxy Alkaline treatment-β-phenethyl of forsythiaside alcohol, which followed were identified by acid hydrolysis via comparison gave caffeic with acidau- thenticand 3,4-dihydroxy- samples by βgas-phenethyl chromatography alcohol, which (GC) wereand thin identified-layer chromatography via comparison with (TLC) authen-. D- glucosetic samples and byL-rhamnose gas chromatography were detected (GC) in andthe hydrolysate thin-layer chromatography at a 1:1 ratio by (TLC). GC. TheseD-glucose data suggestand L-rhamnose that forsythiaside were detected bears in a themarked hydrolysate structural at a resemblance 1:1 ratio by GC. to the These acteoside data suggest (3,4- dihydroxythat forsythiaside-β-phenethyl bears-O a- markedα-L-rhamnopyranosyl(1 structural resemblance→3)-4-O to-caffeoyl the acteoside-β-D-glucopyra- (3,4-dihydroxy- noside)β-phenethyl-, isolatedO-α from-L-rhamnopyranosyl(1 Syringa vulgaris (Oleaceae).→3)-4-O-caffeoyl- β-D-glucopyranoside), isolated fromTheSyringa 13C-NMR vulgaris spectrum(Oleaceae). of forsythiaside was compared with that of known com- pounds,The i.e.,13C-NMR 3,4-dihydroxy spectrum-β- ofphenethyl forsythiaside alcohol, was rutin compared bearing with a rutinose that of knownmoiety com-and chlorogenicpounds, i.e., 3,4-dihydroxy-acid bearing aβ caffeate-phenethyl moiety. alcohol, Based rutin on bearing the 13 aC rutinose-NMR data, moiety namely and chloro- the 13 chemicalgenic acid shifts bearing of athe caffeate rutinose moiety. moiety Based of on forsythiaside the C-NMR data,relative namely to that the chemicalof rutin, shifts the of the rutinose moiety of forsythiaside relative to that of rutin, the glycosidation shift of the glycosidation shift of the α-carbon of forsythiaside relative to that of 3,4-dihydroxy- α-carbon of forsythiaside relative to that of 3,4-dihydroxy-β-phenethyl alcohol, and the βcalculated-phenethyl chemical alcohol shift, and of the the calculatedα-carbon chemical of forsythiaside shift of relative the α-carbon to that of 3,4-dihydroxy-forsythiaside relativeβ-phenethyl to that alcohol, of 3,4- thedihydroxy structure-β of-phenethyl forsythiaside alcohol, was establishedthe structure as of 3,4-dihydroxy- forsythiasideβ - wasphenethyl- establishedO-α-L -rhamnopyranosyl-(1as 3,4-dihydroxy-β-→phenethyl6)-4-O-caffeoyl--O-α-Lβ-rhamnopyranosyl-D-glucopyranoside-(1 as→ shown6)-4-O in- caffeoylFigure2.-β-D-glucopyranoside as shown in Figure 2.

FiFiguregure 2. 2. ChemicalChemical structure structure of of forsythiaside. forsythiaside.

3.3. The The A Anti-Obesitynti-Obesity Effects Effect ofs of Forsythia Forsythia Leaves Leaves Forsythia leaf water extract (FLE) was prepared from the leaves of F. suspensa. The con- Forsythia leaf water extract (FLE) was prepared from the leaves of F. suspensa. The con- tents of the main compounds in FLE were determined using HPLC as follows: pinoresinol tents of the main compounds in FLE were determined using HPLC as follows: pinoresinol β-D-glucoside 1.74 g/100 g, phillyrin 3.28 g/100 g, and forsythiaside 26.62 g/100 g [19]. The β-D-glucoside 1.74 g/100 g, phillyrin 3.28 g/100 g, and forsythiaside 26.62 g/100 g [19]. The anti-obesity effect of 0 (control), 2.5, and 5% FLE was examined in male Sprague–Dawley anti-obesity effect of 0 (control), 2.5, and 5% FLE was examined in male Sprague–Dawley (SD) rats fed an HFD. The average body weights of the groups were nearly identical at the (SD) rats fed an HFD. The average body weights of the groups were nearly identical at the start of the experiment, but body weight was significantly lower in the treatment groups start of the experiment, but body weight was significantly lower in the treatment groups than in the control group after four weeks of feeding as shown in Figure3. The effect of the thanextract in the was control dose-dependent. group after Foodfour weeks intake of was feeding not significantly as shown in different Figure 3. among The effect the groups. of the extract was dose-dependent. Food intake was not significantly different among the groups.

Molecules 2021,, 26,, x 2362 FOR PEER REVIEW 4 4of of 20 21

FigureFigure 3. 3. EffectsEffects of of FLE FLE on on the the body body weight weight of of rats. rats. White adipose tissue (WAT) weight was dose-dependently decreased by FLE supple- White adipose tissue (WAT) weight was dose-dependently decreased by FLE sup- mentation and the ratios of peritoneal WAT (WATp) and epididymal WAT (WATe) weight plementation and the ratios of peritoneal WAT (WATp) and epididymal WAT (WATe) to body weight were also reduced by treatment. Brown adipose tissue (BAT) weight was weigsignificantlyht to body lower weight in the were two FLE also groups reduced than by in treatment. the control Brown group, adipose but the tissue ratio of (BAT) BAT weightweight was to body significantly weight was lower not in different the two among FLE groups the groups. than in These the control findings group, suggest but thatthe ratiofat accumulation of BAT weight in WATto body was weight decreased was not and different the function among for the thermogenesis groups. These in BATfindings was suggeinvariable,st that giving fat accumulation an effective in anti-obesity WAT was effect.decreased Liver and weight the function was significantly for thermogenesis lower in inthe BAT two was FLE invariable, groups than giving in the an control effective group. anti Plasma-obesity triglyceride effect. Liver (TG) weight and free was fatty signifi- acid cantly(FFA) levelslower werein the significantly two FLE groups reduced than by in FLE the treatment control group. as shown Plasma in Table triglyceride2. (TG) and free fatty acid (FFA) levels were significantly reduced by FLE treatment as shown in TableTable 2. Effects of FLE on physical and plasma parameters after four weeks of HFD feeding.

Table 2. Effects of FLE on physical and plasma parametersDiet after four weeks of HFD feeding. Control 2.5% FLE 5.0% FLE Diet (n = 9) (n = 8) (n = 8) Control 2.5% FLE 5.0% FLE Food intake (g/rat/day) (n = 9) (n = 8) (n = 8) ± ± ± Food intake (g/rat/day) The first week 13.3 0.25 12.6 0.61 12.9 0.26 The first week The fourth week13.3 ± 0.25 15.6 ± 0.7112.6 14.9± 0.61± 0.8812. 14.49 ±± 0.26073 The fourth weekBody weight and organ weight15.6 ± 0.71 14.9 ± 0.88 14.4 ± 073 Body weight and organ weight (g/rat)(g/rat) Initial body weight Initial body weight74.7 ± 0.58 74.7 ± 0.5875.3 75.3± 1.40± 1.4074. 74.88 ±± 00.91.91 Final body weight Final body weight255.1 ± 7.41 255.1 ± 7.41167.8 167.8 ± 1.77± 1.77* *148. 148.22 ±± 33.74.74 * * Perirenal white adiposePerirenal tissue white adipose tissue1.44 ± 0.22 1.44 ± 0.220.47 0.47± 0.10± 0.10* *0.3 0.333 ±± 00.07.07 * * Epididymal white adiposeEpididymal tissue white adipose tissue3.14 ± 0.21 3.14 ± 0.211.22 1.22± 0.07± 0.07* *1.0 1.022 ±± 00.09.09 * * Brown adipose tissue Brown adipose tissue1.02 ± 0.04 1.02 ± 0.040.61 0.61± 0.02± 0.02* *0.5 0.599 ±± 00.03.03 * * Liver 2.05 ± 0.05 1.55 ± 0.09 * 1.51 ± 0.06 * Liver 2.05 ± 0.05 1.55 ± 0.09 * 1.51 ± 0.06 * Plasma parameters Plasma parameters Triglyceride (mg/dL) 124.4 ± 15.1 65.5 ± 11.4 * 47.4 ± 4.7 * ± ± ± Free fatty acid (μEq/L) Triglyceride (mg/dL)474.3 ± 29.5 124.4 15.1318.8 65.5± 35.411.4 * *28 47.40 ± 18.64.7 * * HDL-cholesterol (mg/dL)Free fatty acid (µEq/L)38.0 ± 1.3 474.3 ± 29.546. 318.81 ± 2.4± 35.4 * 28047.8± ± 18.61.6 * LDL-cholesterol (mg/dL)HDL-cholesterol (mg/dL)14.0 ± 0.8 38.0 ± 1.317.0 46.1± 0.9± 2.415. 47.84 ±± 01.6.7 Total cholesterol (mg/dL)LDL-cholesterol (mg/dL)102.6 ± 3.7 14.0 ± 0.8121.9 17.0 ± 5.0± 0.911 15.45 ±± 3.90.7 Glucose (mg/dL) Total cholesterol (mg/dL)168.4 ± 4.3 102.6 ± 3.7174.8 121.9 ± 16.6± 5.0168. 1151 ±± 43.9.9 Each value representsGlucose the (mg/dL)mean ± S.E. Sinificantly 168.4 different± 4.3 from 174.8Control:± 16.6 * p < 0.05. 168.1 ± 4.9 Each value represents the mean ± S.E. Sinificantly different from Control: * p < 0.05. To further explore the mechanism of the anti-obesity effect of FLE, gene expression analysisTo furtherby real- exploretime PCR the on mechanism the fatty acid of the uptake anti-obesity by the liver effect were of FLE, measured gene expression [19]. The geneanalysiss were by selected real-time according PCR on theto metabolic fatty acid function, uptake by namely the liver fatty were acid measured β-oxidation, [19]. fatty The

Molecules 2021, 26, 2362 5 of 21

genes were selected according to metabolic function, namely fatty acid β-oxidation, fatty acid transport, and glucose metabolism. The gene expression of fatty acid transport protein (Fatp), a regulator of fatty acid transport, was significantly higher in the two FLE groups than in the control group. The expression of carnitine palmitoyltransferase 1A (Cptla) and ACADVL, which are related to the activation of β-oxidation, was also significantly upregulated by FLE exposure as shown in Table3. The expression of the glycolysis gene glucokinase (Gck) was only increased in the 2.5% FLE group.

Table 3. Gene expression analysis by real-time PCR in the liver after four weeks of HFD feeding.

Fold Change to Control Gene Name (Accession No) 2.5% FLE 5.0% FLE Glycolitic system Gck (NM012565) 1.66 ± 0.14 * 1.53 ± 0.05 TCA cycle Cs (NM130755) 1.03 ± 0.07 1.21 ± 0.10 Ogdh (AI412142) 0.89 ± 0.05 0.96 ± 0.04 Electron transfer system Comp. I (CB5449004) 0.99 ± 0.02 1.03 ± 0.07 Comp. IV (NM017202) 1.84 ± 0.07 * 1.98 ± 0.05 * Fatty acid synthesis Fasn (NM017332) 1.19 ± 0.13 1.24 ± 0.08 Fatty acid transporter Fatp (NM053580) 1.91 ± 0.11 * 1.92 ± 0.04 * Fatty acid β-oxidation Cpt1o (NM031559) 1.84 ± 0.03 * 1.81 ± 0.15 * Acadvl (NM012891) 1.94 ± 0.67 * 1.91 ± 0.10 * Each value represents the mean ± S.E. (n = 6). Sinificantly different from Control: * p < 0.05.

