The Effect of Terpenoids of Dipsacus Asperoides Against Alzheimer's Disease and Development of Simultaneous Analysis by High Performance Liquid Chromatography

Abstract

Dipsaci Radix has been proved to represent an effective treatment strategy for Alzheimer's disease (AD), but the potential active components in Dipsaci Radix have not been evaluated by an AD-related bioassay. In this study, water fraction of Dipsaci Radix had been shown to have highest inhibitory effect on the AD-related activity tests, including inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), β-site amyloid precursor protein cleaving enzyme 1 (BACE1), and advanced glycation end-product (AGE) formation. Therefore, a bioassay-guided approach yielded eleven terpenoid compounds (DR1-11) isolated from water fraction of Dipsaci Radix. Compounds (DR2,3,7 and 11) were stronger inhibitors of AChE. The BChE inhibitory effect of saponins was more positive than that of iridoids (DR1,4). Compounds (DR6,7,9-11) were good inhibitors of BACE1. Compounds (DR7-11) were more effective than positive control on the inhibition of AGE formation. Moreover, we firstly suggested concomitant new connections between the structure of Dipsaci Radix's isolated terpenoid compounds with AChE inhibitory activities and AGE formation. The study also represented that Dipsaci Radix was therapeutic for treating AD and dipsacus saponin XI (DR11) might be one of its active components. Our findings significantly provided the new foundation of the potential capacity bioactive components from Dipsaci Radix for AD remedy.

Keywords

Dipsaci Radix terpenoids Alzheimer’s disease bioactivities HPLC analysis

Introduction

Alzheimer's disease (AD), a disseminated neurodegenerative disorder of the central nervous system, is mainly manifested by progressive memory loss and severe cognitive impairment.^1‐3 BACE1, as a kind of transmembrane aspartic acid protease, is concentrated in the Golgi apparatus and the inner membranes. Plenty of extracellular Aβ aggregation is one of the important pathological features of AD.^4,5 The main point of cholinergic nervous system degeneration is the reduction of ACh level in AD patients’ brains and the decrease of cholinergic nerve impulse transmission. Previous studies also showed that butyrylcholinesterase (BChE) was involved in the pathological process of AD and affected the formation of Aβ plaques.^6,7 Advanced glycation end-products (AGEs) participate in neuronal death-causing direct (chemical) and indirect (cellular) free radical production and consequently increase oxidative stress.^8,9

Dipsaci Radix, historically used as a traditional Chinese medicine, originates from the root of Dipsacus Asperoides C. Y. Cheng et T. M. Ai belonging to the Dipsacaceae family.¹⁰ Based on researches, Dipsaci Radix mainly contains triterpenoid saponins, iridoids, phenols, alkaloids.^11‐19 The study reported that Dipsaci Radix can promote osteoblastic proliferation, bone healing and have anti-inflammatory, antiallergic, anti-AD effects.²⁰ Nevertheless, there is little research on its constituents and their biological activities, which limits its use in medicine. Firstly, most studies focused on the extract and fraction of Dipsaci Radix rather than its constituents.^21,22 Secondly, there is a lack of scientific research on the effects of constituents in Dipsaci Radix.²³ Thirdly, AD has always been a global concern as a geriatric disease. Although the extract and fraction of Dipsaci Radix have been proved to represent an effective treatment strategy for AD, the potential active components in Dipsaci Radix have not been evaluated by an AD-related bioassay. In addition, quality control standards are difficult because the standard marker terpenoid compounds of Dipsaci Radix have not been established.

Thus, in this study, we aimed to isolate compounds from Dipsacus Radix and evaluated their potential inhibition efficiency of these compounds on AD by acetylcholinesterase (AChE), BChE, BACE1, and AGE formation tests. On the other hand, we made use of compounds to develop the quantification analysis method by HPLC and to establish potential quality control standards with the synthesis of the results of repeated measurements of Dipsaci Radix samples. This study is expected to make an effective contribution to the treatment of AD.

Results

Structural Identification of Compounds Isolated from Dipsaci Radix

Chromatographic separation was applied to the isolation of compounds, followed by ¹H-NMR, ¹³C-NMR, and LC-MS for the identification of compounds. Compared with previous studies, loganic acid (DR1), akebia saponin D (DR2), dipsacus saponin X (DR3), sweroside (DR4), cauloside A (DR5), dipsacus saponin A (DR6), dipsacus saponin IV (DR7), dipsacus saponin V (DR8), HN saponin F (DR9), dipsacus saponin C (DR10), dipsacus saponin XI (DR11) were identified as terpenoid compounds. The m/z data and retention time of each compound were provided in Table 1. The structures of isolated compounds were provided in Figure 1.

