Aphrodisiac Principles and other Constituents from the Roots of Panax quinguefolium and Panax ginseng

Bioassay-guided fractionation of P. quinquefolium and P. ginseng root extracts afforded six compounds. Among these, two bioactive compounds ginsenoside Re (1) and (20S)-ginsenoside Rg2 (5) exhibiting significant relaxation in rabbit corpus cavernosum with EC50 values of 95.1 and 114.7 μM, respectively. In addition, the phytochemical composition of the water extract of the roots of P. quinquefolium was investigated, and thirty-one compounds were characterized, including four undescribed compounds panajaponol B (18) and panaxjapynes D-F (21–23). Moreover, the spectral characteristics and biosynthetic pathway of Panax triterpene saponins were discussed according to our results and some previous reports.

Panax roots (Araliaceae) have been extensively applied as Chinese herbal medicine or healthy food in East Asian for a long period of time due to their well-known medicinal properties.The pharmacological studies of Panax species are extensively reported including the anticancer, 1,2 immunomodulatory, 3,4 antiinflammatory, 5 antiallergic, 6 neuroprotective, 7 antihypertensive, 8 and antidiabetic effects. 9,10The phytochemical research previously performed characterize various chemical constituents, and among these triterpenoid saponins further classified into two subgroups based on their aglycons′ skeletons, namely, dammarane-and oleanane-type, is eminent for the broadband bioactivities.The dammarane-type triterpenoid saponins are well-known as ginsenosides, including (20S)-protopanaxadiol and (20S)-protopanaxatriol.Oleanolic acid type ginsenosides seem to be the typical principles of ginseng species such as P. ginseng, P. japonicus (Japanese ginseng), and P. pseudoginseng subsp.himalaicus (Himamayan ginseng), P. vietnamensis (Vietnamese ginseng), and P. zingiberensis (ginger ginseng). 11In our previous publication, the rarely presented polyacetylenes and their inhibitory activities on α-glucosidase were reported from P. japonicus C. A. Meyer var.major. 12For the purpose of exploring new lead compounds from natural sources, this study aimed to investigate the bioactive constituents of the Panax roots.
Nowadays most of the research results of male sexual dysfunction were mainly focused on rapid ejaculation (RE) and erectile dysfunction (ED). 13The goal for RE therapy was usually to improve patient control over ejaculation timing.Since the walls of the vas deferens, seminal vesicles, ejaculatory ducts, and prostate were lined with smooth muscle cells, the previously published research results indicated that the relaxant agent of smooth muscle could treat RE significantly. 14In comparison, the penile arteries and erectile tissue (corpus cavernosum) had to dilate as the erection took place, thereby the blood flow into the penis was increased. 15The extents of corpus cavernosal smooth muscle contraction determined the functional states of penile flaccidity (or detumescence) and erection (tumescence).Therefore, exploring some relaxing principles of the corpus cavernosum could be a potential strategy to induce penile erection.In the preliminary bioactivity examination, the water extract of P. quinquefolium and methanol extract of P. ginseng displayed the significant relaxation of corpus cavernosum with EC 50 values of 1.67 and 0.26 mg/mL, respectively.Therefore, successive fractionation of the extracts and Diaion HP-20 column chromatography isolation afforded several fractions, and all of the fractions were examined for their relaxation activity of corpus cavernosum.Further purification of the bioactive fractions resulted in several bioactive principles (1−6).In addition, the phytochemical composition of the water extract of P. quinquefolium roots was investigated, and four undescribed compounds panajaponol B (18) and panaxjapynes D-F (21−23) were characterized by the spectroscopic and spectrometric analytical methods.These observations are helpful for the further development of new natural aphrodisiac principles.

