Comprehensive profiling of phenolic compounds and triterpenoid saponins from Acanthopanax senticosus and their antioxidant, α-glucosidase inhibitory activities | Scientific Reports

Scientific Reports volume 14, Article number: 26330 (2024) Cite this article

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Acanthopanax senticosus belongs to Araliaceae family and is traditionally used as a tonic. The roots and stems are mainly used as treatments for hypodynamia, rheumatism, and hypertension, but their frequent use may lead to extinction. However, comprehensive and simultaneous analysis of the remaining parts were still limited. There is a need to reorganize them for standardization of functional foods. In this study, 50 phenolic compounds and 82 triterpenoid saponins from the shoots, leaves, fruits, and stems of were characterized using UPLC-QTOF-MS and UPLC-QTRAP-MS/MS. Among them, 52 compounds were newly determined as the cis and malonyl-bound phenolic acids and were found to be structural isomers of Acanthopanax flavonoids and saponins. All compounds were absolutely/relatively quantified, and shoots had the highest content. Peroxynitrite and α-glucosidase inhibitory activities were performed, followed by evaluation of structure-activity relationships. Particularly, hederasaponin B and ciwujianoside B showed remarkable efficacy, which were affected by the C-23 hydroxylation, the C-20(29) double bond, and the presence of rhamnose. These detailed profiling can be used as fundamental data for increasing the utilization of A. senticosus and developing them into functional foods.

Acanthopanax is a thorny shrub belonging to the Araliaceae family and is distributed in Korea, China, Japan, and Russia. Traditionally, it has been used as an adaptogen, and its rhizomes and roots are recorded in the official pharmacopeias of Asian countries as treatments for hypodynamia, rheumatism, and hypertension1,2. Acanthopanax senticosus is named to reflect its long, thin, and densely thorned characteristics and is called Siberian ginseng due to its ginseng-like activity3,4. All parts are used as crude medicines and dietary supplements in the forms of tablets, syrups, capsules, and fluid extracts, and can be consumed as seasoned vegetables, salads, functional tea, and wine5,6. The stems and roots mainly contain phenylpropanoids, lignans, and coumarins, while the fruits are rich in polyphenols, flavonoids, and essential oils, and the major components of leaves are triterpenoid saponins, flavonoids, caffeoylquinic acids, and polysaccharides7,8. Based on these compounds, it has been reported that A. senticosus has biological activities such as antioxidant9, anti-inflammatory10,11, anti-Alzheimer’s disease12,13,14, anti-obesity15,16, anti-diabetic17,18,19, and anti-osteoporosis20,21.

LC-MS is considered a useful tool for comprehensive qualitative and quantitative analyses of food and medicinal plants. High-resolution MS, quadrupole-time of flight mass spectroscopy (QTOF-MS), ion trap-TOF-MS, and Q-orbitrap-MS, are commonly utilized for identification of non-targeted compounds, biomarkers, and fingerprinting. Also, quantification using extracted ion chromatograms (XIC) in MS1 mode is possible22,23. On the other hand, triple Q-MS/MS and hybrid triple Q-linear ion trap (QTRAP)-MS/MS coupled with multiple-reaction monitoring (MRM) are advantageous for fast and sensitive quantification of targeted compounds and therapeutic drug monitoring24. However, their low resolution makes it difficult to identify and quantify metabolites without standards, and calibration curves for all metabolites cannot always be used. Thus, relative quantification may be a realistic method22,24.

Although all parts of the plants are used, the main parts are still the stems and roots, which may cause their extinction. Their comprehensive and simultaneous analysis was still limited. Therefore, we aimed to increase the utilization of remaining parts and reorganize them for the standardization of functional foods by characterizing and quantifying the shoots, leaves, and fruits components along with the stem. Moreover, we evaluated the α-glucosidase and peroxynitrite inhibitory activities of their extracts and triterpenoid saponins, which are main compounds, and attempted to determine the structure-activity relationship of saponins. In the present study, a total of 132 compounds, including 50 phenolic compounds and 82 triterpenoid saponins, were identified and quantified from the shoots, leaves, fruits, and stems of A. senticosus using UPLC-QTOF-MS coupled with UPLC-QTRAP-MS/MS.

Based on retention time, UV, and MS spectra from the literature, a total of 132 compounds consisting of 28 phenolic acids (peaks P1-28), 22 flavonoids (peaks F1-22), and 82 saponins (peaks S1-82), were identified or tentatively estimated in and supported by comparison with 37 reference standards (Table 1, Supplementary Figs. S1, S2). The molecular formulas were confirmed through the protonated and adduct ions in positive mode, and their basic structures are shown in Fig. 1. Additionally, the negative analysis was performed to overcome the difficulty in distinguishing phenolic acid isomers. Moreover, the relative abundance (RA %) of adduct ions was detected differently depending on the compounds. For phenolic compounds, [M + H]+ and [M + Na]+ ions were mainly identified, whereas for saponins, [M + H]+ and [M + NH4]+ were main ions. Previous studies have reported that [M + NH4]+ ion tends to become more prominent than [M + H]+ ion as the proton affinity of the analytes decreases25,26. Phenolic compounds and triterpenoid saponins can also be distinguished by the adduct ions bonded to them.

The basic chemical structures of 132 compounds identified from Acanthopanax senticosus.

Peaks P1-28 showed the characteristic UV absorption of phenolic acids (around 320 nm), and fragment ions were observed with m/z 163, 147, and 177, corresponding to the loss of caffeic acid, p-coumaric acid, and ferulic acid, respectively, in positive mode. On the other hand, the 22 flavonoids were detected with maximum absorption wavelengths of approximately 280 nm (flavanone) and 350 nm (flavonol), and their aglycone ions were observed at m/z 273, 287, 303, and 317. Thus, they were identified as naringenin, kaempferol, quercetin, and isorhamnetin derivatives, respectively.