These results indicated that, in HFD-fed rats, fatty acid uptake by the liver was increased by FLE, followed by an increase in fatty acid β-oxidation, which may have decreased plasma FFA levels. Peroxisome proliferator-activated receptor ã (PPARγ) is a key transcription factor that regulates adipogenesis and lipid metabolism [20] and uncoupling protein 1 (UCP1) in BAT is a mediator of thermogenesis and regulator of lipid levels [21]. The gene expressions of PPARγ and adiponectin were significantly upregulated in WATp in the two FLE groups as shown in Table4. The gene expression of UCP1 was significantly enhanced in BAT in the two FLE groups. Chronic administration of FLE induced PPARγ and adiponectin gene expression, which depends on the accumulation of visceral fat in WAT to improve hyperlipidemia and their upregulation might increase non-shivering thermogenesis in BAT via UCP1. Adiponectin level in BAT was not checked as adiponectin is secreted by WAT [22]. It is known that polyphenolic compounds can regulate obesity in rats fed an HFD [23]. et al. reported that phillyrin suppressed nutritive obesity in mice, including re- ductions of body weight, wet fat weight, the fat index, and the diameter of fat cells, and decreases of serum TG and cholesterol levels [24]. Phillyrin is a selective PDE4 in- hibitor [10]. PDE4 inhibition is therapeutic strategy for metabolic disorders and results in elevated cAMP concentrations in adipose tissues as shown in Figure4. The direct effects of cAMP on lipolysis in WAT and thermogenesis via UCP1 upregulation in BAT increase the thermogenic capacity of BAT and decrease adiposity [11]. Molecules 2021, 26, 2362 6 of 21

Table 4. Gene expression analysis in adipose tissue by real-time PCR after four weeks of HFD feeding.

Molecules 2021, 26, x FOR PEER REVIEW Fold Change to Control 6 of 20

Gene Name (Accession No) 2.5% FLE 5.0% FLE Perirenal white adipose tissue UCP1 (NM012682)Fatty acid receptor and adipocytokine 1.88 ± 0.02 * 1.92 ± 0.11 * Each value represents the mean ± S.E. (n = 6). Signicantly different from Control: * p < 0.05, ** p < 0.01. PPARγ (NM013124) 2.74 ± 0.04 ** 2.85 ± 0.13 ** It is knownAdiponectin that polyphenolic (NM144744) compounds can 1.93 regulate± 0.13 * obesity in rats 1.97 fed± 0.03an HFD * [23]. Zhao et Resistinal. reported (AJ555618) that phillyrin suppressed 1.56 nutr± 0.10itive obesity in mice 1.33, ±including0.13 reductions ofBrown body adiposeweight, tissuewet fat weight, the fat index, and the diameter of fat cells, and decreases of serum TG and cholesterol levels [24]. Phillyrin is a selective PDE4 inhibitor Uncopling ATP synthesis from oxidative [10]. PDE4 inhibition is therapeutic strategy for metabolic disorders and results in ele- metabolism vated cAMP concentrations in adipose tissues as shown in Figure 4. The direct effects of ± ± cAMP on lipolysisUCP1 in (NM012682) WAT and thermogenesis via 1.88UCP1 0.02upregulation * in BAT 1.92 increase0.11 * the ± thermogenicEach value represents capacity the of mean BAT andS.E. (dn ecrease= 6). Signicantly adiposity different [11]. from Control: * p < 0.05, ** p < 0.01.

NE NE

β-AR β-AR

ATP cAMP PDE4 ATP cAMP PDE4 inhibitor inhibitor

PKA PKA FA TG TG HSL FFA HSL UCP-1 Mitochondria FA

energy expanditure White adipose tissue Brown adipose tissue

FigureFigure 4. Mechanism 4. Mechanism of energy of energy expenditure expenditure in adipose in adipose tissues tissues induced induced by norepinephrine by norepinephrine (NE). (NE).â- AR:â -AR:â-Adrenergicâ-Adrenergic receptor; receptor; ATP: ATP: Adenosine Adenosine triphosphate; triphosphate; PKA; PKA; Protein Protein kinase kinase A; HSL; A; HSL; Hormone Hormone sensitivesensitive lipas lipase;e; TG; TG; Triglyceride; Triglyceride; FA; FA; Fatty Fatty acid; acid; FFA: FFA: Free Free fatty fatty acd, acd, UCP1: UCP1: Uncouping Uncouping protein protein 1. 1.

In Inaddition, addition, Stephen Stephen et etal. al. demonstrated demonstrated that, that, in in the the absence absence of of an an adrenergic adrenergic stim stimulus,u- lus,a a combination combination of of twotwo PDEPDE inhibitor inhibitorss is is required required to to fully fully upregulate upregulate UCP1 UCP1 and and incre increasease lipolysislipolysis in inBAT BAT [25 [25]. ].Thus, Thus, pinoresinol pinoresinol β-Dβ--glucosideD-glucoside and and phillyrin phillyrin in inforsythia forsythia leaves leaves mightmight be beresponsible responsible for for the the observed observed anti anti-obesity-obesity effects. effects. These These results results indicate indicatedd that that FLE FLE hashas potential potential value value as asa health a health material material for for reducing reducing obesity obesity similarly similarly as asforsythia forsythia fruit fruit in in thethe Kampo Kampo medicine medicine BOFU. BOFU.

4. Combination4. Combinationss of ofForsythia Forsythia Leaves Leaves and and Other Other Materials Materials BOFU,BOFU, which which is widely is widely used used as asan ananti anti-obesity-obesity pharmaceutical pharmaceutical,, consists consists of of18 18crude crude drugdrug components. components. Yoshida Yoshida et al. et al.reported reported that that the the mixture mixture of ofephedrine ephedrine and and extracts extracts of of glycyrrhiza,glycyrrhiza, schizonepeta schizonepeta spike spikes,s, and andforsythia forsythia fruit fruitalso exerted also exerted an anti an-obesity anti-obesity effect cor- effect respondingcorresponding to the toeffect the effectof BOFU of BOFU[3]. This [3]. result This indicated result indicated that the that combination the combination of both of theboth crude the drugs crude as drugs adrenergic as adrenergic stimuli stimuliand PDE and inhibitors PDE inhibitors is needed is neededto enhance to enhance the anti the- obesityanti-obesity effect of effect forsythia of forsythia leaves leaves.. However, However, most mostcrude crude drugs drugs in BOFU in BOFU are arebarred barred from from use in health foods in Japan. We attempted to develop a simple and beneficial anti-obesity use in health foods in Japan. We attempted to develop a simple and beneficial anti-obesity health food by combining forsythia leaves with other commonly available herbal materials. health food by combining forsythia leaves with other commonly available herbal materi- als.4.1. Gardenia Fruit, Glycyrrhiza, and Immature Orange Forsythia leaves, gardenia fruit, glycyrrhiza, and immature orange (Citrus aurantium) 4.1. Gardenia Fruit, Glycyrrhiza, and Immature Orange were selected [26]. Forsythia leaves and glycyrrhiza contain PDE inhibitors [27]. Immature Forsythia leaves, gardenia fruit, glycyrrhiza, and immature orange (Citrus aurantium) were selected [26]. Forsythia leaves and glycyrrhiza contain PDE inhibitors [27]. Immature orange, which contains synephrine, is a replacement for ephedra [28]. Gardenia fruit was selected because of its reported anti-obesity effects in the literature [29]. In the first experiment, the anti-obesity effect of a mixture of herbal extracts (MHE) was investigated in SD rats fed a normal diet (ND) or HFD, both or without MHE

Molecules 2021, 26, 2362 7 of 21

orange, which contains synephrine, is a replacement for ephedra [28]. Gardenia fruit was selected because of its reported anti-obesity effects in the literature [29]. In the first experiment, the anti-obesity effect of a mixture of herbal extracts (MHE) was investigated in SD rats fed a normal diet (ND) or HFD, both or without MHE supple- mentation. MHE consisted of forsythia leaf, immature orange, glycyrrhiza, and gardenia fruit extracts at a ratio of 3. 3. 5. 3. This ratio was determined by referencing the weight ratio of the four extracts in BOFU (150. 150. 250. 150 (mg/day)). Rats fed the ND with MHE for 10 weeks exhibited decreases in WATp and WATe weight and a significant decrease in plasma TG levels compared to the findings in rats fed the ND alone as shown in Table5. These effects of MHE were similar to those in a previous report using mice fed an ND plus BOFU [30]. The decrease of plasma TG levels was attributed to the inhibition of TG synthesis in the liver and decomposition of TG in the visceral adipose tissue by MHE. The decrease of plasma HDL-cholesterol level was exhibited in the 1% MHE group compared with the ND-control group. In a case using mice fed an ND plus 1.5% BOFU, the tendency of decrease of plasma total cholesterol value was also exhibited after three weeks [30]. So far, these reasons for the decrease are unknown.

Table 5. Effects of MHE on physical and plasma parameters in ND-fed rats after 10 weeks.

Diet (ND) Control 0.2% MHE 1% MHE 5% MHE (n = 4) (n = 4) (n = 4) (n = 4) Food intake (g/rat) Final day 43.2 ± 1.2 44.1 ± 0.3 40.2 ± 0.8 44.2 ± 0.1 Body weight (g/rat) Final body weight 491 ± 13 445 ± 14 454 ± 16 416 ± 15 Organ weight/(g/100 g body weight) Perirenal white adipose tissue 0.67 ± 0.16 0.36 ± 0.06 0.36 ± 0.06 0.31 ± 0.05 * Epididymal white adipose tissue 1.71 ± 0.21 1.14 ± 0.14 * 1.29 ± 0.13 1.03 ± 0.06 * Brown adipose tissue 0.13 ± 0.01 0.15 ± 0.01 0.18 ± 0.01 0.19 ± 0.02 Plasma parameters Triglyceride (mg/dL) 232 ± 38 128 ± 19 * 89.0 ± 3.8 * 81.5 ± 4.0 * Free fatty acid (µEq/L) 355 ± 46 362 ± 30 262 ± 22 313 ± 68 HDL-cholesterol (mg/dL) 32.0 ± 1.7 27.3 ± 1.1 25.8 ± 1.2 * 31.5 ± 2.2 LDL-cholesterol (mg/dL) 11.0 ± 0.7 8.50 ± 0.29 8.50 ± 0.87 8.25 ± 0.25 Total cholesterol (mg/dL) 93.5 ± 7.7 72.0 ± 2.3 68.0 ± 4.2 * 81.5 ± 5.8 Glucose (mg/dL) 150 ± 7 151 ± 3 138 ± 5 154 ± 4 Insulin (ng/mL) 1.27 ± 0.31 1.49 ± 0.06 1.49 ± 0.23 1.90 ± 0.15 Adiponectin (ng/mL) 3881 ± 461 4749 ± 1111 3223 ± 360 3717 ± 544 Each value represents the mean ± S.E. Significantly different from ND-control: * p < 0.05.

Rats fed the HFD containing 5% MHE for 10 weeks exhibited significant decreases in body, WATp, and WATe weight and significant decreases in plasma TG, FFA, total cholesterol, glucose, and insulin levels compared to the results in the HFD control group as shown in Table6. In addition, the 5% MHE group exhibited a marked increase in plasma adiponectin levels under the HFD condition. These effects in the 5% MHE group were similar to those in the previous reports that used mice who were fed an HFD containing BOFU [3,31,32]. Molecules 2021, 26, 2362 8 of 21

Table 6. Effects of MHE on physical and plasma parameters in HFD-fed rats after 10 weeks.

Diet (HFD) Control 0.2% MHE 1% MHE 5% MHE (n = 6) (n = 6) (n = 6) (n = 6) Food intake (g/rat) Final day 35.5 ± 0.4 31.8 ± 0.8 32.9 ± 0.5 39.3 ± 3.7 Body weight (g/rat) Final body weight 588 ± 6 † 561 ± 23 539 ± 13 410 ± 5 * Organ weight (g/100g body weight) Perirenal white adipose tissue 1.64 ± 0.06 † 1.42 ± 0.11 1.40 ± 0.15 0.74 ± 0.11 ** Epididymal white adipose tissue 3.41 ± 0.07 † 3.03 ± 0.22 2.53 ± 0.10 * 1.53 ± 0.17 ** Brown adipose tissue 0.20 ± 0.01 † 0.21 ± 0.02 0.28 ± 0.02 0.24 ± 0.02 Plasma parameters Triglyceride (mg/dL) 229 ± 6 164 ± 36 207 ± 47 65.3 ± 16.4 ** Free fatty acid (µEq/L) 530 ± 25 † 387 ± 5 * 396 ± 26 * 454 ± 51 HDL-cholesterol (mg/dL) 32.2 ± 2.5 28.7 ± 1.2 34.0 ± 2.1 40.7 ± 1.3 * LDL-cholesterol (mg/dL) 15.7 ± 1.4 † 11.3 ± 1.0 * 14.3 ± 1.2 13.7 ± 1.7 Total cholesterol (mg/dL) 125 ± 7 † 83.0 ± 2.5 * 101 ± 10 * 110 ± 7 Glucose (mg/dL) 177 ± 5 † 160 ± 6 * 162 ± 3 * 153 ± 3 * Insulin (ng/mL) 2.62 ± 0.07 † 1.00 ± 0.13 ** 1.49 ± 0.30 * 1.31 ± 0.28 * Adiponectin (ng/mL) 3332 ± 200 6342 ± 998 * 4079 ± 237 5194 ± 757 * Each value represents the mean ± S.E. Significantly different from HFD-control: * p < 0.05, ** p < 0.01. Significantly different from ND-control: † p < 0.05.