Figure 1.

Structures of compounds.

Table 1.

Identification of Compounds by UPLC-ESI/LTQ-Orbitrap-HRMS Analysis.

Compound	Rt(min)	Formula	Mass mode	Theoretical mass	Observed mass	Mass accuracy (ppm)	Mass error (Da)
DR1 (loganic acid)	26.620	C₁₆H₂₄O₁₀	Negative	375.129	375.128	0.720	0.00027
DR2 (akebia saponin D)	14.240	C₄₇H₇₆O₁₈	Negative	927.495	927.495	0.108	0.00010
DR3 (dipsacus saponin X)	15.610	C₇₆H₁₂₄O₄₀	Negative	1675.759	1675.758	0.054	0.00009
DR4 (sweroside)	2.330	C₁₆H₂₂O₉	Negative	357.118	357.118	0.084	0.00003
DR5 (cauloside A)	19.050	C₃₅H₅₆O₈	Negative	603.389	603.389	0.116	0.00007
DR6 (dipsacus saponin A)	15.110	C₄₂H₆₈O₁₄	Negative	795.453	795.453	0.214	0.00017
DR7 (dipsacus saponin IV)	15.060	C₄₉H₇₈O₁₉	Negative	969.505	969.506	0.330	0.00032
DR8 (dipsacus saponin V)	16.260	C₄₇H₇₆O₁₇	Negative	911.500	911.500	0.000	0.00000
DR9 (HN saponin F)	15.110	C₄₁H₆₆O₁₃	Negative	765.442	765.442	0.496	0.00038
DR10 (dipsacus saponin C)	18.900	C₆₄H₁₀₄O₃₀	Negative	1351.653	1351.653	0.118	0.00016
DR11 (dipsacus saponin XI)	18.440	C₅₉H₉₆O₂₆	Negative	1219.611	1219.610	0.115	0.00014

Inhibition of Dipsaci Radix Extract and Fractions Against AChE, BChE, BACE1 Activity, and AGE Formation

In the study, we operated the Dipsaci Radix extract and fractions by AChE, BchE, BACE1, and AGEs tests to evaluate their anti-AD validity. The results were summarized in Table 1. The IC₅₀ values of berberine, quercetin, aminoguanidine hydrochloride were judged as positive control groups. Water fraction possessed the strongest activity inhibition with the AchE inhibition IC₅₀ of 4.200 ± 1.711 µg/mL, the BchE inhibition IC₅₀ of 5.361 ± 1.184 µg/mL, the BACE1 inhibition IC₅₀ of 27.5421 ± 3.793 µg/mL, the AGE-inhibition IC₅₀ of 80.909 ± 2.188 µg/mL. Although it slightly inhibited AGE formation, the Dipsaci Radix extract was strongly inhibited the activity of AchE (IC₅₀ of 15.293 ± 1.757 µg/mL), BchE (IC₅₀ of 17.436 ± 0.406 µg/mL), and BACE1 (IC₅₀ of 68.292 ± 16.866 µg/mL). In addition, dichloromethane (DCM) fraction not only showed no inhibition on AchE activity and BACE1 activity but also had low BchE activity inhibition (IC₅₀ of 231.392 ± 57.028 µg/mL) and slightly inhibited AGE formation (319.087 ± 25.904 µg/mL) (Table 2).

Table 2.

IC₅₀ of the Dipsaci Radix Extract, Fractions Against AchE, BchE, BACE1 Activity, and AGE Formation.

Sample	IC₅₀^a (µg/mL)
	AChE	BChE	BACE1	AGE formation
Extract	15.293 ± 1.757***	17.436 ± 0.406***	68.292 ± 16.866***	200.517 ± 1.722***
DCM fraction	ND^e	231.392 ± 57.028*	ND^e	319.087 ± 25.904**
Water fraction	4.200 ± 1.711***	5.361 ± 1.184*	27.5421 ± 3.793***	80.909 ± 2.188***
Berberine^b	57.171 ± 2.568***	22.199 ± 0.790***
Quercetin^c			182.755 ± 71.192***
Aminoguanidine Hydrochloride^d				110.415 ± 6.031***

Note: Data were presented as the mean ± SD (n = 3).