Peaks P1-5 were identified as caffeoylquinic acids (CQAs) with a molecular formula C16H18O9 by analyzing their protonated ion at m/z 355 (Supplementary Fig. S3). Comparison with elution order and reference standards, peaks P1-4 were confirmed as 1-O-caffeoylquinic acid, neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid, respectively27,28,29. Peak P5 was their isomer and eluted after 5CQA. The MS/MS analysis in negative mode revealed a product ion at m/z 191[QA-H]− from the precursor ion at m/z 353.1[M-H]− (Fig. 2a). According to previous studies30,31, the product ions of CQAs were different depending on the position where caffeic acid was acylated to quinic acid. The m/z 191 ion was generated as the predominant ion for 1 or 5CQA, while the m/z 191, 179, and 135 ions were mainly detected for 3CQA. Also, the acylation of the 4-OH position of quinic acid tended to produce a large m/z 173 ion. By comparing the MS/MS spectra of the reference compounds with literature, peak P5 was estimated to be an isomer of 1 or 5CQA (Fig. 2b, Supplementary Fig. S4). Additionally, it was reported that the trans-CQAs were eluted before the cis forms and were isomerized to the cis forms in plant leaves under UV irradiation32. Peak P5 was proposed to be 5-O-cis-caffeoylquinic acid (5cisCQA) and was first reported in this source. Peaks P11 and P13, generated by dehydration or methylation of peak P5, were tentatively identified as 5-O-cis-p-coumaroylquinic acid and 5-O-cis-feruloylquinic acid, respectively and were first assumed from this source (Supplementary Figs. S3, S4).

(-) ESI-MS/MS spectra of phenolic acids acquired in IDA mode at CE -15 or -35. Caffeoylquinic acids with m/z 353[M-H]−, (a) peak P5, 5-cis-O-caffeoylquinic acid, (b) peak P3, chlorogenic acid; dicaffeoylquinic acids with m/z 515[M-H]−, (c) peak P19, 3-O-cis-caffeoyl-5-O-caffeoylquinic acid, (d) peak P22, 1-O-caffeoyl-5-cis-O-caffeoylquinic acid, (e) peak P18, isochlorogenic acid A, and (f) peak P17, 1,5-di-O-caffeoylquinic acid.

Peaks P15-19, P21, and P22 were identified as dicaffeoylquinic acids (diCQAs) by analyzing its protonated ion at m/z 517 (Supplementary Fig. S3). Compared to standards and literature, peaks P15-18, and P21 were confirmed as 1,4-di-O-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, 1,5-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid, respectively. In the MS2 analysis, the precursor ion at m/z 515.1 of peak P19 gave the product ions similar to 3,5diCQA at m/z 191, 179, 173, and 135, while peak P22 showed a prominent product ion at m/z 191, similar to 1,5diCQA (Fig. 2c-d). Thus, it was confirmed that two compounds were derived from 3,5diCQA and 1,5diCQA, respectively. It was reported that 3CQA and its cis form were eluted closer to each other than the elution intervals of 5CQA and cis-5CQA33. In this study, the elution time interval between 5CQA and 5cisCQA was found to be 1.5 min, which was similar to the interval between peak P22 and 1,5diCQA. However, peak P19 was eluted relatively close to 3,5diCQA. Therefore, peaks P19 and P22 were tentatively identified as 3-O-cis-caffeoyl-5-O-caffeoylquinic acid and 1-O-caffeoyl-5-O-cis-caffeoylquinic acid (1C,5cisCQA), respectively.

Peaks P20, P23-25, which have 86 Da more than diCQAs, showed protonated ions at m/z 603[M + H]+ with the molecular formula C28H26O15 (Supplementary Fig. S3). They showed specific ions at m/z 261 and 243, confirming that the malonyl group (Mal) was bound to quinic acid (Supplementary Fig. S5). The MS3 analysis in negative mode revealed that peaks P20, P23, and P24 were originated from 1,5diCQA or 1C,5cisCQA, and peak P24 was derived from 3,5diCQA, based on the RA % of their product ions (especially m/z 191 and 179 ions) from the precursor ion at m/z 515.1 (Fig. 3a-f). According to Clifford et al.34, CQAs and diCQAs eluted at different times because the increase in the free equatorial of OH group of quinic acid makes the components more hydrophilic than vice versa (1 > 3 > > 5 > 4). In addition, Liao et al.31 conducted the structural analysis of Mal-diCQA isomers through quasi-MS3 analysis. These two compounds showed the product ion pattern similar to 3,5diCQA, and were tentatively identified as 1-O-malonyl-3,5diCQA and 4-O-malonyl-3,5diCQA, respectively. Therefore, considering the RT and MS3 data of the Mal-diCQAs detected in this study, peaks P20, P23- P25 were estimated as 3-O-Mal-1,5diCQA, 3-O-Mal-1 C,5cisdiCQA, 4-O-Mal-1,5diCQA, and 4-O-Mal-3,5diCQA, respectively.

(-) ESI-MS3 spectra of malonyl-dicaffeoylquinic acids with m/z 601[M-H]−, (a) peak P20, 3-O-malonyl-1,5-di-O-caffeoylquinic acid, (b) peak P23, 3-O-malonyl-1-O-caffeoyl-5-cis-O-caffeoylquinic acid, (c) peak P24, 4-O-malonyl-1,5-di-O-caffeoylquinic acid, (d) peak P25, 4-O-malonyl-1,5-di-O-caffeoylquinic acid; standard with m/z 515[M-H]−, (e) 1,5-di-O-caffeoylquinic acid, and (f) isochlorogenic acid A.