■ RESULTS AND DISCUSSION
The roots of P. quinquefolium were extracted with water under reflux and concentrated in vacuo to obtain a deep brown syrup.The extract was partitioned with ethyl acetate and water to obtain two soluble layers and two batches of precipitates, respectively.The ethyl acetate soluble fraction was subjected to Diaion HP-20 column chromatography to produce four subfractrions.Similarly, the methanol extract of P. ginseng was partitioned with ethyl acetate and water to obtain two soluble layers.The successive isolation of this ethyl acetate soluble layer by Diaion HP-20 column chromatography afforded four subfractions.All the resulting layers and subfractions were examined for their relaxation activity of corpus cavernosum of rats, and the precipitates from ethyl acetate layer of P. quinquefolium water extracts (PQEP) and the ethyl acetate subfraction of ethyl acetate layer of P. ginseng methanol extracts (PGEE) exhibited the most significant relaxation of corpus cavernosum with EC 50 values of 0.06 and 0.38 mg/mL, respectively (see Supporting Information, Figure S1).Continuous conventional column chromatographic resolution (Figure S2) of these bioactive fractional samples (PQEP and PGEE) totally resulted in six compounds, including ginsenoside Re (1), 16 ginsenoside Rg1 (2), 17 ginsenoside Rf (3), 18 notoginsenoside R2 (4), 19 (20S)- ginsenoside Rg2 (5), 20 and (20S)-ginsenoside Rg3 (6) 21 (Figure 1).Their chemical structures were identified by comparison of their physical and spectral data with those published in the literature.These purified compounds were examined for their relaxation activity of corpus cavernosum, and among the tested compounds, 1 and 5 displayed the most significant relaxation potential with EC 50 values of 95.1 and 114.7 μM, respectively (Table 1).It was evident that these bioactive principles were responsible for the aphrodisiac activity of the root extracts of P. quinquefolium and P. ginseng.
The above experimental data were further compared with those afforded by molecular docking.Phosphodiesterases (PDE) are enzymes that catalyze the cleavage of the phosphodiester bond of cyclic adenosine monophosphate (cAMP) and cyclic guanidine monophosphate (cGMP).These enzymes control intracellular concentrations of cAMP and cGMP, which play a vital role in mediating the activation of protein kinase to phosphorylate substrates responsible for regulating smooth muscle contraction.cGMP specific phosphodiesterase-5 (PDE5) isoform is expressed in smooth muscle tissue, specifically in the corpus cavernosum. 22Many PDE5 inhibitors were clinically approved to be marketed as drugs for treatment of human male erectile dysfunction, including sildenafil (Viagra), vardenafil (Levitra), tadalafil (Cialis), and udenafil (Zydena).Thus, following the relaxation activity of corpus cavernosum examination, a molecular docking study was conducted to determine the binding abilities of purified compounds to PDE5.
In the present molecular docking study, we used ligand vardenafil (a clinically approved PDE5 inhibitor) as a positive control.The calculated binding energy of positive control was −8.9 kcal/mol.Among the molecular docking results (Table 2), all the binding energy values were below −6.0 kcal/mol, which was common value for the selection of potential candidates that are currently accepted in drug design. 23The docking results showed that ginsenosides purified from the root extracts of P. quinquefolium and P. ginseng have similar inhibitory effects on PDE5 as vardenafil.Compounds 1 and 5 revealed the lowest binding energy values of −8.3 and −7.8 kcal/mol, respectively.These in silico evaluations were consistent with our aphrodisiac bioactivity examination.By exploring the binding mode interactions of purified compounds and PDE5, the aglycone part of ligands linked proteins with alkyl or pi-alkyl interactions (Figure 2).Compounds 3, 4, and 5 showed similar interactions with Phe786, Phe820, Tyr612, Ala767, Ile768, and Val782.Moreover, the residues Asn662, His657, and Asp764 formed hydrogen bond with R1 sugar of compound 1, probably enhanced the binding affinity between compound 1 and PDE5.
In vivo and in silico studies indicated that compounds 1 and 5 have the most significant potential and lowest binding energy values.Ginsenoside Re (1) and (20S)-ginsenoside Rg2 (5)  have two polysaccharides at the C-3 position.These data suggested that two polysaccharides linked to the aglycone at C-3 contribute to aphrodisiac bioactivity and protein binding affinity.In addition, substitution of the C-3 position by α-Lrhamnopyranosyl-(1 → 2)-β-D-glucopyranoside has the advantage in bioactivity and binding affinity.The present results showed a few discrepancies as compare with the literature previously published, 24 among which ginsenosides Re (1), Rg2 (2), and Rf (3) were not active according to those in silico calculations.These differences may originate from the pocket size of the binding protein, and these results should be clarified through more detailed animal experiments.
Compound 23 displayed very similar UV absorption maxima, IR characteristic absorption peaks, and typical 1 Hand 13 C NMR signals (Figures S32 and S33) as those of 22.The most significant variation of its spectral data should be the disappearance of the terminal C−C double bond, which was replaced by a saturated carbon chain indicative of the methyl triplet [δ 0.88 (1H, J = 6.4 Hz, H-17)], and this was also supported by its ESI-MS analytical data (m/z 363 for [M + Na] + ) (Figure S34).The 2D NMR analyses (Figures S35 and  S38), including the COSY and HMBC spectra, both suggested that the basic structure of 23 was the same as that of 22, and only the terminal olefinic group was saturated in 23.Conclusively, 23 was determined as shown and named trivially as panaxjapyne F; similarly, the absolute configurations remained undeteremined.