(IC₅₀) was calculated from the least-squares regression line of the logarithmic concentrations plotted against the residual activity.

(Berberine) was used as a positive control of AChE and BChE inhibitory activity.

(Quercetin) was used as a positive control of BACE1 inhibitory activity.

(Aminoguanidine Hydrochloride) was used as a positive control for the inhibition of the formation of AGE.

(ND) was not detectable.

*states a significant difference from control; *P < .05, **P < .005, ***P < .001.

Inhibition of Isolated Compounds Against AChE, BChE, BACE1 Activity, and AGE Formation

DR1-11 was tested for their ability to inhibit AChE, BChE, BACE1 activity, and AGE formation. The results were summarized in Table 3. The IC₅₀ values of berberine, quercetin, aminoguanidine hydrochloride were also judged as positive control groups. DR2 (IC₅₀ of 0.768 ± 0.002 mmol/L) and DR4 (IC₅₀ of 1.237 ± 0.095 mmol/L) had mild inhibitory activity toward AChE and DR3, 7, 11 more significantly inhibited AChE activity than other compounds with IC₅₀ values of 0.167 ± 0.006, 0.086 ± 0.010, 0.115 ± 0.002 mmol/L, respectively. DR1-11 showed either high or low inhibitory activity of BChE and DR5, 10, 11 showed stronger BChE activity inhibition with IC₅₀ values of 0.028 ± 0.004, 0.042 ± 0.008, 0.045 ± 0.008 mmol/L, respectively. DR6-7, 9-11 were stronger BACE1 inhibitors than quercetin with IC₅₀ values of 0.316 ± 0.048, 0.241 ± 0.077, 0.287 ± 0.025, 0.445 ± 0.033, 0.082 ± 0.028 mmol/L, respectively. DR7-11 inhibited AGE formation more than aminoguanidine hydrochloride, the positive control. DR10 was the best inhibitor of AGE formation with an IC₅₀ of 0.121 ± 0.014 mmol/L.

Table 3.

IC₅₀ of the Compounds Against AChE, BChE, BACE1 Activity, and AGE Formation.

Compound	IC₅₀^a (mmol/L)
	AChE	BChE	BACE1	AGE
DR1 (loganic acid)	ND^e	1.466 ± 0.103**	3.904 ± 1.390***	ND^e
DR2 (akebia saponin D)	0.768 ± 0.002***	0.196 ± 0.021***	ND^e	ND^e
DR3 (dipsacus saponin X)	0.167 ± 0.006*	0.167 ± 0.042***	ND^e	ND^e
DR4 (sweroside)	1.237 ± 0.095**	1.953 ± 0.484*	ND^e	ND^e
DR5 (cauloside A)	ND^e	0.028 ± 0.004***	ND^e	ND^e
DR6 (dipsacus saponin A)	ND^e	0.359 ± 0.049**	0.316 ± 0.048***	ND^e
DR7 (dipsacus saponin IV)	0.086 ± 0.010**	0.288 ± 0.029**	0.241 ± 0.077***	0.704 ± 0.093**
DR8 (dipsacus saponin V)	ND^e	0.133 ± 0.022*	ND^e	1.140 ± 0053***
DR9 (HN saponin F)	ND^e	0.146 ± 0.014***	0.287 ± 0.025***	1.148 ± 0.203***
DR10 (dipsacus saponin C)	ND^e	0.042 ± 0.008***	0.445 ± 0.033***	0.121 ± 0.014***
DR11 (dipsacus saponin XI)	0.115 ± 0.002 ***	0.045 ± 0.008***	0.082 ± 0.028***	0.400 ± 0.014***
Berberine^b	0.243 ± 0.011***	0.077 ± 0.003***
Quercetin^c			0.605 ± 0.236***
Aminoguanidine Hydrochloride^d				1.818 ± 0.348***

Note: Data were presented as the mean ± SD (n = 3).

(IC₅₀) was calculated from the least-squares regression line of the logarithmic concentrations plotted against the residual activity.

(Berberine) was used as a positive control of AChE and BChE inhibitory activity.

(Quercetin) was used as a positive control of BACE1 inhibitory activity.

(Aminoguanidine Hydrochloride) was used as a positive control for the inhibition of the formation of AGE.

(ND) was not detectable.

*states a significant difference from control; *P < .05, **P < .005, ***P < .001.