Peaks F1, F3, and F4 were confirmed to be the same protonated ion at m/z 627. Peaks F1 and F3 showed a large intensity of the m/z 465[M + H-Glu]+ ion. This suggested that, unlike F4 (quercetin 3-O-sophoroside)28, peaks F1 and F3 had two glucose bound separately (Supplementary Fig. S6). And quercetin derivative with glucose bound to the 7-OH position eluted before the 3-OH conjugate35. Thus, peaks F1 and F3 were derived from hyperoside and isoquercitrin, and were assumed to be quercetin 3-O-galactoside-7-O-glucoside and quercetin 3,7-di-O-glucoside, respectively. Additionally, peak F2 eluted between the two compounds and showed the ion [M + H]+ at m/z 773.2147, which was attributed to one additional glucose (Supplementary Fig. S6). Thus, it was tentatively identified as quercetin 3-O-robinobioside-7-O-glucoside.

Unlike phenolic compounds, triterpenoid saponins are difficult to analyze by UV spectrum due to the absence of chromophore or fluorophore. Thus, saponins analysis through LC-MS is a method with high sensitivity and specificity36. Acanthopanax senticosus includes aglycones such as mesembraynthemoidic acid (m/z 473, MA) echinocystic acid (m/z 473, EA), hederagenin (m/z 473, Hed), serratagenic acid (m/z 487, SA), 11-deoxy-anhydrochiisanogenoic acid (m/z 487, 11-DAA), akebonic acid (m/z 441, Ake), oleanlolic acid (m/z 457, OA), and chiisanogenin (m/z 485, Chii)14,37,38. Among them, MA, EA, and Hed need to be distinguished. For the relative proportions of aglycone ions under the same MS conditions, the MA derivatives were detected with fragment ions in the ratios at m/z 455[aglycone + H-H2O]+ and 437[aglycone + H-2H2O]+ as about 100% and 3%, respectively, while those of EA or Hed were measured as about 100% and 30% or 80% and 100%, respectively (Fig. 4a-c). These results were supported by the fragmentation pattern of the standards and the literature38,39. Based on these results, a total of 82 triterpenoid saponins were composed of 17 MA derivatives, 8 SA derivatives, 15 EA derivatives, 5 Hed derivatives, 11 Ake derivatives, 20 OA derivatives, and 6 other saponins (Table 1).

(+) ESI-MS spectra of hydroxyoleanolic acid derivatives with m/z 1221[M + H]+ (a–c) and 1075[M + H]+ (d–i). Hederagenin derivatives, (a) hederacoside C (standard), (d) peak S22, Ara-Hed-GGR (cauloside D), and (g) dipsacoside B (standard); mesembryanthemoidigenic acid derivatives, (b) peak S6, RA-MA-GGR (ciwujianoside A3), (e) peak S7, Ara-MA-GGR, (h) peak S9, RA-MA-Gen; echinocystic acid derivatives, (c) peak S23, RA-EA-GGR (tauroside H1), (f) peak S29, Ara-EA-GGR, and (i) peak S36, RA-EA-Gen. Ara, arabinosyl; RA; rhamnosyl-(1→2)-arabinosyl; Gen, gentiobioside; GGR, rhamnosyl-(1→4)-glucosyl-(1→6)-glucoside.

Peaks S6 and S23, which had a protonated ion at m/z 1221, were identified as isomers of hederacoside C. Three compounds were confirmed to be isomers with the same sugar moieties via the detection of the same fragment ions, but the RA % of each fragment ion were different. For hederacoside C, the m/z 1221[M + H]+ ion and m/z 1075[M + H-Rham]+, 943[M + H-Rham-Ara]+, and 751[M + H-Rham-2Glu]+ ions were prominently detected (Fig. 4a). On the other hand, peaks S6 and S23 showed high intensity of the m/z 1238[M + NH4]+ or m/z 1092[M + NH4-Rham]+, 960[M + NH4-Ara]+, and 768[M + NH4-GGR]+ ions generated by dissociation of the sugar moieties (Figs. 4b-c). However, peaks S6 and S23 had differences in the RA % of aglycone ions and were tentatively identified as ciwujianoside A3 (MA derivative)27,28,38 and tauroside H1 (EA derivative)15,38,40, respectively.

Peaks S7, S9, S22, S29, and S36 were determined to have the molecular formula C53H86O22 by detection of ions at m/z 1075[M + H]+ and 1092[M + NH4]+ with fragment ions, indicating structures with one less rhamnose unit than peaks S6 and S23. Based on the RA % of aglycone ions, peak S22 was identified as cauloside D and confirmed by comparison with the standard (Fig. 4d). Peaks S7 and S9 were confirmed to be MA derivatives (Fig. 4e, h), and peaks S29 and S36 were EA derivatives (Figs. 4f, i). Fragment ions at m/z 960[M + NH4-Ara]+ in S7 and S29 were observed, while m/z 946[M + NH4-Rham]+ and 814[M + NH4-Rham-Ara]+ ions were detected in S8 and S36. The sugar moieties of the C-3 position of Acanthopanax saponins dissociated one by one from the terminal sugar, but the ester linkage at the C-28 position tended to break easily regardless of the positive or negative mode14,27, supported by MS spectra of dipsacoside B (Fig. 4g). Therefore, S7, S9, S29, and S36 were tentatively identified as 3-O-arbinosyl-MA 28-O-rhamnoyl-(1→4)-glucosyl-(1→6)-glucoside27,41, 3-O-rhamnosyl-(1→2)-arabinosyl-MA 28-O-gentiobioside, 3-O-arabinosyl-EA 28-O-rhamnoyl-(1→4)-glucosyl-(1→6)-glucoside38, and 3-O-rhamnosyl-(1→2)-arabinosyl-EA 28-O-gentiobioside, respectively, with peaks S9 and S36 were reported for the first time in this source.