A study by Hioki et al. indicated that BOFU improved visceral adiposity and insulin resistance in obese women with impaired glucose tolerance [33]. Therefore, the results of this experiment suggested that the fat mass-lowering effect of MHE may be attributable to a direct pharmacological action on adipose tissues similar to that of BOFU. In the second experiment, body, WATp, WATe, and BAT weight were compared in HFD-fed rats treated with 5% MHE or 5% BOFU (ALPS Pharmaceutical Ind. Co.) as shown in Figure5 and Table7. Real-time PCR demonstrated that fatty acid uptake by the liver was increased by MHE and BOFU in HFD-fed rats, followed by an increase in fatty acid β-oxidation as shown in Table8.

Table 7. Effects of MHE and BOFU on body and organ weight after four weeks of feeding.

Diet (HFD) Control 5% MHE 5% BOFU (n = 9) (n = 8) (n = 8) Body weight (g/rat) Initial body weight 74.7 ± 0.6 74.8 ± 0.9 74.2 ± 0.9 Final body weight 255.1 ± 7.4 183.3 ± 7.8 * 189.6 ± 5.9 * Organ weight (g/100 g body weight) Perirenal white adipose tissue 0.53 ± 0.08 0.31 ± 0.08 * 0.31 ± 0.07 * Epididymal white adipose tissue 1.2 ± 0.06 0.63 ± 0.09 * 0.69 ± 0.05 * Brown adipose tissue 0.38 ± 0.01 0.35 ± 0.02 0.36 ± 0.02 Each value represents the mean ± S.E. Significantly different from Control: * p < 0.05. Molecules 2021, 26, x FOR PEER REVIEW 8 of 20

Final body weight 588 ± 6 † 561 ± 23 539 ± 13 410 ± 5 * Organ weight (g/100g body weight) Perirenal white adipose tissue 1.64 ± 0.06 † 1.42 ± 0.11 1.40 ± 0.15 0.74 ± 0.11 ** Epididymal white adipose tissue 3.41 ± 0.07 † 3.03 ± 0.22 2.53 ± 0.10 * 1.53 ± 0.17 ** Brown adipose tissue 0.20 ± 0.01 † 0.21 ± 0.02 0.28 ± 0.02 0.24 ± 0.02 Plasma parameters Triglyceride (mg/dL) 229 ± 6 164 ± 36 207 ± 47 65.3 ± 16.4 ** Free fatty acid (μEq/L) 530 ± 25 † 387 ± 5 * 396 ± 26 * 454 ± 51 HDL-cholesterol (mg/dL) 32.2 ± 2.5 28.7 ± 1.2 34.0 ± 2.1 40.7 ± 1.3 * LDL-cholesterol (mg/dL) 15.7 ± 1.4 † 11.3 ± 1.0 * 14.3 ± 1.2 13.7 ± 1.7 Total cholesterol (mg/dL) 125 ± 7 † 83.0 ± 2.5 * 101 ± 10 * 110 ± 7 Glucose (mg/dL) 177 ± 5 † 160 ± 6 * 162 ± 3 * 153 ± 3 * Insulin (ng/mL) 2.62 ± 0.07 † 1.00 ± 0.13 ** 1.49 ± 0.30 * 1.31 ± 0.28 * Adiponectin (ng/mL) 3332 ± 200 6342 ± 998 * 4079 ± 237 5194 ± 757 * Each value represents the mean ± S.E. Significantly different from HFD-control: * p < 0.05, ** p < 0.01. Significantly different from ND-control: † p < 0.05

A study by Hioki et al. indicated that BOFU improved visceral adiposity and insulin resistance in obese women with impaired glucose tolerance [33]. Therefore, the results of this experiment suggested that the fat mass-lowering effect of MHE may be attributable to a direct pharmacological action on adipose tissues similar to that of BOFU. In the second experiment, body, WATp, WATe, and BAT weight were compared in HFD-fed rats treated with 5% MHE or 5% BOFU (ALPS Pharmaceutical Ind. Co.) as shown in Figure 5 and Table 7. Real-time PCR demonstrated that fatty acid uptake by the liver Molecules 2021, 26, 2362 was increased by MHE and BOFU in HFD-fed rats, followed by an increase in fatty acid9 of 21 β-oxidation as shown in Table 8.

FigureFigure 5. 5. EffectsEffects of of MHE MHE and and BOFU BOFU on on body body weight weight in in HFD HFD-fed-fed rats. rats.

TableTable 7. 8. EffectsLiver of gene MHE expression and BOFU analysis on body by and real-time organ weight PCR afterafter fourfour weeks of feeding. MHE and BOFU supplementation. Diet (HFD) Control 5% MHE 5% BOFU Fold Change to Control Gene Name (Accession No)(n = 9 5%) MHE(n = 8) 5% BOFU(n = 8) Body weight (g/rat) Glycolytic system Initial body weight 74.7 ± 0.6 74.8 ± 0.9 74.2 ± 0.9 Gck (NM012565) 0.54 ± 0.05 *** 0.45 ± 0.09 ** TCA cycle Cs (NM130755) 1.08 ± 0.12 0.97 ± 0.09

Ogdh (AI412142) 1.16 ± 0.06 1.18 ± 0.06 Electron transfer system Comp. I (CB5449004) 1.49 ± 0.06 0.89 ± 0.06 Comp. IV (NM017202) 1.89 ± 0.04 * 1.84 ± 0.07 * Fatty acid synthesis Fasn (NM017332) 0.46 ± 0.03 *** 0.80 ± 0.11 Fatty acid transporter Fatp (NM053580) 1.87 ± 0.20 * 1.82 ± 0.17 * Fatty acid β-oxidation Cpt1α (NM031559) 1.94 ± 0.06 * 1.74 ± 0.06 * ACADVL (NM012891) 1.87 ± 0.11 * 1.87 ± 0.09 * Each value represents the mean ± S.E. (n = 6). Significantly different from Control: * p < 0.05, ** p < 0.01, *** p < 0.001.

To compare the anti-obesity effects among 5% MHE and 5% BOFU groups, rats were divided into three groups (0% control, 5% MHE, and 5% BOFU groups) based on their body weight. Gene expressions by fold change to control in 5% MHE and 5% BOFU groups were calculated with that of the control group as value of 1, respectively. Obesity is a common risk factor for type 2 diabetes. Adiponectin gene expression and plasma adiponectin levels were reported to be significantly reduced in obese/diabetic mice and humans [34]. Under HFD conditions, chronic administration of MHE or BOFU induced the gene expression of PPARγ and adiponectin as shown in Table9. These effects were considered to result in the observed increases of plasma adiponectin levels, decreases of visceral fat Molecules 2021, 26, 2362 10 of 21

accumulation in WATp and WATe and reductions of plasma glucose levels in the first experiment, thereby improving insulin resistance. That is, PPARγ gene expression induces an increase of plasma adiponectin levels [35]. In addition, Matsuzawa et al. reported that plasma adiponectin levels decrease with visceral fat accumulation [36]. This finding indicates that the increase of plasma adiponectin levels is related to the decrease of visceral fat accumulation.

Table 9. Gene expression analysis in adipose tissue by real-time PCR following four weeks of MHE and BOFU exposure.

Fold Change to Control Gene Name (Accession No) 5% MHE 5% BOFU Perirenal white adipose tissue Fatty acid receptor and adipocytokine PPARγ (NM013124) 2.22 ± 0.09 * 1.89 ± 0.25 * Adiponectin (NM144744) 2.42 ± 0.05 ** 1.92 ± 0.19 * Resistin (AJ555618) 1.30 ± 0.07 1.60 ± 0.17 Brown adipose tissue Uncoupling ATP synthesis from oxidative metabolism UCP1 (NM012682) 1.86 ± 0.09 * 1.32 ± 0.05 Each value represents the mean ± S.E. (n = 6). Significantly different from Control: * p < 0.05, ** p < 0.01.

It is noteworthy that UCP1 gene expression in BAT was significantly upregulated in the MHE group. UCP1 expression in BAT is known as a significant component of whole body energy expenditure and its dysfunction contributes to the development of obesity [37]. These findings revealed that MHE or BOFU supplementation inhibited visceral fat accumulation in HFD-fed rats, and both supplements upregulated the UCP1 gene, which regulates energy metabolism in WAT. These results suggested that MHE exerts similar anti- obesity effects as BOFU. It can be presumed that gardenoside and geniposide in gardenia fruit, synephrine in immature orange, pinoresinol β-D-glucoside and phillyrin in forsythia leaves, and glabridin in glycyrrhiza are partly responsible for the synergistic effects of MHE, supporting the use of the extract as a simple and beneficial supplement for preventing diet-induced obesity.

4.2. Eucommia Leaves We expected that eucommia leaves would synergistically enhance the effects of forsythia leaves. The leaves of Eucommia ulmoides Oliv. have been used in the health tea “Tochucha” for anti-hypertensive purposes in Japan [38]. The main constituents of the leaves are iridoid glucosides, asperuloside, and geniposidic acid, which have similar chemical structures as iridoid glucosides, gardenoside, and geniposide in gardenia fruit, respectively [39]. We reported the anti-obesity effects in eucommia leaf extract (ELE) [35] in comparison with asperuloside [40]. ELE was comprised of asperuloside 13.7 mg/g and geniposidic acid 69.5 mg/g. As presented in Tables 10 and 11, the oral administration of ELE or asperuloside to HFD-fed rats resulted in anti-obesity effects, confirming that asperuloside is one of the bioactive compounds responsible for the anti-obesity effects of ELE. Molecules 2021, 26, 2362 11 of 21

Table 10. Effects of ELE on physical and plasma parameters in HFD-fed rats after three months.

Diet (HFD) Control 3% ELE 9% ELE (n = 8) (n = 8) (n = 8) Final body weight (g/rat) 548.6 ± 16.8 485.3 ± 13.6 * 422.2 ± 17.7 * Food intake (g/day/rat) 25.3 ± 3.0 19.7 ± 3.6 15.2 ± 1.7 * WAT weight (g/rat) Perirenal white adipose tissue 10.0 ± 0.8 5.6 ± 0.4 *** 3.5 ± 0.6 *** Epididymal white adipose tissue 18.3 ± 0.7 13.9 ± 0.6 *** 5.8 ± 0.4 *** Plasma parameters Glucose (mg/L) 1520 ± 17 1458 ± 5 * 1433 ± 17 * Insulin (ng/mL) 6.6 ± 0.5 4.2 ± 0.5 ** 2.4 ± 0.3 *** Free fatty acid (µEq/L) 610.4 ± 78.8 450.8 ± 33.8 493.4 ± 26.2 Total cholesterol (mg/L) 780 ± 27 655 ± 28 ** 725 ± 15 Adiponectin (µg/L) 27 ± 3 42 ± 4 53 ± 4 ** TNF-α (pg/mL) 178.5 ± 22.6 137.1 ± 15.1 63.5 ± 8.3 * Resistin (ng/mL) 187.6 ± 15.9 175.9 ± 15.9 111.4 ± 11.0 ** Leptin (ng/mL) 6.8 ± 0.4 5.9 ± 0.7 6.7 ± 0.8 Each value represents the mean ± S.E. Significantly different from HFD-control: * p < 0.05, ** p < 0.01, *** p < 0.001(Tukey HSD).

Table 11. Effects of ASP on physical and plasma parameters in HFD-fed rats after three months.