Simultaneous Quantification of Compounds in Dipsaci Radix

Considering together the bioassay and HPLC results, DR1, 2, 3, 4, 5, 6, 8, 10, 11 were selected to be the marker terpenoid compounds of Dipsaci Radix. Even though DR7 and 9 could be detected under the same HPLC condition, their peaks appeared almost at the same time and cannot be separated properly, furthermore, the contents of DR7 and 9 were too minimal to be detected in almost all extracts. To accomplish optimum separation of compounds in the Dipsaci Radix extract within a short time and idealized detection wavelength, DR7 and 9 were ultimately not included. Based on the method that 0.050% phosphoric acid in water and acetonitrile as the mobile phase system and 212 nm wavelength were selected to provide optimum separation and analyze, target compounds in Dipsaci Radix extract were separated without interference. HPLC analysis of extract and target compounds’ mixed solution was shown in Figure 2. The standard calibration curve was constructed using six different concentrations. The linear relationship between peak area and concentration is described in Table 4. The concentrations of the target compounds were calculated using regression equations based on the calibration curves.

Figure 2.

HPLC chromatograms of Dipsaci Radix extract (1) and standard mixture (2).

Table 4.

Calibration Curves and Content Criteria of Compounds.

Compound	Rt (min)	Regression equation	R²	Linear range (μg/mL)	LOD (μg/mL)	LOQ (μg/mL)	Content criteria (mg/g)
DR1 (loganic acid)	9.453	y = 4.316x − 22.462	0.993	10-100	1.058	3.207	0.050
DR2 (akebia saponin D)	41.075	y = 2.904x + 0.368	0.986	10-100	2.478	7.510	0.086
DR3 (dipsacus saponin X)	36.592	y = 1.266x − 1.148	0.996	10-100	2.314	7.012	0.047
DR4 (sweroside)	13.792	y = 5.862x − 26.336	1.000	10-100	1.323	4.010	0.010
DR5 (cauloside A)	26.914	y = 5.648x + 25.507	0.934	10-100	0.690	2.091	0.035
DR6 (dipsacus saponin A)	51.842	y = 1.505x − 10.756	0.982	10-100	1.728	5.235	0.001
DR8 (dipsacus saponin V)	52.486	y = 1.323x − 6.251	0.997	10-100	1.942	5.884	0.004
DR10 (dipsacus saponin C)	59.311	y = 0.968x − 1.983	0.994	10-100	0.952	2.886	0.007
DR11 (dipsacus saponin XI)	59.786	y = 0.954x − 1.664	0.994	10-100	1.756	5.320	0.004

Note: In the regression equation y = ax + b, x refers to the concentration of the compound (µg/mL), y the peak area; R²: the correlation of the equation.

Abbreviations: Rt, retention time; LOD, limit of detection; LOQ, limit of quantification.

To optimize the extraction efficiency, samples were extracted by altering the extraction solvent, solvent composition, time. The extracted samples by 10%, 30%, 50%, 70%, 100% of EtOH and 10%, 30%, 50%, 70%, 100% of MeOH for 30, 60, 120 min were analyzed to uncover the optimal extraction solvent and solvent ratio. The target compounds were quantified by HPLC and comparing the areas of the peaks in each condition. The result exhibited the sample extracted with 70% MeOH for 60 min was containing the highest peak area of target compounds. The contents of target compounds in samples were simultaneously quantified by the validated HPLC method. The data of content criteria were shown in Table 4.

Discussion and Conclusion

The increasing incidence of AD poses a major challenge to its treatment. Although Dipsaci Radix has already been demonstrated to have ameliorative effects on AD, more active components are left to discover. Therefore, we aimed to assess whether any other components of Dipsaci Radix have the potential to treat AD by inhibiting AChE, BChE, BACE1 activity, and AGE formation.

In this study, the potential inhibition of Dipsaci Radix extract and fractions against AChE, BChE, BACE1 activity, and AGE formation were ultimately evaluated. Water fraction was identified to possess the most effective inhibition against AChE, BChE, BACE1 activity, and AGE formation inhibition tests, who not only effectively inhibited the activity of AChE, BChE, BACE1 but also affected the formation of AGE. Therefore, bioassay-guided isolation of compounds was executed from water fraction and eleven isolated terpenoid compounds were identified by ¹H-NMR, ¹³C-NMR, and ESI/LTQ-Orbitrap-HRMS techniques. The structures of triterpenoid saponins in Dipsaci Radix are mainly based on hederagenin with sugar groups attached to carbon 3 (C-3) and carbon 18 (C-28).