Peaks S1, S2, S18, and S20 exhibited the protonated ions at m/z 1237 and were isomers of cauloside F. LC-MS analysis confirmed that glucose and arabinose were bound to the C-3 position by detecting fragment ions at m/z 1092[M + NH4-Glu]+ and 960[M + NH4-Glu-Ara]+. Particularly, the RA % of the m/z 1092 ion of peaks S1, S2, S18, and S20 was observed to be 2.9%, 7.0%, 12.5%, and 10.0%, respectively, indicating that there was a difference in the linkage between the two sugars. Peaks S1 and S2 (Fig. 5a-b); peaks S18 and S20 (Fig. 5c-d) were confirmed to have the similar relationship with begoniifolide A (peak S42) and ciwujianoside A1 (peak S46), (Fig. 5e-f). The MS spectra of peaks S42 (14.5%) and S46 (6.9%) showed different RA % of the m/z 1076[M + NH4-Glu]+ ion due to differences of linkage between glucose and arabinose at the C-3 position (glucosyl-(1→3)-arabinosyl, G3A > glucosyl-(1→2)-arabinosyl, G2A). These results suggested that G3A binding may be weaker than G2A binding. Additionally, compounds of G2A binding were identified as the main components. Thus, peaks S1, S2, S18, and S20 were estimated to be 3-O-G2A-MA 28-O-rhamnoyl-(1→4)-glucosyl-(1→6)-glucoside27, 3-O-G3A-MA 28-O-rhamnoyl-(1→4)-glucosyl-(1→6)-glucoside, 3-O-G3A-EA 28-O-rhamnoyl-(1→4)-glucosyl-(1→6)-glucoside, and 3-O-G2A-EA 28-O-rhamnoyl-(1→4)-glucosyl-(1→6)-glucoside, respectively. And peaks S2, S18, and S20 were detected for the first time from Acanthopanax species. Moreover, compounds (peaks S8, S10, S30, and S35) with an additional 42 Da (acetyl group, Ac) attached to aforementioned compounds (peaks S1, S2, S18, and S20) were identified (m/z 1279[M + H]+), and presumed to be isomers of ciwujianoside A4 (peak S8)27,28,31. These compounds were detected as characteristic ions at m/z 513[Rham + Ac + 2Glu + H]+, 367[Ac + 2Glu + H]+, 351[Rham + Ac + Glu + H]+, and 205[Ac + Glu + H]+, attributed to the presence of acetyl groups (Supplementary Fig. S7), which was consistent with previous reports27,31, and supported by the MS spectra of the standard (acanthopanaxoside B). Thus, peaks S10, S30, and S35 were tentatively identified as 3-O-G3A-MA 28-O-rhamnoyl-(1→4)-(6-O-acetyl)glucosyl-(1→6)-glucoside, 3-O-G3A-EA 28-O-rhamnoyl-(1→4)-(6-O-acetyl)glucosyl-(1→6)-glucoside, and 3-O-G2A-EA 28-O-rhamnoyl-(1→4)-(6-O-acetyl)glucosyl-(1→6)-glucoside, respectively, and reported from this source for the first time.

(+) ESI-MS spectra of hydroxyoleanolic acid derivatives with m/z 1237[M + H]+, (a) peak S1, G2A-MA-GGR, (b) peak S2, G3A-MA-GGR, (c) peak S18, G2A-EA-GGR, and (d) peak S20, G3A-EA-GGR; oleanolic acid derivatives with 1221[M + H]+, (e) peak S42, G3A-OA-GGR (begoniifolide A), (f) peak S46, G2A-OA-GGR (ciwujianoside A1); and dihydroxyoleanolic acid derivatives with m/z 665[M + H]+, (g) peak S17, Gluc-29OH-EA, (h) peak S27, Gluc-29OH-Hed, and (i) peak S65, Gluc-Cau; G2A, glucosyl-(1→2)-arabinosyl; G3A, glucosyl-(1→3)-arabinosyl.

Peaks S3, S21, S26, and S40 contained one or two glucoses at the C-3 position based on the detection of their parent ions and fragment ions (Supplementary Fig. S7). Peak S40 (Rt 25.50) was estimated as 3-O-glucosyl-OA 28-O-rhamnoyl-(1→4)-glucosyl-(1→6)-glucoside28,38, which was the component in which arabinose of ciwujianoside C3 (peak S64, Rt 29.00) was replaced by glucose. Peak S26 (Rt 23.74), which possessed one additional glucose unit compared to peak S40, was assumed to be derived from ciwujianoside A1 (peak S46, Rt 26.33), which had a similar elution time interval of the peaks S40 and S64. Since the elution time interval between peaks S3 vs. S8 and peaks S21 vs. S34 was similar to the above, it was inferred that peak S3 was derived from S8, and peak S21 was formed from S34. Therefore, peak S3 was tentatively identified as 3-O-sophorosyl-MA 28-O-rhamnoyl-(1→4)-(6-O-acetyl)glucosyl-(1→6)-glucoside, peak S21 as 3-O-sophorosyl-MA, and peak S26 as 3-O-sophorosyl-OA 28-O-rhamnoyl-(1→4)-glucosyl-(1→6)-glucoside.

Peak S51 showed a signal for the ion [M + NH4]+ at m/z 1192.6113, which corresponds to an addition of 86 Da to the molecular weight of peak S40. Similar to the MS pattern of Q3(6Mal)Glu (peak F14), the characteristic ions were observed at m/z 944[M + H-MalGlu]+ and 231[MalGlu + H-H2O]+. Therefore, peak S51 was tentatively identified as 3-O-(6-O-malonyl)glucosyl-OA 28-O-rhamnoyl-(1→4)-glucosyl-(1→6)-glucoside and was first reported in this source. Peak S82 had a protonated ion at m/z 705.4225[M + H]+, corresponding to the loss of the GGR unit from peak S51. Thus, the structure of peak S82 was assumed to be 3-O-(6-O-malonyl)glucosyl-OA and was reported for the first time from Acanthopanax species (Supplementary Fig. S7).