Diet (HFD) Control 0.03% ASP 0.1% ASP 0.3% ASP (n = 6) (n = 6) (n = 6) (n = 6) Initial body weight (g/rat) 71 ± 1.0 71.2 ± 1.5 72.5 ± 0.5 71 ± 0.6 Food intake (g/day/rat) 27.8 ± 2.2 21.3 ± 3.2 * 17.7 ± 2.7 * 14.9 ± 2.0 * Final body weight (g/rat) 564 ± 9 516 ± 19 * 465 ± 8 * 461 ± 7 * Body weight gain (g/rat) 493 ± 10 445 ± 18 * 393 ± 8 * 390 ± 7 * Relative WAT weight (%) Perirenal white adipose 2.7 ± 0.3 1.5 ± 0.2 * 1.4 ± 0.1 * 1.3 ± 0.1 * tissue Epididymal white adipose 2.6 ± 0.2 2.5 ± 0.2 2.2 ± 0.1 2.0 ± 0.1 tissue Relative BAT weight (%) 0.24 ± 0.02 0.31 ± 0.01 * 0.33 ± 0.02 * 0.37 ± 0.02 * Relative Sol. M. weight (%) 0.07 ± 0.01 0.07 ± 0.01 0.07 ± 0.01 0.07 ± 0.01 Plasma parameters Glucose (mg/L) 1621 ± 71 1501 ± 37 * 1394 ± 42 * 1338 ± 55 * Insulin (ng/mL) 7.7 ± 0.6 5.2 ± 1.1 * 3.9 ± 0.8 * 3.3 ± 0.6 * Free fatty acid (µEq/L) 639.1 ± 33.7 449 ± 56.0 * 402.7 ± 21.6 * 397.3 ± 20.9 * Total cholesterol (mg/L) 880 ± 34 721 ± 25 * 708 ± 24 * 664 ± 26 * Adiponectin (µg/L) 29 ± 5 39 ± 6 * 48 ± 4 * 53 ± 3 * TNF-α (pg/mL) 198.3 ± 18.2 136.5 ± 13.1 * 98.7 ± 9.2 * 70.6 ± 8.9 * Each value represents the mean ± S.E. Significantly different from HFD-control: * p < 0.05. Sol. M.: Soleus Muscle. ASP: Asperuloside.

We revealed for the first time that oral asperuloside administration to rats increased the bile acid pool in the intestine [38]. Gardenoside from gardenia fruit is known to Molecules 2021, 26, x FOR PEER REVIEW 11 of 20

TNF-α (pg/mL) 178.5 ± 22.6 137.1 ± 15.1 63.5 ± 8.3 * Resistin (ng/mL) 187.6 ± 15.9 175.9 ± 15.9 111.4 ± 11.0 ** Leptin (ng/mL) 6.8 ± 0.4 5.9 ± 0.7 6.7 ± 0.8 Each value represents the mean ± S.E. Significantly different from HFD-control: * p < 0.05, ** p < 0.01, ***p < 0.001(Tukey HSD).

Table 11. Effects of ASP on physical and plasma parameters in HFD-fed rats after three months.

Diet (HFD) Control 0.03%ASP 0.1% ASP 0.3%ASP

(n = 6) (n = 6) (n = 6) (n = 6) Initial body weight (g/rat) 71 ± 1.0 71.2 ± 1.5 72.5 ± 0.5 71 ± 0.6 Food intake (g/day/rat) 27.8 ± 2.2 21.3 ± 3.2 * 17.7 ± 2.7 * 14.9 ± 2.0 * Final body weight (g/rat) 564 ± 9 516 ± 19 * 465 ± 8 * 461 ± 7 * Body weight gain (g/rat) 493 ± 10 445 ± 18 * 393 ± 8 * 390 ± 7 * Relative WAT weight (%) Perirenal white adipose tissue 2.7 ± 0.3 1.5 ± 0.2 * 1.4 ± 0.1 * 1.3 ± 0.1 * Epididymal white adipose tissue 2.6 ± 0.2 2.5 ± 0.2 2.2 ± 0.1 2.0 ± 0.1 Relative BAT weight (%) 0.24 ± 0.02 0.31 ± 0.01 * 0.33 ± 0.02 * 0.37 ± 0.02 * Relative Sol. M. weight (%) 0.07 ± 0.01 0.07 ± 0.01 0.07 ± 0.01 0.07 ± 0.01 Plasma parameters Glucose (mg/L) 1621 ± 71 1501 ± 37 * 1394 ± 42 * 1338 ± 55 * Insulin (ng/mL) 7.7 ± 0.6 5.2 ± 1.1 * 3.9 ± 0.8 * 3.3 ± 0.6 * 397.3 ± 20.9 Free fatty acid (µEq/L) 639.1 ± 33.7 449 ± 56.0 * 402.7 ± 21.6 * * Total cholesterol (mg/L) 880 ± 34 721 ± 25 * 708 ± 24 * 664 ± 26 * Adiponectin (µg/L) 29 ± 5 39 ± 6 * 48 ± 4 * 53 ± 3 * TNF-α (pg/mL) 198.3 ± 18.2 136.5 ± 13.1 * 98.7 ± 9.2 * 70.6 ± 8.9 * Each value represents the mean ± S.E. Significantly different from HFD-control: * p < 0.05. Sol. M.: Molecules 2021, 26, 2362 Soleus Muscle. ASP: Asperuloside. 12 of 21

We revealed for the first time that oral asperuloside administration to rats increased the bile acid pool in the intestine [38]. Gardenoside from gardenia fruit is known to protect protectthe leaves the against leaves againstinsect feeding. insect feeding. Regarding Regarding the mechanism, the mechanism, it was demonstrated it was demonstrated that gar- thatdenoside gardenoside is easily ishydrolyzed easily hydrolyzed to aglyco tone aglycone in the digestive in the digestive tract by β tract-glucosidase, by β-glucosidase, followed followedby its binding by its with binding proteins with proteinsof transport of transportersers to cause tolethal cause damage lethal damage (Figure (Figure6) [41].6 )[ 41].

FigureFigure 6.6. Chemical structure ofof asperuloside.asperuloside.

ItIt hashas longlong beenbeen knownknown thatthat thethe leavesleaves of EucommiaEucommia are hardlyhardly damageddamaged by insects. ThisThis suggests that the the leaves leaves contain contain the the protective protective compound. compound. In Inthethe same same manner manner,, as- asperulosideperuloside is iseasily easily hydrolyzed hydrolyzed to toaglycone aglycone by byβ-glucosidase.β-glucosidase. It has It hasbeen been reported reported that thataglycone aglycone binds binds with withamino amino acids acidsto produce to produce resinous resinous substances substances in vitroin similar vitro similarlyly as gar- as gardenoside [42]. Meanwhile, asperuloside plasma levels are extremely low in rats Molecules 2021, 26, x FOR PEER REVIEWdenoside [42]. Meanwhile, asperuloside plasma levels are extremely low in rats (Cmax12 of 20= (Cmax198 ng/mL = 198 at ng/mL50 mg/kg at 50p.o mg/kg., unpublished p.o., unpublished data). In addition, data). In a addition,review article a review stated article that statedmetformin that metforminincreases the increases bile acid the pool bile within acid pool the intestine, within the pr intestine,edominantly predominantly through re- throughduced ileal reduced absorption ileal absorption [43]. Therefore, [43]. Therefore, it is assumed it is that assumed the increase that the of increase the bile of acid the pool bile acidin the pool intestine in the intestinefollowing following asperuloside asperuloside administration administration in rats is in also rats isattributable also attributable to the toreduction the reduction of ileal of absorption ileal absorption of bile of acid bile acidby the by transport the transporterer bound bound by t byhe theaglycone aglycone of as- of asperuloside.peruloside. Further Further research research is isrequired required to to clarify clarify the the mechanism. mechanism. PriorPrior researchresearch recordedrecorded increasesincreases ofof thethe bilebile acidacid poolpool inin thethe intestineintestine andand choliccholic acidacid levelslevels in in plasma plasma after after oral oral cholic cholic acid acid treatment treatment in HFD-fed in HFD mice-fed [ 44mice]. An [4 increased4]. An increase plasmad LDLplasma level LDL is alsolevel observed, is also observed following, following increases increases of the bileof the acid bile pool acidin pool the in intestine the intestine [45]. The[45]. increasing The increasing cholic cholic acid content acid content in the bloodin the actsblood on acts TGR5 on receptors TGR5 receptors of BAT inof rodentsBAT in androdent muscles and skeletal muscle inskeletal humans in human to exerts itsto exert anti-obesity its anti- effectobesity (Figure effect7 )[(Figure46]. Therefore, 7) [46]. There- it is assumedfore, it is thatassumed the anti-obesity that the anti effect-obesity of asperuloside effect of asperuloside in rodents mightin rodent depends might on andepend indirect on effectan indirect of cholic effect acid, of ascholic opposed acid, as to aopposed direct effect to a direct of asperuloside. effect of asperuloside.

FigureFigure 7.7. MechanismMechanism byby whichwhich gardenosidegardenoside inducesinduces bindingbinding withwith proteinsproteins ofof transporters.transporters.

Furthermore,Furthermore, thethe increaseincrease ofof thethe bilebile acidacid poolpool followingfollowing metforminmetformin waswas accompa-accompa- niednied byby aa markedmarked increaseincrease ofof AkkermansiaAkkermansia muciniphilamuciniphila countscounts inin thethe gutgut gastrointestinalgastrointestinal tracttract [[4433],], andand similarsimilar increasesincreases inin A.A. muciniphilamuciniphila countscounts inin thethe gastrointestinalgastrointestinal tracttract werewere inducedinduced byby asperulosideasperuloside [[4477].]. InIn addition,addition, itit waswas recentlyrecently reportedreported thatthat BOFUBOFU containingcontaining gardenosidegardenoside fromfrom gardeniagardenia fruitfruit cancan induceinduce markedmarked increasesincreases ofof A.A. muciniphilamuciniphila countscounts inin A. muciniphila thethe gastrointestinalgastrointestinal tracttract [[4488].]. A. muciniphila isis expected toto emerge asas a beneficialbeneficial microbe,microbe, which produces active short chain fatty acids for anti-obesity effects from the dietary fiber in the gut [49]. Conversely, the anti-obesity effects of the ELE product were weak in humans unlike the case of rodents [50]. This finding is believed to be associated with the lack of BAT activation by cholic acid via TGR5 in humans [51]. Thermogenesis in skeletal muscle is low in humans compared to that in BAT (skeletal muscle:BAT = 1:100) [52]. A prior report indicated that mitochondrial activity in skeletal muscle is enhanced by PDE4 inhibition as shown in Figure 8 [53]. BOFU contains the active iridoid glucosides, gardenoside and geniposide from gar- denia fruit, and polyphenol, phillyrin from forsythia fruit. Similarly, eucommia leaves contain the active iridoid glucosides, asperuloside, and geniposidic acid, and forsythia leaves contain polyphenol, phillyrin.

Molecules 2021, 26, 2362 13 of 21

which produces active short chain fatty acids for anti-obesity effects from the dietary fiber in the gut [49]. Conversely, the anti-obesity effects of the ELE product were weak in humans unlike the case of rodents [50]. This finding is believed to be associated with the lack of BAT activation by cholic acid via TGR5 in humans [51]. Thermogenesis in skeletal muscle is low in humans compared to that in BAT (skeletal muscle:BAT = 1:100) [52]. A prior report Molecules 2021, 26, x FOR PEER REVIEW 13 of 20 indicated that mitochondrial activity in skeletal muscle is enhanced by PDE4 inhibition as shown in Figure8[53].

BA

TGR5

PDE4 cAMP inhibitor

D2 Epac1

T4 T3 AMPK

TR PGC1α

Mitochondrial function

energy expanditure Brown adipose tissue（Rodents) Skeletal muscle (Humans) FigureFigure 8. 8. MechanismMechanism of of energy energy expenditure expenditure induced induced by by bile bile acid acid () (BA) via via brown brown adipose adipose tissue tissue or or skeletalskeletal muscle. muscle. TGR5: TGR5: G Gprotein protein-coupled-coupled receptor receptor 5; D: 5; D:Iodothyronine Iodothyronine deiodinase; deiodinase; T: Thyroxine; T: Thyroxine; TRTR:：Thyroido Thyroido hormone hormone receptor; receptor; Epac Epac 1: 1:Exchange Exchange protein protein activated activated by cyclic by cyclic AMP AMP 1; AMPK: 1; AMPK: AMPAMP-activated-activated kinase; kinase; PGC PGC 1á; 1á ;Peroxisome Peroxisome proliferators proliferators-activated-receptor-activated-receptor ãã coco-activator-1-activator-1á.á .