Although studies reported that akebia saponin D had the capacity to antagonize Aβ_25-35-induced cytotoxicity, the discovery of anti-AD bioactivity of other potential active components in Dipsaci Radix is still insufficient. Accordingly, in this study, we applied isolated terpenoid compounds to AChE, BChE, BACE1 activity, and AGE formation inhibition assays, which were significant to have access to the anti-AD capacity of components in Dipsaci Radix.

DR3, 7, and 11 possessed much more powerful AChE inhibitory activities, compared with other terpenoid compounds, furthermore, concomitant new connections have also been revealed among the terpenoid compounds’ structures and their AChE inhibitory activities: (1) Based on the same two sugar groups at C-28, those triterpenoid saponins showed stronger the AChE inhibition activity with more sugar groups attached to C-3 (DR2, 3, 6); (2) Triterpenoid saponin with an extra acetyl group attached to the 3-O-sugar moiety (DR7) inhibited AChE activity more than those without an extra acetyl group attached to the 3-O-sugar moiety (DR2).

Triterpenoid saponins (DR2-3, 5-11) much more inhibited BChE activity than iridoids (DR1, 4). Compared with DR10 and DR11 which had relatively strong inhibition and DR5 exhibited the strongest BChE inhibitory activity among the terpenoid compounds.

DR6, 7, 9, 10 reacted intensively to BACE1 inhibition. DR11 showed higher inhibitory activities than others as well. In addition, saponin with an extra acetyl group attached to the 3-O-sugar moiety (DR7) still maintained their inhibitory advantage over those without an extra acetyl group attached to the 3-O-sugar moiety (DR2) in the BACE1 inhibition test.

The results suggested that DR 7-11 were strong inhibitors to effect AGE formation, even more inhibitory than aminoguanidine hydrochloride (the positive control). Oppositely, the absorbance values of DR 1-6 were too high to be detected, declaring that they may have low or no activity inhibition of AGE formation. We suggested the following structure-activity relationship for AGE formation inhibition by terpenoid compounds: (1) Triterpenoid saponin with an extra acetyl group attached to the 3-O-sugar moiety (DR7) inhibited AGE formation more than those without an extra acetyl group attached to the 3-O-sugar moiety (DR2); (2) Based on the same carboxy group at C-28, those triterpenoid saponins showed higher AGE formation inhibition activity with more sugar groups attached to C-3 (DR5, 10, 11).

DR11 showed remarkably high inhibitory activities against AChE, BChE, BACE1 activity, and AGE formation, whose inhibitory activity was even higher than that of the positive control in each group, indicating dipsacus saponin XI (DR11) may present the potential in the treatment of AD.

Owing to the developed simultaneous HPLC analysis method, target components were quantified in Dipsaci Radix samples under optimized extraction conditions (with 70% methanol for 60 min). In addition, we established potential quality control standards using the developed HPLC method that simultaneously analyzed the active target components with the synthesis of the results of repeated measurements of Dipsaci Radix samples.

Overall, in this study, we applied bioassay-guided isolation to 11 terpenoid compounds from Dipsaci Radix. We evaluated the potential AD-inhibitory activities of these compounds by AChE, BChE, BACE1 activity inhibition assays, and AGE formation inhibition assays. In addition, we suggested a structure-activity relation for AChE inhibitory activities and AGE formation inhibition. All the above results of this study represented that Dipsaci Radix was therapeutic for treating AD and dipsacus saponin XI (DR11) might be one of its active components. Potential quality control standards of target compounds from Dipsaci Radix were simultaneously identified through the HPLC technique.

With the development of science and technology, the selection of effective components in medicinal plants is mostly based on molecular, cell, and animal models to evaluate the pharmacodynamics in vivo and in vitro. The pathogenesis of AD is varied and complex. In this study, some compounds are low in content and not easy to obtain. In addition, this study is only based on the anti-AD model constructed in vitro to screen the active ingredients of Dipsaci Radix, which has certain limitations. Therefore, continuing to study and establish anti-AD cell models and animal models is also of great significance for the study of AD.