Peaks S17, S27, and S65 with the molecular formula C36H56O11 (m/z 665[M + H]+), were confirmed to be components with a glucuronide bound to the C-3 position. The predominant ions of three compounds were detected at m/z 453 and 471, generated by H2O loss from the aglycone ion (m/z 489). The RA % of the three compounds at m/z 453 ion were 100%, while their RA % at m/z 471 were 97.1%, 75.0%, and 60.8%, respectively (Fig. 5g-i). Similar to the different aglycone ion patterns of the MA, EA, and Hed derivatives, it is possible that components with an additional hydroxyl group may have different fragment ion patterns depending on the binding position. Similarly, caulophyllogenin (16α,23-dihydroxy-OA) type saponins from Aralia eleta leaves (Araliaceae) were characterized by aglycone ions at m/z 471 and 453 39, and they were reported to exhibit higher anti-cancer and anti-diabetic activities than Hed, E, and OA-type saponins42,43,44. Additionally, 29-hydroxyhederagenin derivatives were identified in the stem bark of A. brachypus and leaves of A. nipponicus45,46. Thus, peak S17, S27, and S65 were tentatively identified as 3-O-glucuronyl-29-hydoxyechinocystic acid, 3-O-glucuronyl-29-hydoxyhederagenin, and 3-O-glucuronyl-caulophyllogenin, respectively, and these compounds were first reported from A. senticosus fruits.

A total content (mg/100 g dry weight, DW) of 132 compounds ranged from 16.33 to 4621.20 and is summarized in Table 2. Triterpenoid saponin derivatives accounted for more than 70% of the total compounds in shoots, leaves, and fruits, but no saponins were identified in the stems. Also, the total phenolic acids content (TPAC) was higher than the total flavonoids content (TFC) in all parts except for the shoots, which was consistent with the previous data9,47,48,49. Similarly, triterpene saponins were reported as the main components of leaves, fruits, and roots, while stems seemed to contain other compounds, including lignans, coumarins, and phenylpropanoid glycosides8,50.

For phenolic compounds, a total content of 28 phenolic acids by parts was distributed from 15.82 to 646.56 mg/100 g DW, with the leaves containing the most phenolic acids, while stems had the lowest content. On the other hand, a total content of 22 flavonoids was highest in shoots, followed by leaves, fruits, and stems. These results were inconsistent with the total phenolic content (TPC) and TFC values of leaves, fruits, and stems of A. senticosus reported by Heo et al.9 and Kim et al.48. This is probably due to differences in the experimental methods (extraction solvent and quantitative method) and the exclusion of the content of other components (lignans, coumarins, etc.) that may be included in the colorimetric quantification. However, it was similar to the above studies in that TPC and TFC were confirmed in the order of leaves > fruits > stems. Comparing shoots and leaves, the TPAC of leaves was about twice as high as that of shoots, while the TFC of shoots was approximately 1.7 times greater than that of the leaves, which was consistent with the previous report51. Sithisarn et al.52 found that the leaves of A. trifoliatus contained more phenolic acids in June and July than the leaves harvested in April. Also, it was confirmed that the stems of Korean A. senticosus harvested in winter contained the most phenolic compounds53. Acanthopanax phenolic compounds appeared to be affected by harvested season and temperature. Studies on other plants have shown similar results, with the TPC and TFC of summer Moringa oleifera leaves found to be higher than those in spring. It was recorded that immature leaves produced primary metabolites rather than secondary metabolites for growth and survival54. In contrast, an increase in respiration rate via the abundant chlorophyll in young tea leaves was associated with an increase in the synthesis of secondary metabolites55,56. It was reported that during the rainy season when precipitation increases, the catechin content decreased, but the concentration of phenols and antioxidant capacity increased. Likewise, quercetin 3-O-glucuronide and nicotiflorin in raspberry leaves were contained more in spring and fall leaves than in summer one, and were mainly accumulated in apical leaves57. Since these secondary metabolites are influenced by various factors, such as genes, maturity, temperature, precipitation, amount of sunlight, bacteria, virus, and insects, rather than a single factor58, the variation in the phenolics content in this study may be related to these factors.

A total content of 82 triterpenoid saponins from shoots, leaves, and fruits ranged from 630.04 to 3576.67 mg/100 g. Proportions of saponins in shoots, leaves, and fruits were 77.7%, 71.5%, and 71.6%, respectively. Among them, OA derivatives appeared to be the most abundant type. The shoots and leaves contained more MA and EA derivatives than fruits, and Ake derivatives ranked second. While, Hed derivatives accounted for 39.8% in the fruits. According to previous studies59,60, saponins are one of the plant defense compounds and tended to accumulate more in young leaves than in mature leaves to protect against bacteria, insects, and herbivores. In addition, previous studies61,62 reported that the expression of the 3-hydroxy-3-methylglutaryl-CoA reductase gene is important for saponin synthesis, with high expression in the somatic embryonic state and stems, and the lowest expression level during leaf senescence. These synthetases can be affected by drought stress, and the decrease in DNA methylation caused by low rainfall was negatively correlated with the saponin content63. Also, saponin content is affected by other abiotic factors such as solar radiation, temperature, soil, and sunshine duration60,64. In this study, the saponin content of each part was different, and particularly, shoots collected in April contained more saponins than leaves harvested in June, indicating that several factors were applied.