FromBOFU the contains aforementioned the active results, iridoid the glucosides, simple combination gardenoside of andforsythia geniposide leaves fromand eu- gar- commiadenia fruit, leaves and as an polyphenol, herbal tea are phillyrin expected from to have forsythia synergistic fruit. effects Similarly, on obesity eucommia in humans. leaves contain the active iridoid glucosides, asperuloside, and geniposidic acid, and forsythia 5.leaves Other contain Biological polyphenol, Activities phillyrin. 5.1. EffectFroms for the Forsythia aforementioned Leaves on Atopic results, Dermatitis the simple combination of forsythia leaves and eucommiaIt has been leaves known as an for herbal more than tea are40 years expected that tohigh have number synergistics of Staphyloc effectsoccus on obesityaureus growin humans. when samples from the skin of patients with atopic dermatitis are cultured. It has long been5. Other debated Biological whether Activities S. aureus, which grows on the skin, is the cause of inflammation or the result of chronic inflammation. Kobayashi et al. revealed using ADAM17 cKO mice that 5.1. Effects for Forsythia Leaves on Atopic Dermatitis when atopic dermatitis becomes severe, the diversity of bacteria on the skin surface is sig- nificantlyIt has reduced, been known and S. for aureus more becomes than 40 years dominant that high [54]. numbers A causal of relationshipStaphylococcus between aureus atopicgrow dermatitis when samples and S. from aureus the has skin not of been patients estab withlished atopic to date dermatitis because of are the cultured. lack of a Itsuit- has S. aureus ablelong animal been debatedmodel. whether , which grows on the skin, is the cause of inflammation or the result of chronic inflammation. Kobayashi et al. revealed using ADAM17 cKO mice Since colonization by S. aureus is the cause of inflammation in atopic dermatitis, it is that when atopic dermatitis becomes severe, the diversity of bacteria on the skin surface is suggested that a component with antibacterial activity against S. aureus could suppress significantly reduced, and S. aureus becomes dominant [54]. A causal relationship between inflammation. We previously reported that forsythia extract (MIC = 6% w/v) and its main atopic dermatitis and S. aureus has not been established to date because of the lack of a constituent forsythiaside (MIC = 3.2 mM) have strong antibacterial effects against S. aureus suitable animal model. Terashima [55,56]. Qu et al. evaluated the anti-microbial activity of forsythiaside and phil- Since colonization by S. aureus is the cause of inflammation in atopic dermatitis, it is lyrin against Escherichia coli-10B, Pseudomonas aeruginosa and S. aureus Rn4220 using the suggested that a component with antibacterial activity against S. aureus could suppress microtiter plate method. Forsythiaside exhibited strong antibacterial activity against all inflammation. We previously reported that forsythia extract (MIC = 6% w/v) and its main three bacteria, and it was more effective against S. aureus (MIC = 76.67 μg/mL) than tetra- constituent forsythiaside (MIC = 3.2 mM) have strong antibacterial effects against S. aureus cycline (MIC = 119.05 μg/mL). However, the activity of phillyrin was not remarkable [57]. Terashima [55,56]. Qu et al. evaluated the anti-microbial activity of forsythiaside and Thesephillyrin results against supportEscherichia the use coli of-10B, forsythiaPseudomonas as an herbal aeruginosa medicineand S.for aureus skin diseases.Rn4220 using the microtiterFurthermore, plate method. Sung et Forsythiasideal. evaluated the exhibited in vivo strongand in antibacterialvitro therapeutic activity effects against of For- all sythia suspensa fruit extract (FSE) in an NC/Nga mouse model exposed to Dermatophagoides farinae crude extract (DfE). Topical application of FSE on lesional skin of DfE-induced atopic dermatitis mice effectively alleviated the development of atopic dermatitis-like lesions by suppressing the expression of chemokines, cytokines, and adhesion molecules in keratino- cytes. In addition, among the components of FSE, forsythiaside and phillyrin inhibited the production of thymus- and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC), and regulated activation, normal T cell expressed, and secreted (RANTES) in human keratinocytes [12]. These results suggested that forsythia fruit or leaves might be also useful candidate for treating allergic skin inflammatory disorders.

Molecules 2021, 26, 2362 14 of 21

three bacteria, and it was more effective against S. aureus (MIC = 76.67 µg/mL) than tetra- cycline (MIC = 119.05 µg/mL). However, the activity of phillyrin was not remarkable [57]. These results support the use of forsythia as an herbal medicine for skin diseases. Furthermore, Sung et al. evaluated the in vivo and in vitro therapeutic effects of Forsythia suspensa fruit extract (FSE) in an NC/Nga mouse model exposed to Dermatophagoides farinae crude extract (DfE). Topical application of FSE on lesional skin of DfE-induced atopic dermatitis mice effectively alleviated the development of atopic dermatitis-like lesions by suppressing the expression of chemokines, cytokines, and adhesion molecules in ker- atinocytes. In addition, among the components of FSE, forsythiaside and phillyrin inhibited the production of thymus- and activation-regulated chemokine (TARC), macrophage- Molecules 2021, 26, x FOR PEER REVIEW 14 of 20 derived chemokine (MDC), and regulated activation, normal T cell expressed, and secreted (RANTES) in human keratinocytes [12]. These results suggested that forsythia fruit or leaves might be also useful candidate for treating allergic skin inflammatory disorders. AhluwaliaAhluwalia etet al.al. summarizedsummarized the effectseffects ofof PDE4PDE4 inhibitorsinhibitors onon atopicatopic dermatitisdermatitis inin aa reviewreview articlearticle [[5588].]. InIn particular,particular, increasedincreased PDE4PDE4 activityactivity isis correlatedcorrelated withwith inflammatoryinflammatory dysregulationdysregulation inin patientspatients withwith atopicatopic dermatitisdermatitis [[5959––6161].]. IncreasedIncreased PDE4PDE4 activityactivity waswas alsoalso detecteddetected inin cordcord bloodblood cellscells fromfrom newbornsnewborns withwith atopicatopic parents.parents. leadingleading toto speculationspeculation aboutabout aa geneticgenetic abnormalitya[bnormality [ 61[6].1]. MultipleMultiple in vitro assays have have revealed revealed decreased decreased production production of of PGE2, PGE2, IL IL-4,-4, and and IL IL--10 10in inatopic atopic leukocytes leukocytes exposed exposed to high to high-potency-potency PDE4 PDE4 inhibitors inhibitors [62]. [These62]. These predictions predictions were werelater confirmed later confirmed in vivoin by vivo theby finding the finding that PDE4 that PDE4 inhibitor inhibitorss reduc reduceded cytokine cytokine and mediator and me- diatorrelease, release, either by either directly by directly acting actingon IL-4 on production IL-4 production or the Th2 or the cell Th2 balance cell balance [59]. This [59 clinical]. This clinicalanti-inflammatory anti-inflammatory effect serves effect as serves the rational as the rationalee for develop for developinging PDE4 inhibitor PDE4 inhibitorss for atopic for atopicdermatitis. dermatitis. Currently, Currently, several several PDE4 PDE4inhibitors inhibitors including including topical topical and oral and formulation oral formulation,, have havebeen beendeveloped developed to target to target the infla themmatory inflammatory cascade cascade of atopic of atopicdermatitis dermatitis [58]. [58]. FromFrom thesethese results,results, forsythiaforsythia leaves are expected to have efficacyefficacy against atopic der- matitismatitis asas indicatedindicated inin thethe clinicalclinical photosphotos sentsent fromfrom Dr.Dr. O.O. KatayamaKatayama (Figure(Figure9 ).9).

FigureFigure 9.9. ImprovementImprovement ofof atopicatopic dermatitisdermatitis by the treatment withwith forsythiaforsythia leaf decoction between 1010 JuneJune 20192019 andand 66 JanuaryJanuary 20202020 (photos(photos sentsent fromfrom Dr.Dr. O.O. Katayama).Katayama).

5.2.5.2. EffectsEffects of Forsythia Leaves againstagainst InfluenzaInfluenza AA VirusVirus InfectionInfection DengDeng etet al.al. reportedreported thatthat forsythiasideforsythiaside controlled influenzainfluenza A virus infection and im- provedproved the course after infection infection in in SPF SPF C57BL/6j C57BL/6j mice mice [14 [14]. Specifically,]. Specifically, body body weight weight was was re- reducedduced by by virus virus infection, infection, and and this this change change was was alleviated alleviated in forsythiaside in forsythiaside and and oseltamivir oseltamivir--ad- administeredministered mice. mice. The Thedrugs drugs also suppressed also suppressed pathological pathological damage damage in the lungs in the and lungs reversed and reversedthe upregulation the upregulation of TLR7 mRNA of TLR7 expression. mRNA These expression. findingsThese suggested findings that forsythiaside suggested that can forsythiasideimprove the outcome can improve of influenza the outcome A virus of infection influenza by A inhibiting virus infection replication by inhibiting of the virus. replica- tion ofThe the anti virus.-viral effect of phillyrin against influenza A virus infection in vivo was investi- gated to identify a novel anti-viral drug [13]. The administration of phillyrin at a dose of 20 mg/kg/day for three days significantly prolonged the mean survival time, reduced the lung index, decreased viral titers and IL-6 levels, reduced lung hemagglutinin levels, and attenu- ated lung tissue damage in mice infected with influenza A virus. PDE4 inhibition has been demonstrated to ameliorate acute lung injury caused by influenza A virus in mice [10]. Thus, PDE4 inhibitors such as phillyrin may specifically ameliorate airway and lung inflammation. From these reports, forsythia leaves are expected to have effects against influenza A virus infection. Table 12 presents the results of a preliminary test of the preventive effects of for- sythia leaf tea against influenza A virus in a Japanese middle school between December 2018 and February 2019. The experiment indicated that forsythia leaf tea may be effective for pre- venting influenza virus infection. More detailed intake tests are needed in the future.

Molecules 2021, 26, 2362 15 of 21

The anti-viral effect of phillyrin against influenza A virus infection in vivo was inves- tigated to identify a novel anti-viral drug [13]. The administration of phillyrin at a dose of 20 mg/kg/day for three days significantly prolonged the mean survival time, reduced the lung index, decreased viral titers and IL-6 levels, reduced lung hemagglutinin levels, and attenuated lung tissue damage in mice infected with influenza A virus. PDE4 inhibition has been demonstrated to ameliorate acute lung injury caused by influenza A virus in mice [10]. Thus, PDE4 inhibitors such as phillyrin may specifically ameliorate airway and lung inflammation. From these reports, forsythia leaves are expected to have effects against influenza A Molecules 2021, 26, x FOR PEER REVIEWvirus infection. Table 12 presents the results of a preliminary test of the preventive effects15 of of20 forsythia leaf tea against influenza A virus in a Japanese middle school between December 2018 and February 2019. The experiment indicated that forsythia leaf tea may be effective for preventing influenza virus infection. More detailed intake tests are needed in the future. Table 12. Preliminary test of the preventive effect of forsythia leaf tea against influenza A virus infection. Table 12. Preliminary test of the preventive effect of forsythia leaf tea against influenza A virus infection. Non-Intake Number (%) Intake Number (%) Non-Intake Number (%) Intake Number (%) Infected 15 (12.2) 6 (7.9) NonInfected-infected 108 15 (12.2)(87.8) 70 6 (7.9)(92.1) Non-infectedTotals 108123 (87.8) 70 (92.1)76 Totals 123 76 5.3. Function of Phillyrin as a Phytoestrogen 5.3. FunctionPhytoestrogens of Phillyrin, also as termed a Phytoestrogen plant estrogens, are exogenous estrogens that have the samePhytoestrogens, function as female also hormones termed plant. As the estrogens, major phytoestrogens, are exogenous soy estrogens isoflavone that, haveits intes- the sametinal bacterial function metabolite, as female hormones.equol, and enterolactone, As the major a phytoestrogens, plant lignan metabolite soy isoflavone, generated its intestinalby intestinal bacterial bacteria, metabolite, have been equol, described. and enterolactone, These compounds a plant lignanhave estrogen metabolite-like generated activity, byand intestinal they are bacteria,expected have to have been effects described. such Theseas preventi compoundsng breast have cancer, estrogen-like menopause activity, and andosteoporosis they are in expected women to and have thyroid effects cancer such in as men preventing [63]. breast cancer, menopause and osteoporosisWe conducted in women joint and research thyroid on cancer metabolites in men of [63 lignans]. produced by gastrointestinal flora We[6,6 conducted4], confirming joint that research enterolactone on metabolites was excreted of lignans in urine produced when phillyrin by gastrointestinal was orally floraadministered [6,64], confirming to male SD that rats enterolactone as shown in Figure was excreted 10. The in results urine demonstrated when phillyrin that was the orally risk administeredof breast cancer to was male decreased SD rats as shown shown in in Figure Figure 11 10 and. The menopausal results demonstrated symptoms were that sup- the riskpressed of breast as the cancerurinary was excretion decreased of enterolactone as shown in increases Figure 11 in andhumans menopausal [65]. It was symptoms clarified werethat phillyrin suppressed is metabolized as the urinary by intestinal excretion bacteria of enterolactone to the phytoestrogen increasesin enterolactone. humans [65 ].The It wasestimated clarified metabolic that phillyrin pathway is by metabolized intestinal bacteria by intestinal is presented bacteria in Figure to the 12 phytoestrogen [6]. enterolactone.Based on these The findings, estimated forsythia metabolic leaves pathway are expected by intestinal to be beneficial bacteria material is presenteds based in Figureon the phytoestrogen12[6]. content.