According to the present research results, we can clarify the spectral characteristics of these purified triterpenoid saponins.These natural ginsenoside derivatives could be classified into three basic skeletons, i.e. dammarane, ocotillol, and oleanolic acid (Figure S39).The first dammarane type compounds exhibited 30 carbon signals including eight methyls and one set of double bonds resonated at the region of δ 125.0−132.0.Two common subtypes of dammarane basic skeleton were reported, including the protopanaxtriol and protopanaxdiol.For the protopanaxtriol subtype, the C-3, -6, and -20 were hydroxylated and even more glycosylated, but usually only one of C-3 and C-6 would be linked with a sugar moiety.In comparison, in the protopanaxdiol subtype usually only C-3 and -20 were hydroxylated or glycosylated.These two subtypes could be differentiated from the chemical shift of CH 3 -28 and C-5, among which the downfield shift of CH 3 -28 (∼δ 2.00) and C-5 (>δ 60.0) were the indication of protopanaxtriol derivatives.The chemical shift of glycosylated C-3 was close to δ 89.0 compared to the unglycosylated carbon at δ 78.0.In comparison, the chemical shift of glycosylated C-20 was close to δ 83.0 compared to that of the unglycosylated carbon at δ 73.0.Another observed significant signal is one quaternary carbon located at δ 70.0−72.0representative of C-20 of the dammarane type triterpenoid.The stereochemistry at C-20 could be trivially deduced by the chemical shift of C-22, in which C-20 was assigned as S if C-22 was located around δ 35.0−36.0,whereas C-20 was assigned as R if C-22 was resonated at the region of δ 42.0−44.0.The second ocotillol type triterpenoids also contained 30 carbon signals including eight methyls; however, there were not any olefinic carbons observed.Usually C-3, C-12, and C-25 were hydroxylated, and sometimes C-6 was also hydroxylated and glycosylated.The absolute configuration at C-24 could be established as S if C-24 was located at δ 85.0.In contrast, C-24 would be assigned as R while C-24 was downfield shifted to δ 88.0.The third skeleton of these natural triterpenoid saponins belongs to the oleanolic acid basic structure.This type of ginsenoside derivatives exhibited 30 carbon signals including seven methyl groups which were one fewer than those of another two types, and one set of double bond resonated at the region of δ 123.0−144.5 characteristic for the C-12/C-13 olefinic functionality.Among most of the examples, only C-3 was hydroxylated or glycosylated.The C-28 was usually oxidized to a carboxylic acid group and further glycosylated in some cases, resulting in the upfield shift of C-28.
The commonly acceptable biosynthetic mechanism of triterpenoid was well studied in the previous report (Figure S40). 46The squalene oxide was arranged in a chair-boat-chairboat conformation and catalyzed by cycloartenol synthase to result in most plant sterols.In contrast, another chair−chair− chair−boat conformation of squalene oxide would afford dammarane-and oleanane-type triterpene saponins.The dammarenediol synthase would produce the dammarenyl cation, and further hydration at C-20 usually resulted in the 20S-dammarenediols while a few 20R-epimer could be observed (Figure S41). 47The successive oxidation at C-12 and C-6 yielded protopanaxdiol and protopanaxtriol, and further glycosylation produced the ginsenoside derivatives.The ocotillol type triterpenoids were biosynthesized through the protopanaxtriol intermediate, which was under C-24/C-25 epoxidation and then ring closure of 20S−OH to form a tetrahydrofuran 5-membered ring (Figure S42).The oleananetype triterpene β-amyrin was resulted from the catalysis of βamyrin synthase, and the oxidation of CH 3 -28 resulted in the commonly observed oleanolic acid which was further glycosylation to produce the oleanolic acid type ginsenoside derivatives (Figure S43).

■ EXPERIMENTAL SECTION
General Experimental Procedures.Optical rotations of purified compounds were measured by using a Jasco P-2000 digital polarimeter.The ultraviolet (UV) spectra were obtained by a Hitachi U-2001 UV/vis spectrometer.The infrared (IR) spectra were examined with a Jasco FT/IR-4000 FTIR spectrometer. 1 H (400 MHz), 13 C (100 MHz), and 2D NMR spectra were recorded on a Bruker Avance III 400 NMR spectrometer using pyridine-d 5 , CD 3 OD, or CDCl 3 as the solvents.Chemical shifts are shown in δ values (ppm) with tetramethylsilane as an internal standard.ESI and HR-ESI mass spectra were measured on a Bruker APEX II mass spectrometer (operated in positive-ion mode).Reversed-phase column chromatography (CC) was accomplished with Diaion HP-20 and Sephadex LH-20 columns.Silica gel column chromatography (SiO 2 CC) was carried out using a Kieselgel 60 (70−230 and 230−400 mesh, Merck).Thin-layer chromatography (TLC) was executed on precoated Kieselgel 60 F 254 plates (Merck), with compounds visualized by UV light or spraying with anisaldehyde in 10% (v/v) H 2 SO 4 followed by charring at 110 °C for 10 min.