Most parts were composed of di-hydroxycinnamoylquinic acid derivatives. Among them, 1,5diCQA accounted for 50.9% of the total phenolic acids in shoots, whereas 3,5diCQA was identified as the predominant compound in leaves (24.5%) and fruits (49.7%). These results are similar to previous data investigated by Zhang et al.65. Compared to the shoots, the proportion of caffeoylshikimic acids and Mal-diCQAs was approximately two times higher in leaves. Hyperoside was the most abundant compound, followed by isoquercitrin, which were consistent with previous data reported by Zhang et al.65, isoquercitrin content in leaves: 123.0 ~ 248.4 mg/100 g; Hu et al.17, hyperoside content in leaves: 147 ~ 3656 mg/100 g; Lee et al.66, hyperoside content in fruits: 49.4 mg/100 g. Especially, naringenin is the precursor of cyanidin, the main anthocyanin in the Acanthopanax fruits67, which may be related to the relatively high proportion of naringenin 7-O-glucoside in the fruits. Meanwhile, ciwujianosides C2 and C4, hederasaponin B, copteroside B, and calenduloside E were identified as the major components accounting for more than 10% of the total saponin content. Hederasaponin B and ciwujianosides C4 were mainly identified in shoots and leaves, which was similar to the previous data17. On the other hand, copteroside B and calenduloside E accounted for 35.2% and 23.8% of fruit saponins, respectively. According to Hwang et al.68, the A. senticosus leaves contained genes related to the synthase of β-amyrin (the precursor of OA) and among the identified cytochrome P450 genes, CYP-18 (β-amyrin 28-oxidase) was particularly likely to be involved in the synthesis of OA types. In addition, Han et al.69 found that CYP72A397 (23-hydroxylase) of Kalopanax septemlobus (Araliaceae) shoots induced the synthesis of hederagenin. In this study, it was assumed that the differences in saponin composition of shoots, leaves, and fruits was due to the presence of different genes in each part, and further research on these is necessary.

The α-glucosidase and ONOO− inhibitory activities of the shoots, leaves, fruits, and stems of were examined. All parts showed significantly greater inhibitory activity against α-glucosidase than positive control, acarbose (Table 3). In particular, the leaves extracts exhibited the most potent activity toward α-glucosidase, followed by the stems, shoots, and fruits. These results suggested that the relatively low activity of the fruits was affected by the low content of phenolic compounds and saponins. These compounds may be potent contributors in the anti-diabetic activity, which were supported by previous results showing that the phenolic and saponin fractions of leaves exhibited higher α-glucosidase inhibitory activity than the 70% ethanol extract17. Also, the diCQAs had a higher influence on the inhibition of this enzyme than the CQAs, and in particular, substitution of the caffeic acid at the OH-4 position of quinic acid was crucial factor. Furthermore, hyperoside, isoquercitrin, quercitirin, and quercetin 3-O-glucuronide were reported to inhibit α-glucosidase more than acarbose70,71,72,73,74. However, the stems showed a high inhibitory capacity despite its low content of these components, which may be related to other components within the stems. In fact, the stem extracts and eleutheroside E, the main component in stems, showed potent anti-diabetic activity in mouse models of type 1 or 2 diabetes75,76,77.

On the other hand, these extracts showed moderate scavenging activity against ONOO− compared to the positive control. The stem extracts were confirmed to have a higher antioxidant capacity than those of other parts, which was similar to the findings of Heo et al.9 (DPPH scavenging activity, stems > leaves > fruits). Compared to shoots, leaves exhibited significantly higher antioxidant activity, which appears to be particularly related to the efficacy of diCQAs and quercetin derivatives identified in the samples78. Similarly, despite the low phenolic compound content in the stems, they exhibited high antioxidant capacity, which was presumed to be due to the lignans and phenylpropanoid glycosides50,79.

The structure-activity relationship of triterpenoid saponins, which occupied most of the components, was evaluated, and the 50% inhibition concentration (IC50) of these saponins for both activities is shown in Table 3. All tested compounds showed significantly higher α-glucosidase inhibitory activity, and hederasaponin B exhibited the most potent activity against α-glucosidase with an IC50 value of 0.47 µM, while hederacoside C showed the lowest activity. The hydroxylation at the C-23 position affected to reduce in the α-glucosidase activity, which was supported by previous studies80,81. Additionally, the presence of rhamnose at C-3 position was assumed to be an important point in α-glucosidase inhibition, and Wang et al.19 and Liu et al.82 also reported similar results. Meanwhile, several compounds exhibited moderate inhibitory activities against ONOO−. Ciwujianoside B showed the most potent activity against ONOO−, whereas, hederacoside C, ciwujianosides A1 and C3 exhibited no scavenging activity at 50 µg/mL. The formation of a double bond between C-20 and C-29 in ciwujianosie B was confirmed to increase ONOO− scavenging activity, which was consistent with previous data showing higher ONOO− radical inhibition for 30-norgypsogenin saponin than for the gypsogenin type83. Furthermore, comparison of cauloside D and dipsacoside B suggested that the presence of the rhamnosyl group at the C-3 position is likely important. The importance of these sugar moieties was also mentioned in anticancer evaluation studies84,85.

Siberian ginseng (A. senticosus) was obtained from a plateau farm in Jinan-gun, Jeollabuk-do, Republic of Korea (latitude/longitude: 35°38′07″N/127°27′20″E), in April, June, September, and November 2020, respectively (Supplementary Fig. S8). The samples were collected with permission of Rural Development Administration (RDA, Republic of Korea), and identified by the authors (Heon-Woong Kim and Sang Hoon Lee) of this article. The voucher specimens (shoots, RDASPCF040; leaves, RDASPCF041; fruits, RDASPCF042; stems, RDASPCF043) by plant parts were deposited in the Department of Agrofood Resources, National Institute of Agricultural Sciences, RDA. Experimental research, including the collection of plant material, complied with relevant institutional, national, and international guidelines and legislation. All samples were lyophilized, crushed with a grinding machine, and sieved through a 30 mesh sieve.