Materials and Methods

Materials

Samples of D Asperoides C. Y. Cheng et T. M. Ai were purchased from Kyung-Dong market, Seoul, Korea, botanically identified by Prof. Whang Wan Kyunn (College of Pharmacy, Chung-Ang University, South Korea). The compounds applied to HPLC analysis were obtained by the isolation of this study. 1 mg of each compound was dissolved in 1 mL of HPLC-grade methanol and was filtered through 0.200 µm PVDF membrane filters. Standard solutions for HPLC analysis were prepared by diluting the stock solutions with HPLC-grade methanol to obtain concentration ranges of 10 to 100 µg/mL (10, 25, 35, 50, 70, and 100). All the standard solutions were stored at room temperature.

Equipment and Reagents

DCM, distilled water (DW), methanol (MeOH), and ethanol (EtOH) were applied to the experiment of extraction, fractionation, open column chromatography, and anti-AD activity. DIAION® HP-20 (Supelco, Bellefonte, PA, USA), MCI GEL CHP 20P (Supelco, St. Louis, MO, USA), ODS GEL (400-500 mesh; Waters, Milford, MA, USA), and SEPHADEX LH-20 (25-100 μm; Pharmacia, Stockholm, Sweden) were used for the experiment of open column chromatography. Thin-layer chromatography (TLC) was performed with the TLC siliga gel 60 F₂₅₄ plates made up of silica gel on aluminum sheets of 0.250 mm layer (Merck Co., Darmstadt, Germany). Prior to TLC plates heated at 120°C, 10% sulfuric acid (in 100% ethanol) was sprayed for later detection under U.V. radiation (Vilber Co., France). ¹H-NMR at 600 MHz, ¹³C-NMR at 150 MHz were analyzed with the application of the JEOL ECZ600R spectrometer (JEOL, Tokyo, Japan). Methonal-d₄ (CD₃OD) and dimethyl sulfoxide-d₆ (DMSO-d₆) were used as NMR solutions. The separation column in the experiment of high-performance liquid chromatography (HPLC) was the Waters Sunfire™ C₁₈ column (4.600 × 250 mm, 5 μm) followed with YL9101 Vacuum Degasser, YL9110 Quaternary Pump, and YL9120 UV/Vis Detector (Younglin Co., Korea). HPLC-grade acetonitrile and HPLC-grade distilled water were bought from J. T. Baker® (Phillipsburg, PA, USA). HPLC-grade phosphoric acid was purchased from Sigma Co., Japan. The molecular weight of isolated compounds was analyzed by ultra-high-performance liquid chromatography and high-resolution mass spectrometry, conducted with electrospray ionization hybrid linear trap-quadruple-Orbitrap mass spectrometry (UPLC-ESI/LTQ-Orbitrap-HRMS) with the Ultimate 3000 rapid separation liquid chromatography (RSLC) system (Thermo, Darmstadt, Germany). Bovine serum albumin (BSA), D-(+)-glucose, D-(−)-fructose, AChE, acetylthiocholine iodide (ATCh), BChE from equine serum, S-butyrylthiocholine iodide (BTCh), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), berberine, quercetin, and aminoguanidine hydrochloride were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). The β-site amyloid precursor protein cleavage enzyme 1 (BACE1) FRET assay kit was bought from Pan Vera Company (Madison, WI, USA).

Extraction, Fractionation, and Isolation of Dipsaci Radix

Dipsaci Radix was extracted in methanol at room temperature, whose filtrate was concentrated to dryness in vacuo. The dried remains were suspended in distilled water, and after being partitioned in DCM, residual water fraction was obtained about 517g. Open column chromatography of water fraction was performed repeatedly and ultimately it brought out 11 terpenoid compounds. The water fraction was subjected to the Diano HP-20 chromatography and eluted in increasing MeOH: water (20:80 to 100:0) solutions yielding five sub-fractions. Sub-fraction 1 was separated on a Sephadex LH-20 column with 10% MeOH, the MCI gel with 5% MeOH, the ODS column and eluted with 15% MeOH to obtain fraction 1-1-0.5-1.5. Fraction 1-1-0.5-1.5 was separated on a Sephadex LH-20 column with 50% EtOH to yield DR1. Sub-fraction 2 was separated on a Sephadex LH-20 column with 0.5% MeOH to obtain fraction 2-0.5 and an MCI column with 60% MeOH to obtain fraction 3-6. Fraction 2-0.5 was separated on an MCI column with 60% MeOH, the ODS column with 60% MeOH to obtain fraction 2-0.5-6-6, sequentially. Fraction 2-0.5-6-6 was separated on a Sephadex LH-20 column with 50% EtOH to isolate DR2 and with 10% EtOH to obtain fraction 2-0.5-6-6-1. Fraction 2-0.5-6-6-1 was separated on the MCI gel with 60% MeOH and the ODS column with 70% MeOH to obtain fraction 2-0.5-6-6-1-6-7 and DR3 was isolated from fraction 2-0.5-6-6-1-6-7. Sub-fraction 5 was separated on ODS (70% MeOH) and Sephadex LH-20 with 70% EtOH to yield DR5. Sub-fraction 4 was separated on an MCI column with 30% MeOH, the ODS column with 60% MeOH, the Sephadex LH-20 with 50% EtOH, sequentially, to obtain DR4. DR6-11 were isolated from Sub-fraction 6 by MCI (80% MeOH), ODS (60% MeOH) and Sephadex LH-20 eluted with 50% EtOH.