180.0

160.0

140.0

120.0

100.0

80.0

60.0

40.0

20.0 U rinary excretion of enterolactone(μｇ） of excretion U rinary 0.0 Control 1st day 2nd day Figure 10. Urine excretion of enterolactoneenterolactone ((µg)µg) inin malemale SDSD ratsrats followingfollowing the the oral oral administration administration of phillyrinof phillyrin (25 (25 mg/kg) mg/kg) (data (data from from Prof. Prof. H. H. Adlercreutz). Adlercreutz).

Figure 11. Breast cancer risk and urinary excretion of enterolactone in 144 subjects (data from Prof. H. Adlercreutz).

Molecules 2021, 26, x FOR PEER REVIEW 15 of 20

Table 12. Preliminary test of the preventive effect of forsythia leaf tea against influenza A virus infection.

Non-Intake Number (%) Intake Number (%) Infected 15 (12.2) 6 (7.9) Non-infected 108 (87.8) 70 (92.1) Totals 123 76

5.3. Function of Phillyrin as a Phytoestrogen Phytoestrogens, also termed plant estrogens, are exogenous estrogens that have the same function as female hormones. As the major phytoestrogens, soy isoflavone, its intes- tinal bacterial metabolite, equol, and enterolactone, a plant lignan metabolite generated by intestinal bacteria, have been described. These compounds have estrogen-like activity, and they are expected to have effects such as preventing breast cancer, menopause and osteoporosis in women and thyroid cancer in men [63]. We conducted joint research on metabolites of lignans produced by gastrointestinal flora [6,64], confirming that enterolactone was excreted in urine when phillyrin was orally administered to male SD rats as shown in Figure 10. The results demonstrated that the risk of breast cancer was decreased as shown in Figure 11 and menopausal symptoms were sup- pressed as the urinary excretion of enterolactone increases in humans [65]. It was clarified that phillyrin is metabolized by intestinal bacteria to the phytoestrogen enterolactone. The estimated metabolic pathway by intestinal bacteria is presented in Figure 12 [6]. Based on these findings, forsythia leaves are expected to be beneficial materials based on the phytoestrogen content.

180.0

160.0

140.0

120.0

100.0

80.0

60.0

40.0

20.0 U rinary excretion of enterolactone(μｇ） of excretion U rinary 0.0 Control 1st day 2nd day Molecules 2021, 26, 2362 16 of 21 Figure 10. Urine excretion of enterolactone (µg) in male SD rats following the oral administration of phillyrin (25 mg/kg) (data from Prof. H. Adlercreutz).

Molecules 2021, 26, x FOR PEER REVIEW 16 of 20

Figure 11. Breast cancer risk and urinary excretion excretion of of enterolactone enterolactone in in 144 144 subjects subjects (data (data from from Prof. Prof. H. Adlercreutz).

EnterolactoneEnterolactone

PhillyrinPhillyrin PhillygeninPhillygenin

Figure 12. Estimated pathway of the metabolism of phillyrin to enterolactone by intestinal bacteria. Figure 12. Estimated pathway of the metabolism of phillyrin to enterolactone by intestinal bacteria. Based on these findings, forsythia leaves are expected to be beneficial materials based 6.on Toxicity the phytoestrogen and Safety content. of Forsythia Leaves. We examined the toxicity of FLE following a single oral dose and repeated administra- tion6. Toxicity for 14 days and in Safety male ddY of Forsythia mice [66]. LeavesFollowing a single oral dose of as much as 6.67 g/kg/day FLE (equivalentWe examined to 18.4 the g/kg toxicity forsythia of FLE leaves following), some mice a single displaye orald dose slight and sedation repeated and admin- subse- quentistration excitement for 14 days immediately in male ddYafter miceadministration, [66]. Following but this a effect single was oral not dose observed of as much on sub- as sequent6.67 g/kg/day days. The FLE treatment (equivalent did not to 18.4influence g/kg body forsythia weight leaves), as shown some in mice Table displayed 13. slight sedation and subsequent excitement immediately after administration, but this effect was Table 13. Influencenot of observed a single oral on subsequent dose of FLE days.(6.67 g/kg The/day) treatment to male did ddYmice not influence on body weight. body weight as shown in Table 13. Days 0 1 2 3 7 14 Cont (5) 32.9 ± 3.15Table 13.29.Influence1 ± 3.59 of a single33. oral0 ± 3 dose.26 of FLE33. (6.674 ± g/kg/day)2.80 to35. male1 ± 1 ddYmice.88 on37 body.0 ± weight.1.58 FLE (5) 33.5 ± 3.35 29.1 ± 3.15 34.1 ± 2.97 33.7 ± 3.13 36.1 ± 1.89 37.6 ± 1.67 Days 0 1 2 3 7 14 Each value represents the mean ± S.D. (n =5) (g). Cont (5) 32.9 ± 3.15 29.1 ± 3.59 33.0 ± 3.26 33.4 ± 2.80 35.1 ± 1.88 37.0 ± 1.58 FLERepeated (5) 33.5oral± administration3.35 29.1 ± 3.15 of 0.166 34.1 g/kg/day± 2.97 FLE 33.7 (0.46± 3.13 g/kg 36.1 equivalent± 1.89 to 37.6 forsythia± 1.67 leavesEach value) for represents 14 days the did mean not± causeS.D. ( nany= 5) obvious (g). changes to general symptoms, body weight, and organ weight as shown in Table 14. Repeated oral administration of 0.166 g/kg/day FLE (0.46 g/kg equivalent to forsythia Table 14. Influence of repeatedleaves) oral administration for 14 days of did FLE not (0.166 cause g/kg/day) any obvious to male changesddY mice toon generalbody weight symptoms, and organ body weight. weight, and organ weight as shown in Table 14. Adrenal Thymus Hypophysi (n) BW (g) Spleen (g) Liver (g) Renal (g) Lung (g) Heart (g) Testis (g) Brain (g) (mg) (mg) s (mg) Cont (5) 37.1 ± 1.00 0.12 ± 0.019 1.8 ± 0.12 0.63 ± 0.023 0.18 ± 0.012 0.17 ± 0.012 5.3 ± 0.87 50.1 ± 2.23 0.23 ± 0.013 0.48 ± 0.025 1.2 ± 0.42 FLE (5) 38.2 ± 1.31 0.12 ± 0.026 1.9 ± 0.13 0.59 ± 0.043 0.19 ± 0.018 0.17 ± 0.008 5.3 ± 0.88 51.9 ± 14.55 0.24 ± 0.027 0.48 ± 0.021 1.3 ± 0.56 Each value represents the mean ± S.D. (n = 5).

There were no deaths in mice that received a single dose, and the LD50 of FLE was estimated to be at least 10 g/kg or more, suggesting that its toxicity was low at the moment. Prior research identified no acute toxicity of FLE or the ethanol extract of forsythia leaves in mice, even at a daily dose of 61.60 g/kg [67]. Conversely, forsythiaside displayed cytotoxicity in PK-15, Mark-145, and CHK cells with IC50 values of 0.138, 0.087, and 0.384 mg/mL, respectively [68] and also caused acute toxicity in mice (IC50 = 1.98 g/kg) [69]. et al. reported the toxicity and safety of phillyrin, following a single oral dose of 18,100 mg/kg in NIH mice [70]. Mortality was not observed after 14 days, and no clinically relevant adverse effects or variations in body weight or food consumption were observed. The maximum tolerated dose of phillyrin was determined to exceed 18,100 mg/kg. Fur- thermore, sub-chronic toxicity was evaluated, following the oral administration of 0, 540, 1620, and 6480 mg/kg phillyrin for 30 days in SD rats. After 30 days, no mortality or sig- nificant toxicological effects such as decreased food consumption, body weight, or changes of biochemical parameters in serum and vital signs were observed. The no-

Molecules 2021, 26, 2362 17 of 21

Table 14. Influence of repeated oral administration of FLE (0.166 g/kg/day) to male ddY mice on body weight and organ weight.

Hypophysis (n) BW (g) Spleen (g) Liver (g) Renal (g) Lung (g) Heart (g) Adrenal (mg) Thymus (mg) Testis (g) Brain (g) (mg)

Cont (5) 37.1 ± 1.00 0.12 ± 0.019 1.8 ± 0.12 0.63 ± 0.023 0.18 ± 0.012 0.17 ± 0.012 5.3 ± 0.87 50.1 ± 2.23 0.23 ± 0.013 0.48 ± 0.025 1.2 ± 0.42

FLE (5) 38.2 ± 1.31 0.12 ± 0.026 1.9 ± 0.13 0.59 ± 0.043 0.19 ± 0.018 0.17 ± 0.008 5.3 ± 0.88 51.9 ± 14.55 0.24 ± 0.027 0.48 ± 0.021 1.3 ± 0.56 Each value represents the mean ± S.D. (n = 5).

There were no deaths in mice that received a single dose, and the LD50 of FLE was estimated to be at least 10 g/kg or more, suggesting that its toxicity was low at the moment. Prior research identified no acute toxicity of FLE or the ethanol extract of forsythia leaves in mice, even at a daily dose of 61.60 g/kg [67]. Conversely, forsythiaside dis- played cytotoxicity in PK-15, Mark-145, and CHK cells with IC50 values of 0.138, 0.087, and 0.384 mg/mL, respectively [68] and also caused acute toxicity in mice (IC50 = 1.98 g/kg) [69]. Han et al. reported the toxicity and safety of phillyrin, following a single oral dose of 18,100 mg/kg in NIH mice [70]. Mortality was not observed after 14 days, and no clinically relevant adverse effects or variations in body weight or food consumption were observed. The maximum tolerated dose of phillyrin was determined to exceed 18,100 mg/kg. Further- more, sub-chronic toxicity was evaluated, following the oral administration of 0, 540, 1620, and 6480 mg/kg phillyrin for 30 days in SD rats. After 30 days, no mortality or significant toxicological effects such as decreased food consumption, body weight, or changes of biochemical parameters in serum and vital signs were observed. The no-observed-adverse- effect level after 30 days was 6480 mg/kg body weight. These results indicated that the oral phillyrin has low or no toxicity. The additional biochemical and histopathological examinations will be required for further evaluation of the toxicity and safety of forsythia leaves by us. Regarding human intake, no adverse events have been reported for forsythia leaf tea even though it has been on the market in Niigata Prefecture, Japan for more than 17 years.

7. Limitations of the Study In this review, we demonstrated that forsythia leaves have potential value as a health material for reducing obesity in SD rats fed an HFD. However, anti-obesity effects in humans are still to be determined.