Astragalin, avicularin, caffeic acid, chlorogenic acid methyl ester, p-coumaric acid, ferulic acid, guaijaverin, hyperoside, isoquercitrin, nicotiflorin, quercetin 3-O-glucronide, and quercitrin were supplied from Extrasynthese (Genay, France). 1,5-di-O-caffeoylquinic acid, 5-O-feruloylquinic acid, chlorogenic acid, cryptochlorogenic acid, cynarin, hederacoside C, isochlorogenic acids A-C, neochlorogenic acid, and rutin were obtained from PhytoLab GmbH & Co. (Vestenbergsgreuth, Germany). 1-O-caffeoylquinic acid, acanthopanaxoside B, asperosaponin VI, ciwujianoside C3, and dipsacoside B were provided by MedChemExpress (Monmouth Junction, USA). Caulosides D and F, ciwujianoside A1, and hederasaponin B supplied from TargetMol (Boston, MA, USA). Calenduloside E and α-hederin were obtained from ChemFaces (Biochemical Co., Wuhan, China). 1,4-di-O-caffeoylquinic acid, ciwujianoside B, and quercetin 3-O-(6″-O-malonyl)glucoside were obtained from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China), ALB Materials Inc. (Nevada, USA), and Sigma Aldrich Co. (St. Louis, MO, USA), respectively. Formic acid was purchased from Junsei Chemical (Tokyo, Japan) and LC-MS grade solvents: acetonitrile (ACN) and methanol (MeOH) were purchased from Fisher Scientific (Fair Lawn, NJ, USA).

For in vitro assays, Acarbose, diethylenetriaminepentaacetic acid (DTDA), dihydrorhodamine 123 (DHR 123), p-nitrophenyl α-d-glucopyranoside (pNPG), and yeast α-glucosidase from Saccharomyces cerevisiae were obtained from Sigma Aldrich Co. (St. Louis, MO, USA). Peroxynitrite (ONOO−) was purchased from Bio Rad Laboratories Calbiochem (San Diego, CA, USA). Potassium chloride, sodium carbonate anhydrous, sodium chloride, sodium hydroxide, sodium phosphate dibasic anhydrous, and sodium phosphate monobasic dihydrate were supplied from SAMCHUM (Seoul, Korea).

For UPLC-QTOF-MS and UPLC-QTRAP-MS/MS analyses, a 0.1 g of the powdered samples were extracted twice with 1.5 mL of 70% MeOH using an ultrasonic extractor for 30 min (POWERSONIC 520, Hwasin Technology, Seoul, Korea), and then centrifuged for 15 min at 2,016×g (LABOGENE 1580R, Bio-Medical Science Co., Seoul, Korea). The collected supernatants were filtered through a 0.2 µm PVDF syringe filter (Thermo Fisher Scientific Inc., Waltham, MA, USA) and concentrated using N2 gas, then re-dissolved in 5 mL of distilled water (DW). In order to remove undesirable components and improve separation and detection in UPLC-MS23, the solid phase extraction was performed and the process was as follows. A C18 cartridge (Hypersep C18 500 mg, Thermo Fisher Scientific Inc.) was conditioned with MeOH (3 mL) and DW (6 mL), then the samples were loaded into cartridge and washed with DW (6 mL). Finally, the loaded samples were eluted with 70% MeOH (5 mL) and concentrated using N2 gas, then re-dissolved in 70% MeOH (1 mL).

For in vitro assays, a 5 g of the powdered samples were extracted with 70% MeOH (30 mL, twice) using an ultrasonic extractor for 30 min (POWERSONIC 520, Hwasin Technology, Seoul, Korea), and then centrifuged for 15 min at 2,016×g (LABOGENE 1580R, Bio-Medical Science Co., Seoul, Korea). The collected supernatants were filtered through a 0.2 µm PVDF syringe filter (Thermo Fisher Scientific Inc., Waltham, MA, USA) and concentrated using N2 gas, and MeOH extracts of shoots, leaves, fruits, and stems were dissolved in 10% DMSO to prepare stock solutions (10 mg/mL).

The conditions for UPLC system (Shimadzu, Kyoto, Japan) coupled with QTOF-MS (SCIEX X500R, SCIEX Co., MA, USA) and UPLC-QTRAP-MS/MS (SCIEX QTRAP 4500, SCIEX Co.) were set up as follows. Chromatographic conditions: pre-column, CORTECS UPLC Vanguard T3, 2.1 × 50 mm, 1.6 µm, (Waters Co., Milford, MA, USA); column, CORTECS UPLC T3, 2.1 × 150 mm, 1.6 µm (Waters Co.); column temperature, 30℃; flow rate, 0.30 mL/min; sample injection volume, 1 µL; mobile phase, 0.1% formic acid in DW (A), 0.1% formic acid in ACN (B). Gradient conditions: 0–10 min, 15% B; 10 min, 25% B; 40–45 min, 50% B; 50–60 min, 15% B.

For qualitative analysis of overall compounds, mass spectra were multi-scanned in the range of m/z 100–2000 of positive mode through an electrospray ionization (+ ESI) source, with the corresponding parameters were: ion source temperature, 500℃; spray voltage, 5500 V; ion source gas, 50 psi; curtain gas, 30 psi; declustering potential (DP), 80 V; collision energy (CE), 15 ± 10 V. In order to identify the structure of phenolic compounds, TOF MS/MS analysis was performed in negative and information-dependent acquisition (IDA) mode and the parameters were as follow: ion source temperature, 500℃; spray voltage, -4500 V; ion source gas, 50 psi; curtain gas, 30 psi; DP, -80 V; CE, -15 ± 10 V or -35 ± 10 V. The parameters of MS3 analysis in negative mode using QTRAP 4500 were as follow: Ion source temperature, 500℃; spray voltage, -4500 V; ion source gas, 50 psi; curtain gas, 30 psi; DP, -40 V; CE, -15 ± 10 V; excitation energy, 0.1.