Plant Materials.The extracts of the roots of P. quinquefolium and P. ginseng were provided by Chuang Song Zong Pharmaceutical Co., Ltd. in Kaohsiung, Taiwan, in 2006.A voucher specimen (TSWu 2006-CSZ-001 and 002) were deposited in Chuang Song Zong Pharmaceutical Co., Ltd. and School of Pharmacy, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Extraction and Isolation.The water extracts of the roots of P. quinquefolium (PQ, 850 kg) were chromatographed on Diaion HP-20 gel eluted by a water step gradient with ethanol to produce the supernatant (PQEE) and precipitate (PQEP) fractions.A part of the precipitate fraction (PQEP, 46 mg) was isolated by high performance liquid chromatography (HPLC) eluted with water and acetonitrile (15:85) to yield 1 (3.2 mg) and 5 (2.0 mg).The methanol extracts of the roots of P. ginseng (PG, 630 g) were partitioned between ethyl acetate and water to obtain the corresponding layers.The ethyl acetate layer (PGEE, 85 g) was eluted on Diaion HP-20 gel by ethyl acetate to afford the eluents, which was further chromatographed on silica gel eluted by ethyl acetate step gradient with methanol to produce nine fractions.The fifth and sixth fractions were isolated by SiO 2 CC eluted by ethyl acetate step gradient with methanol (19:1 to 9:1), and further purification of the minor fractions by HPLC (water: acetonitrile = 15:85) to yield 2 (59.0 mg), 3 (33.5 mg), and 4 (17.2 mg), respectively.Fractions 7 and 9 were chromatographed on Sephadex LH-20 eluted by a water step gradient with methanol, and the resulting minor fractions were further purified to afford 3 (4.0mg), 4 (12.1 mg), 5 (10.2 mg), and 6 (31.8 mg), respectively.Compounds 1−6 purified from these bioactive fractions were subjected to aphrodisiac bioactivity examination.
Panajaponol    Bioactivity Examination.The relaxation activity of corpus cavernosum of rats were examined according to the previously reported method. 48The male Sprague−Dawley rats (250−300 g) were used and housed in a light-controlled room with a 12 h day/night cycle and given free access to food and water.Experiments were approved by the Animal Care Committee of the National Research Institute of Chinese Medicine (No. 97-P-06, 10/22/2008).Tissue preparation and endothelium disruption were performed as described. 48Briefly, after equilibration, the cavernosal strip contractions were evoked by PE (3 μM) or KCl (40 mM).When the contractile response was stabilized, the tested compounds were added to examine the relaxation against different PE doses.The relaxation induced was expressed as a percentage of relaxation against PE-, KCl-evoked contractions running from 0 to 100% and the EC 50 was calculated by PCS 4.0 software (Pharmacological Calculation System, Springer-Verlag, New York, NY, USA).
Molecular Docking Study.The in silico evaluation was conducted on AutoDock Vina software. 49The crystal structure of the 3′,5′ -cyclic phosphodiesterase enzyme (PDE5) and its cocrystallized ligand vardenafil has been characterized, 50 and a. PDB file was downloaded from the Protein Databank (PDB ID: 1XP0).Vardenafil (a clinically approved PDE5 inhibitor) was first computed to determine the accuracy of the present docking model.The 3D structures of the ligands were constructed in the Chem3D program.The hydrogen supplement, Gasteiger charge measurement for protein atoms, and selection of flexible torsions for ligands were conducted by AutodockTools (ADT ver.1.5.6).The size of the grid was designed by ligand size and a grid center at dimensions (x, y, and z, respectively): − 22.4, 28.6, 62.7 was determined.The binding affinity energy was provided as docking scores and is shown in kcal/mol.The best interaction was considered to be only the top-scoring pose.The visualization of the best docking interactions was analyzed in Biovia Discovery Studio client 2021. 51■ ASSOCIATED CONTENT * sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.4c04965.

Table 1 .
EC 50 Values of Relaxation of Corpus Cavernosum of Rats for Purified Compounds

Table 2 .
Binding Energies of Purified Compounds 1-5 And Vardenafil Calculated In Silico