8. Conclusions Forsythia fruit, the dried fruit of F. suspensa, is listed in Japanese, Chinese, and Korean Pharmacopoeias. Up to now, many compounds have been identified in the fruit. The major compounds are phillyrin, pinoresinol α-D-glucoside, and forsythiaside. Many pharmacological studies have confirmed that forsythia fruit possesses anti-inflammatory, antioxidant, antiviral, antivomiting, and antitumor activities, as well as hepatoprotective, neuroprotective, and cardiovascular protective effects [71]. Forsythia fruit is used in many Kampo medicines in Japan as a principal drug. How- ever, there are a number of factors that limit the wide use of the forsythia fruit. Fruits cannot be harvested in Japan because F. suspensa growing in Japan does not have fruits. Forsythia fruit used in Kampo medicines is imported from the People’s Republic of . In addition, the use of forsythia fruit as a health food is prohibited in Japan. We found that the major compounds in forsythia leaves are also phillyrin, pinoresinol α-D-glucoside, and forsythiaside, similar to those of forsythia fruit [5,6]. Therefore, we studied the biological effects of forsythia leaves as an alternative to forsythia fruit. In particular, phillyrin was reported as a novel selective PDE4 inhibitor [10]. PDE4 in- hibition is a therapeutic strategy for metabolic disorders [11]. Increased PDE4 activity is correlated with inflammatory dysregulation in patients with atopic dermatitis [59–61]. Furthermore, PDE4 inhibition has been demonstrated to ameliorate acute lung injury caused by influenza A virus in mice [10]. We reported that phillyrin is metabolized to enterolactone as phytoestrogen by gastrointestinal flora. Molecules 2021, 26, 2362 18 of 21

In this review, we summarized our studies on the biological effects of forsythia leaves containing phillyrin and other polyphenolic compounds, particulary against obesity, atopic dermatitis, and influenza A virus infection, and its potential as a phytoestrogen. In conclusion, forsythia leaves were revealed to be useful and safe as a health food containing a PDE4 inhibitor, supporting its use in the treatments of metabolic disorders and inflammatory dysregulation. Furthermore, we expect that forsythia leaves will also be used as one of the new drug sources in the future.

Author Contributions: Conceptualization, S.N.; investigation and resources, T.F., K.M.-S., and S.N.; data curation, S.N., K.M.-S., and T.F.; writing—original draft preparation, S.N.; writing—review and editing, S.N., K.M.-S., J.S., and T.F.; visualization, S.N. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: The studies in this review were conducted according to the “Guide for the Care and Use of Laboratory Animals” (NIH Publication revised in 1996) and the experimental protocols were approved by the Institutional Animal Care and Use Committes at the Suzuka University of Medical Science (N0. 56). Informed Consent Statement: The photos in the clinical treatment were sent from Dr. Osamu Katayama of Katayama Clinic under informed consent with patient. Data Availability Statement: The copyright permissions were obtained for uses of Figures and Tables in this review from Japan Society of Medical and Pharmaceutical Sciences for Traditional Medicine. Acknowledgments: We thank the late Herman Adlercreutz of the University of Helsinki for sending us data of enterolactone, and O. samu Katayama of Katayama Clinic for sending us clinical photos. We are grateful to Mariko Mikame of Suzuka University of Medical Science, Sigeharu Yamaguchi and Hideki Chuma for their technical assistance. We also thank Ihsan Calis of Near East University for the helpful advice on our paper. Conflicts of Interest: The authors declare no conflict of interest.

References 1. Urek, R.; Crncevic-Urek, M.; Cubrilo-Turek, M. Obesity—A global public health problem. Acta Med. Croat. Cas. Hravatske Akad. Med. Znan. 2007, 61, 161–1642. 2. The Japanese Pharmacopoeia, 17th ed.; Minister of Health, Labour and Welfare: Tokyo, Japan, 2016; p. 1934. (In Japanese) 3. Yoshida, T.; Sakane, N.; Wakabayashi, Y.; Umekawa, T.; Kondo, M. Thermogenic, anti-obesity effects of bofu-tsusho-san in MSG-obese mice. Int. J. Obes. Relat. Metab. Disord. J. Int. Assoc. Study Obes. 1995, 19, 717–722. 4. Wang, Z.; Xia, Q.; Liu, X.; Liu, W.; , W.; Mei, X.; , J.; Shan, M.; Lin, R.; Zou, D.; et al. Phytochemistry, pharmacology, quality control and future research of Forsythia suspensa (Thunb.) Vahl: A review. J. Ethnopharmacol. 2018, 210, 318–339. [CrossRef] 5. Noro, Y.; Hisata, Y.; Okuda, K.; Kawamura, T.; Tanaka, T.; Nishibe, S. Phenylethanoid glycosides in the leaves of Forsythia spp. Shoyakugaku Zasshi 1992, 46, 254–256. (In Japanese) 6. Nishibe, S.; Kawamura, T.; Tanaka, T.; Aldercreutz, H. On the contents of lignans in Forsythia Fruit and in Forsythia leaf. Nat. Med. 2001, 55, 300–303. (In Japanese) 7. Murase, T.; Nagasawa, A.; Suzuki, J.; Hase, T.; Tokimitsu, I. Beneficial effects of tea catechins on diet-induced obesity: Stimulation of lipid catabolism in the liver. Int. J. Obes. Relat. Metab. Disord. 2002, 26, 1459–1464. [CrossRef] 8. Wang, L.; Yamasaki, M.; Katsube, T.; Sun, X.; Yamasaki, Y.; Shiwaku, K. Antiobesity effect of polyphenolic compounds from molokheiya (Corchorus olitorius L.) leaves in LDL receptor-deficient mice. Eur. J. Nutr. 2011, 50, 127–133. [CrossRef] 9. Nikaido, T.; Ohmoto, T.; Kinoshita, T.; Sankawa, U.; Nishibe, S.; Hisada, S. Inhibition of cyclic AMP phosphodiesterase by lignans. Chem. Pharm. Bull. 1981, 29, 3586–3592. [CrossRef][PubMed] 10. Coon, T.A.; Mckelvey, A.C.; Weathington, N.M.; Birru, R.L.; Lear, T.; Leikauf, G.D.; , B.B. Novel PDE4 inhibitors derived from Chinese medicine Forsythia. PLoS ONE 2014, 9, e115937. [CrossRef] 11. , C.; Rajagopalan, S. Phosphodiesterase-4 inhibition as a therapeutic strategy for metabolic disorders. Obes. Rev. 2016, 17, 429–441. [CrossRef] 12. Sung, Y.Y.; Yoon, T.; Jang, S.; Kim, H.K. Forsythia suspensa suppresses house dust mite extract-induced atopic dermatitis in NC/Nga mice. PLoS ONE 2016, 11, e0167687. 13. Qu, X.Y.; Li, Q.J.; Zhang, H.M.; Zang, X.J.; Shi, P.H.; Zhang, X.J.; , J.; Zhou, Z.; Wang, S.Q. Protective effects of phillyrin against influenza A virus in vivo. Arch. Pharm. Res. 2016, 39, 998–1005. [CrossRef][PubMed] Molecules 2021, 26, 2362 19 of 21

14. , L.; Pang, P.; , K.; Nie, J.; , H.; Wu, S.; Chen, J.; Chen, X. Forsythoside A controls influenza A virus infection and improves the prognosis by inhibiting virus replication in mice. Molecules 2016, 21, 524. [CrossRef][PubMed] 15. Kitagawa, S.; Hisada, S.; Nishibe, S. Phenolic compounds from Forsythia leaves. Phytochemistry 1984, 23, 1635–1636. [CrossRef] 16. Chiba, M.; Hisada, S.; Nishibe, S. The position of glucose linkage in phillyrin. Chem. Pharm. Bull. 1977, 25, 3435–3436. [CrossRef] 17. Pelter, A.; Ward, R.S.; Rao, V.; Sastry, K.V. Revised structures for pluviatilol, methyl pluviatilol and xanthoxylol: General methods for the assignment of stereochemistry to 2,6-diaryl- 3,7-dioxabicyclo[3.3.0]octane lignans. Tetrahedron 1976, 32, 2783–2788. [CrossRef] 18. Nishibe, S.; Okabe, K.; Tsukamoto, H.; Sakushima, A.; Hisada, S. The structure of Forsythiaside isolated from Forsythia suspensa. Chem. Pharm. Bull. 1982, 30, 1048–1050. [CrossRef] 19. Nishibe, S.; Yamaguchi, S.; Hasegawa, M.; Oba, K.; Fujikawa, T. Anti-obesity effect of Forsythia leaf extract containing polypheno- lic compounds and its mechanism. J. Trad. Med. 2012, 29, 149–155. 20. Rosen, E.D.; Walkey, C.J.; Puigserver, P.; Spiegelman, B.M. Transcriptional regulation of adipogenesis. Genes Dev. 2000, 14, 1293–1307. 21. Ricquier, D.; Thibault, J.; Bouillaud, F.; Kuster, Y. Molecular approach to thermogenesis in brown dipose tissue. J. Biol. Chem. 1983, 258, 6675–6677. [CrossRef] 22. Musovic, S.; Olofsson, C.S. Adrenergic stimulation of adiponectin secretion in visceral mouse adipocytes is blunted in high-fat diet induced obesity. Sci. Rep. 2019, 9, 10680. [CrossRef] 23. Han, L.K.; Sumiyoshi, M.; Zhang, J.; Liu, M.X.; Zhang, X.F.; Zheng, Y.N.; Okuda, H.; Kimura, Y. Anti-obesity action of Salix matsudana leaves (Part 1). Anti-obesity action by polyphenols of Salix matsudana in high fat-diet trated rodent animals. Phytother. Res. 2003, 17, 1188–1194. [CrossRef] 24. Zhao, Y.; Li, F.Y.; An, X.; Zhou, M. Effect of phillyrin on the anti-obesiy in nutritive obesity mice. Zhong Yao . 2005, 28, 123–124. (In Chinese) 25. Kraynik, S.M.; Miyaoka, R.S.; Beavo, J.A. PDE3 and PDE4 isozyme-selective inhibitors are both required for synergistic activation of brown adipose tissue. Mol. Pharmacol. 2013, 83, 1155–1165. [CrossRef][PubMed] 26. Nishibe, S.; Yamaguchi, S.; Hasegawa, M.; Oba, K.; Fujikawa, T. Anti-obesity effect of the mixture of herbal extracts containing a new material, Forsythia leaf extract. J. Trad. Med. 2013, 30, 82–90. 27. Kusano, A.; Nikaido, T.; Kuge, T.; Ohmoto, T.; Monache, G.D.; Botta, B.; Botta, M.; Saitoh, T. Inhibition of adenosine 30,50-cyclic monophosphate phosphodiesterase by flavonoids from Licorice roots and 4-arylcoumarins. Chem. Pharm. Bull. 1991, 39, 930–933. [CrossRef] 28. Kubo, K.; Kiyose, C.; Ogino, S.; Saito, M. Suppressive effect of Citrus aurantium against body fat accumulation and its safety. J. Clin. Biochem. Nutr. 2005, 36, 11–17. [CrossRef] 29. Kojima, K.; Shimada, T.; Nagareda, Y.; Watanabe, M.; Ishizaki, J.; Sai, Y.; Miyamoto, K.; Aburada, M. Preventive effect of geniposide on metabolic disease status in spontaneously obese type 2 diabetic mice and free fatty acid-treated HepG2 cells. Biol. Pharm. Bull. 2011, 34, 1613–1618. [CrossRef][PubMed] 30. Ohno, A.; Sakata, M.; Morimoto, Y.; Tajima, S. Effects of Bofu-tsusho-san (Fang-Feng-Tong- Sheng-San) on body weight and fat content in normal mice. J. Trad. Med. 2001, 18, 33–38. (In Japanese) 31. Akagiri, S.; Naito, Y.; Ichikawa, H.; Mizushima, K.; Takagi, T.; Handa, O.; Kokura, S.; Yoshikawa, T. Bofutsushosan, an oriental herbal medicine, attenuates the weight gain of white adipose tissue and the increased size of adipocytes associated with the increase in their expression of uncoupling protein 1 in high-fat diet-fed male KK/Ta mice. J. Clin. Biochem. Nutr. 2008, 42, 158–166. [CrossRef][PubMed] 32. Nakayama, T.; Suzuki, S.; Kudo, H.; Sassa, S.; Nomura, M.; Sakamoto, S. Effects of three Chinese herbal medicines on plasma and liver lipids in mice fed a high-fat diet. J. Ethnopharmacol. 2007, 109, 236–240. [CrossRef] 33. Hioki, C.; Yoshimoto, K.; Yoshida, T. Efficacy of bofu-tsusho-san, an oriental herbal medicine, in obese Japanese women with impaired glucose tolerance. Clin. Exp. Pharmacol. Physiol. 2004, 31, 614–619. [CrossRef] 34. Hu, E.; , P.; Spiegelman, B.M. AdipoQ is a novel adipose-specific gene dysregulated in obesity. J. Biol. Chem. 1996, 271, 10697–10703. [CrossRef] 35. Fujikawa, T.; Hirata, T.; Wada, A.; Kawamura, N.; Yamaguchi, Y.; Fujimura, K.; Ueda, T.; Yurugi, Y.; Soya, H.; Nishibe, S. Chronic administration of eucommia leaf stimulates metabolic function of rats across several organs. Br. J. Nutr. 2010, 104, 1868–1877. [CrossRef] 36. Matsuzawa, Y.; Funahashi, T.; Kihara, S.; Shimomura, I. Adiponectin and metabolic syndrome. Arter. Thromb. Vasc. Biol. 2004, 24, 29–33. [CrossRef] 37. Lowell, B.B.; Susulic, V.S.; Hamann, A.; Lawitts, J.A.; Hagen, J.H.; Boyer, B.B.; Kozak, L.P.; Flier, J.S. Development of obesity in transgenic mice after genetic ablation of brown adipose tissue. Nature 1993, 366, 740–742. [CrossRef] 38. Hirata, T.; Ikeda, T.; Fujikawa, T.; Nishibe, S. The chemistry and boactivity of Eucommia ulmoides Oliver leaves. In Studies in Natural Products Chemistry, 1st ed.; Atta-ur-Rahman, F.R.S., Ed.; Elsevier: Amsterdam, The Netherlands, 2014; Volume 41, pp. 235–240, 256–257. 39. Hosoo, S.; Koyama, M.; Kato, M.; Hirata, T.; Yamaguchi, Y.; Yamasaki, H.; Wada, A.; Wada, K.; Nishibe, S.; Nakamura, K. The restorative effects of Eucommia ulmoides Oliver leaf extract on vascular function in spontaneously hypertensive rats. Molecules 2015, 20, 21971–21981. [CrossRef] Molecules 2021, 26, 2362 20 of 21