Identification of phenolic compounds and triterpenoid saponins was performed using UPLC-QTOF-MS (SCIEX X500R, SCIEX Co., MA, USA), referring to the mass fragmentation and retention time (RT, min) in previous literature and confirmed with 37 reference standards (Supplementary Fig. S2). Targeted compounds quantification was conducted by UPLC-QTRAP-MS/MS (SCIEX QTRAP 4500, SCIEX Co.) based on MRM mode. The optimized MRM conditions for the 28 external standards are detailed in Supplementary Table S1. Whereas untargeted compounds were relatively quantified with standards (phenolic compounds: cynarin, rutin, and avicularin; triterpenoid saponins: dipsacoside B, aseprosaponin VI, and α-hederin) according to the structural characteristics of identified compounds using XIC in MS1 mode of UPLC-QTOF-MS (SCIEX X500R, SCIEX CO.). To quantify all analysts, the calibration curve was obtained by plotting the peak areas of standards (major fragment ions or MRM transition ions) and regression equation, R2, linear ranges are described in Supplementary Table S2. The limit of detection (LOD) and quantification (LOQ) were determined using the calibration curve data according to the following equations: \(\:\text{L}\text{O}\text{D}=3.3\times\:\delta\:/S\) and \(\:\text{L}\text{O}\text{Q}=10\times\:\delta\:/S\), where δ and S are the standard deviation of the y-intercept and the slope of the calibration curve, respectively (Supplementary Table S2). The intra- and inter-day precisions were determined by analyzing the mixed standard solution (10 µg/mL, n = 3) on a single day and 3 consecutive days, and the results were expressed as the relative standard deviation (RSD %).

Assessment of α-glucosidase inhibition was carried out using spectrophotometric method as previously described86. In brief, α-glucosidase enzyme (0.1 unit) was added to a plate containing the substrate (2.5 mM pNPG) and samples diluted in 100 µM sodium phosphate buffer (pH 6.8) to a total volume of 80 µL. The plate was incubated at 37 °C for 20 min, and then 0.2 M sodium carbonate solution (80 µL) was added to stop the reaction. In the control or positive control, sample solutions were replaced with the sodium phosphate buffer or acarbose. The absorbance was measured at 405 nm using a microplate spectrophotometer (Molecular Devices., San Jose, CA, USA).

In the ONOO− scavenging activity analysis, the antioxidant capacity of samples was evaluated using the principle that highly fluorescent rhodamine 123 is rapidly converted to non-fluorescent DHR 123 in the presence of ONOO−87. Briefly, the rhodamine buffer (pH 7.4) consisting of 100 µM DTPA and 5 µM DHR 123 was prepared and placed on ice before use. After treating the sample (10 µL) with the same amount of 200 µM authentic ONOO− for 5 min, the fluorescence intensities of the oxidized DHR 123 in the control and sample were measured using a fluorescence microplate reader (VERSA MAX GEMINI XPS, Molecular Devices., CA, USA) at excitation (485 nm) and emission (530 nm) wavelengths. l-Penicillamine was used as the positive control.

All results were expressed as mean ± standard deviation of triplicates. One-way ANOVA was performed using SPSS (version 28.0, SPSS Institute; Chicago, IL, USA) to determine a significant difference between individual means using Duncan’s multiple range test (p < 0.05).

Thirteen phenolic acids, five flavonoids and thirty-one triterpenoid saponins were newly suggested in A. senticosus. Particularly, three aglycones, including 29-hydroxyhederagenin, 29-hydroxyechinocystic acid, and caulophyllogenin, were reported for the first time in the fruits. Quantification revealed that flavonoids and saponins were most abundant in shoots, while phenolic acids were highest in leaves, with diCQAs, quercetin and OA derivatives being the main compounds. In bioassays, all extracts showed effective inhibitory activities against α-glucosidase and ONOO−. Hederasaponin B and ciwujianoside B were newly reported as promising anti-oxidant and anti-diabetic agents. Additionally, both activities were reduced by the C-23 hydroxylation, whereas the presence of rhamnose increased them. Furthermore, the formation of a double bond between C20 and C29 may improve anti-oxidative activity. Our detailed profiling can serve as basic data for utilizing discarded parts of A. senticosus in functional foods and will be helpful in further research on the genomics, metabolomics, and various conditions of A. senticosus.

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

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This research was supported by ‘Cooperative Researcher Program for Agriculture Science and Technology Development (Project No. PJ014176012021) and (Project No. PJ016718012024)’, ‘RDA fellowship Program’ and ‘Collaborative Research Program between University and RDA’ of the National Institute of Agricultural Sciences, Rural Development Administration, Republic of Korea.

Department of Agrofood Resources, National Institute of Agricultural Sciences, Rural Development Administration, Wanju, 55365, Republic of Korea

Ryeong Ha Kwon, Hyemin Na, Ju Hyung Kim, So Ah Kim, Sang Hoon Lee, Chi-Do Wee, Kwang-Sik Lee & Heon-Woong Kim

Department of Food Science and Human Nutrition, Jeonbuk National University, Jeonju, 54896, Korea

Ryeong Ha Kwon, Se Yeon Kim & Hyun-Ah Jung

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R.H.K. and H-W.K. designed the study, R.H.K., H.N., and J.H.K. participated in sample preparation, R.H.K., H.N., J.H.K., and S.A.K. performed the MS analysis, R.H.K. performed the data collection and processing, R.H.K., S.Y.K., and H.-A.J. conducted bioassays, R.H.K. wrote main manuscript, H.N., J.H.K., S.A.K., H.-A.J., S.H.L., and H-W.K. reviewed the manuscript, S.H.L., C.-D.W., K.-S.L., H-W.K. supervised the research. All authors have read and agreed to the published version of the manuscript.

Correspondence to Heon-Woong Kim.

The authors declare no competing interests.

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Kwon, R.H., Na, H., Kim, J.H. et al. Comprehensive profiling of phenolic compounds and triterpenoid saponins from Acanthopanax senticosus and their antioxidant, α-glucosidase inhibitory activities. Sci Rep 14, 26330 (2024). https://doi.org/10.1038/s41598-024-77574-5

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Received: 24 May 2024

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Published: 01 November 2024

DOI: https://doi.org/10.1038/s41598-024-77574-5

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