Identification of Compounds

NMR

Samples were dissolved with Methonal-d₄ (CD₃OD) and dimethyl sulfoxide-d₆ (DMSO-d₆). Dissolved samples were detected via ¹H-NMR at 600 MHz, ¹³C-NMR at 150 MHz spectroscopy. Chemical shifts are presented as parts per million (ppm) on the δ scale and coupling constants (J) are shown in Hertz (Hz). Compared with previous studies, loganic acid (DR1, 1.200g), akebia saponin D (DR2, 2.300g), dipsacus saponin X (DR3, 0.263g), sweroside (DR4, 0.159g), cauloside A (DR5, 0.004g), dipsacus saponin A (DR6, 0.009g), dipsacus saponin IV (DR7, 0.010g), dipsacus saponin V (DR8, 0.006g), HN saponin F (DR9, 0.004g), dipsacus saponin C (DR10, 0.018g), dipsacus saponin XI (DR11, 0.007g) were identified as terpenoid compounds.

UPLC-ESI/LTQ-Orbitrap-HRMS Conditions

The molecular weights of DR1-11 were detected by UPLC-ESI/LTQ-Orbitrap-HRMS. Samples were dissolved with the solution (50%MeOH: H₂O = 1:1). All of the samples were filtered via 0.2 µm PVDF membrane filters. The column (Agilent ZORBRAX SB C₁₈, 2.1 × 50 mm, 1.8 µm) and sampler temperatures were set to 30°C and 15°C, respectively. The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). The gradient conditions were 0-18 min, 10%-50% B; 18-20 min, 50%-100% B; 20-23 min, 100%. The flow rate was 0.3 mL/min, and the sample injection volume was 5.0 µL for the standard solution and 2.0 µL for the extract solution. The optimal analysis conditions were as follows: Spray capillary voltage, 3.0 kV; S-lens RF level, 50.0 V; Capillary temperature, 360°C; Heater temperature, 300°C; Sheath gas flow rate, 45 L/h; Auxiliary gas flow rate, 10 L/h; Full MS resolution, 35 000 (FWHM @ m/z 200); Full MS AGC target, 3e⁶; and Full MS maximum IT, 200 ms.

Identification

Loganic Acid (DR1)

Loganic Acid²⁴: C₁₆H₂₄O₁₀; White amorphous solid; ESI/LTQ-Orbitrap-HRMS m/z: 375.127 [M-H]⁻; ¹H-NMR (600 MHz, DMSO-d₆) δ: 7.30 (1H, s, H-3), 5.09 (1H, d, J = 4.8 Hz, H-1), 4.48 (1H, d, J = 7.2Hz, H-1′), 2.09-2.05 (1H, m, H-6a), 1.83-1.78 (1H, m, H-9), 1.74-1.68 (1H, m, H-8), 1.47-1.41 (1H, m, H-6b), 0.98 (3H, d, J = 7.2 Hz); ¹³C-NMR (150 MHz, DMSO-d₆) δ: 96.1 (C-1), 150.0 (C-3), 112.7 (C-4), 31.0 (C-5), 41.8 (C-6), 73.2 (C-7), 40.6 (C-8), 44.8 (C-9), 13.6 (C-10), 168.2 (C-11), 98.6 (C-1′), 72.2 (C-2′), 76.8 (C-3′), 70.1 (C-4′), 77.2 (C-5′), 61.2 (C-6′).