40. Fujikawa, T.; Hirata, T.; Hosoo, S.; Nakajima, K.; Wada, A.; Yurugi, Y.; Soya, H.; Matsui, T.; Yamaguchi, A.; Ogata, M.; et al. Asperuloside stimulates metabolic function in rats across several organs under high-fat diet conditions, acting like the major ingredient of Eucommia leaves with anti-obesity activity. J. Nutr. Sci. 2012, 1, e10. [CrossRef] 41. Yoshinaga, N.; Mori, N. Diverse insect adaptation strategies for the defensive substance iridoid individual strategy and evolution- ary direction. Kagaku Seibutsu 2018, 56, 454–456. (In Japanese) [CrossRef] 42. Trim, A.R.; Hill, R. The preparation and properties of aucubin, asperuloside and some related glycosides. Biochem. J. 1952, 50, 310–319. [CrossRef] 43. McCreight, L.J.; Bailey, C.J.; Pearson, E.R. Metformin and the gastrointestinal tract. Diabetologia 2016, 59, 426–435. [CrossRef] [PubMed] 44. Watanabe, M.; Horai, Y.; Houten, S.M.; Morimoto, K.; Sugizaki, T.; Arita, E.; Mataki, C.; Sato, H.; Tanigawara, Y.; Schoonjans, K.; et al. Lowering bile acid pool size with a synthetic farnesoid X receptor (FXR) agonist induces obesity and diabetes through reduced energy expenditure. J. Biol. Chem. 2011, 286, 26913–26920. [CrossRef] 45. Kajiyama, G. The effects of bile acid on serum cholesterol. J. Jpn. Atheroscler. Soc. 1998, 26, 47–51. (In Japanese) [CrossRef] 46. Zietak, M.; Kozak, L.P. Bile acids induce uncoupling protein 1-dependent thermogenesis and stimulate energy expenditure at thermoneutrality in mice. Am. J. Physiol. Endocrinol. Metab. 2016, 310, E346–E354. [CrossRef] 47. Nakamura, A.; Yokoyama, Y.; Tanaka, K.; Benegiamo, G.; Hirayama, A.; , Q.; Kitamura, N.; Sugizaki, T.; Morimoto, K.; Itoh, H.; et al. Asperuloside improves obesity and type 2 diabetes through modulation of gut microbiota and metabolic signaling. iScience 2020, 23, 101522. [CrossRef] 48. Fujisaka, S.; Usui, I.; Nawaz, A.; Igarashi, Y.; Okabe, K.; Furusawa, Y.; Watanabe, S.; Yamamoto, S.; Sasahara, M.; Watanabe, Y.; et al. Bofutsushosan improves gut barrier function with a bloom of Akkermansia muciniphila and improves glucose metabolism in mice with diet-induced obesity. Sci. Rep. 2020, 10, 5544. [CrossRef] 49. Cani, P.; Vos, W.M. Next-generation beneficial microbes: The case of Akkermansia muciniphila. Front. Microbiol. 2017, 8, 1765. [CrossRef] 50. Imai, K.; Kobayashi, Y.; Hirata, T.; Yoshikawa, S.; Ueda, T.; Nishibe, S. Effects of a food containing Eucommia leaf extract on body fat reduction. Jpn. Pharmacol. Ther. 2017, 45, 93–102. (In Japanese) 51. Brufau, G.; Bahr, M.J.; Staels, B.; Claudel, T.; Ockenga, J.; Boker, K.H.; Murphy, E.J.; Prado, K.; Stellaard, F.; Manns, M.P.; et al. Plasma bile acids are not associated with energy metabolism in humans. Nutr. Metab. 2010, 7, 73. [CrossRef] 52. Kawada, T. Food function study of obesity and fat energy metabolism. Nippon Eiyo Shokuryo Gakkaishi 2014, 67, 119–125. (In Japanese) [CrossRef] 53. Park, S.J.; Ahmad, F.; Philp, A.; Baar, K.; Williams, T.; Luo, H.; Ke, H.; Rehmann, H.; Taussig, R.; Brown, A.L.; et al. Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases. Cell 2012, 148, 421–433. [CrossRef] 54. Kobayashi, T.; Glatz, M.; Horiuchi, K.; Kawasaki, H.; Akiyama, H.; Kaplan, D.H.; Kong, H.H.; Amagai, M.; Nagao, K. Dysbiosis and Staphylococcus aureus colonization drives inflammation in atopic dermatitis. Immunity 2015, 42, 756–766. [CrossRef] 55. Nishibe, S.; Okabe, K.; Tsukamoto, H.; Sakushima, A.; Hisada, S.; Baba, H.; Akisada, T. Studies on the Chinese crude drug “Forsythiae Fructus” VI. The structure and antibacterial activity of suspensaside isolated from Forsythia suspensa. Chem. Pharm. Bull. 1982, 30, 4548–4553. [CrossRef] 56. Kitagawa, S.; Nishibe, S.; Baba, H. Studies on the Chinese crude drug “Forsythiae Fructus”. VIII. On isolation of phenylpropanoid glycosides from fruits of and their antibacterial activity. Yakugaku Zasshi. 1987, 107, 274–278. (In Japanese) [CrossRef] 57. Qu, H.; Zhang, Y.; Wang, Y.; Li, B.; Sun, W. Antioxidant and antibacterial activity of two compounds (forsythiaside and forsythin) isolated from Forsythia suspensa. J. Pharm. Pharmacol. 2008, 60, 261–266. [CrossRef] 58. Ahluwalia, J.; Udkoff, J.; Waldman, A.; Borok, J.; Eichenfield, L.F. Phosphodiesterase 4 inhibitor therapies for atopic dermatitis: Progress and outlook. Drugs 2017, 77, 1389–1397. [CrossRef] 59. Hanifin, J.M.; Chan, S.S.; Cheng, J.B.; Tofte, S.J.; Henderson, W.R., Jr.; Kirby, D.S.; Weiner, E.S. Type 4 phosphodiesterase inhibitors have clinical and in vitro anti-inflammatory effects in atopic dermatitis. J. Investig. Dermatol. 1996, 107, 51–56. [CrossRef] 60. Grewe, S.R.; Chan, S.C.; Hanifin, J.M. Elevated leukocyte cyclic AMP-phosphodiesterase in atopic disease: A possible mechanism for cyclic AMP-agonist hyporesponsiveness. J. Allergy Clin. Immunol. 1982, 70, 452–457. [CrossRef] 61. Butler, J.M.; Chan, S.C.; Stevens, S.; Hanifin, J.M. Increased leukocyte histamine release with elevated cyclic AMP- phosphodiesterase activity in atopic dermatitis. J. Allergy Clin. Immunol. 1983, 71, 490–497. [CrossRef] 62. Heskel, N.S.; Chan, S.C.; Thiel, M.L.; Stevens, S.R.; Casperson, L.S.; Hanifin, J.M. Elevated umbilical cord blood leukocyte cyclic adenosine monophophate-phosphodiesterase activity in children with atopic parents. J. Am. Acad. Dermatol. 1984, 11, 422–426. [CrossRef] 63. Nishibe, S. Bioactive phenolic compounds for cancer prevention from herbal medicines. In Food Factors for Cancer Prevention; Ohigashi, H., Osawa, T., Terao, J., Watanabe, S., Yoshikawa, T., Eds.; Springer: Tokyo, Japan, 1997; pp. 276–279. 64. Heinonen, S.; Nurmi, T.; Liukkonen, K.; Poutanen, K.; Wahala, K.; Deyama, T.; Nishibe, S.; Adlercreutz, H. In vitro metabolism of plant lignans: New precursors of mammalian lignans enterolactone and enterodiol. J. Agric. Food Chem. 2001, 49, 3178–3186. [CrossRef] 65. Nishibe, S. On the utilization of Forsythia Leaf. Medical Herb. 2015, 34, 30–34. (In Japanese) Molecules 2021, 26, 2362 21 of 21

66. Nishibe, S.; Kawamura, T.; Fujikawa, T. The toxicity test of Forsythia leaf extract by single and repeated oral administration to mice. In Proceedings of the 135th Annual Meeting of the Pharmaceutical Society of Japan, Kobe, Japan, 25–27 March 2015. 27PB-am198 (In Japanese). 67. Ai, X.; Dong, X.T.; Wang, I.J.; Han, X.; Luo, C.R. Acute toxicity test and micronucleus test of Forsythia suspena leaves in mice. J. Anhui Agri. Sci. 2011, 11, 6397–6398. 68. Wu, J.F.; Zhang, Z.W.; Kuang, L.; , H.; , Y.; Wu, G.J. Study on the toxicity of forsythiaside on cell culture in vitro. Chin. J. Vet. Drug. 2009, 6, 4–8. 69. Mao, D.Y.; Zhang, Z.W.; Yang, M.; Wang, H.C.; Wu, G.J. Acte toxicity and the induction on IFN-á of forthythiaside in mice. Prog. Vet. Med. 2009, 6, 15–17. 70. Han, Z.; Lei, X.L.; Zhang, H.; Liu, L.; Chen, Z.S.; Yang, W.; Lun, Z.R. Evaluating the safety of forsythin from Forsythia suspensa leaves by acute and sub-chronic oral administration in rodent models. Asian Pac. J. Trop. Med. 2017, 10, 47–51. [CrossRef] 71. Dong, Z.; Lu, X.; Tong, X.; Dong, Y.; Tang, L.; Liu, M. Forsythiae Fructus: A review on its phytochemistry, quality control, pharmacology and pharmacokinetics. Molecules 2017, 22, 1466. [CrossRef]