Akebia Saponin D (DR2)

Akebia Saponin D²⁵: C₄₇H₇₆O₁₈; White needles; ESI/LTQ-Orbitrap-HRMS m/z: 927.495 [M-H]⁻; ¹H- NMR (600 MHz, CD₃OD-d₄) δ: 0.58, 0.68, 0.86, 0.87, 0.87, 1.08 (3H each, s, CH₃), 4.18 (1H, d, J = 6.0 Hz, H-1″ of 28-O-Glc), 4.20 (1H, d, J = 7.8 Hz, H-1′ of 3-O-ara), 5.16 (1H, brs, H-12), 5.22 (1H, d, J = 8.4 Hz, H-1′ of 28-O-Glc); ¹³C-NMR (150 MHz, CD₃OD-d₄) δ: 39.4 (C-1), 26.2 (C-2), 83.4 (C-3), 43.7 (C-4), 48.1 (C-5), 18.8 (C-6), 33.3 (C-7), 40.5 (C-8), 49.9 (C-9), 37.6 (C-10), 24.5 (C-11), 123.6 (C-12), 144.8 (C-13), 42.9 (C-14), 28.8 (C-15), 23.9 (C-16), 47.9 (C-17), 42.4 (C-18), 47.1 (C-19), 31.5 (C-20), 34.8 (C-21), 33.0 (C-22), 64.8 (C-23), 13.5 (C-24), 16.6 (C-25), 17.8 (C-26), 26.4 (C-27), 177.9 (C-28), 33.6 (C-29), 24.1 (C-30), 106.2 (3-O-sugar moiety, 3-O-Ara-1′), 72.8 (3-O-sugar moiety, 3-O-Ara-2′), 74.3 (3-O-sugar moiety, 3-O-Ara-3′), 69.7 (3-O-sugar moiety, 3-O-Ara-4′), 66.8 (3-O-sugar moiety, 3-O-Ara-5′), 95.5 (28-O-sugar moiety, Inner-Glc-1′), 73.6 (28-O-sugar moiety, Inner-Glc-2′), 77.9 (28-O-sugar moiety, Inner-Glc-3′), 70.7 (28-O-sugar moiety, Inner-Glc-4′), 77.5 (28-O-sugar moiety, Inner-Glc-5′), 69.3 (28-O-sugar moiety, Inner-Glc-6′), 104.4 (28-O-sugar moiety, Outer-Glc-1″), 74.9 (28-O-sugar moiety, Outer-Glc-2″), 77.7 (28-O-sugar moiety, Outer-Glc-3″), 71.3 (28-O-sugar moiety, Outer-Glc-4″), 77.7 (28-O-sugar moiety, Outer-Glc-5″), 62.5 (28-O-sugar moiety, Outer-Glc-6″).

Dipsacus Saponin X (DR3)

Dipsacus Saponin X¹²: C₇₆H₁₂₄O₄₀; White needles; ESI/LTQ-Orbitrap-HRMS m/z: 1675.758 [M-H]⁻; ¹H- NMR (600 MHz, CD₃OD-d₄) δ: 0.70, 0.80, 0.91, 0.94, 0.98, 1.17 (3H each, s, CH₃), 1.22 (3H, d, J = 6.0 Hz, Rha-CH3), 1.25 (3H, d, J = 6.6 Hz, Rha-CH₃), 4.34 (1H, d, J = 8.4 Hz, H-1″ of 28-O-Glc), 4.37 (1H, d, J = 7.2 Hz, H-1′ of Ara), 4.48 (1H, d, J = 6.0 Hz, 4″″of Xyl), 4.50 (2H, dd, J = 8.4, 7.8 Hz, H-1″′,H-1^ of Glc), 5.25 (1H, brs, H-12), 5.35 (1H, d, J = 8.4 Hz, H-1′ of 28-O-Glc); ¹³C-NMR (150 MHz, CD₃OD-d₄) δ: 39.7 (C-1), 26.6 (C-2), 82.4 (C-3), 44.0 (C-4), 49.8 (C-5), 18.8 (C-6), 33.5 (C-7), 40.7 (C-8), 48.2 (C-9), 37.6 (C-10), 24.6 (C-11), 123.8 (C-12), 144.9 (C-13), 43.0 (C-14), 28.9 (C-15), 23.7 (C-16), 48.1 (C-17), 42.6 (C-18), 47.2 (C-19), 31.5 (C-20), 34.9 (C-21), 33.2 (C-22), 64.6 (C-23), 13.8 (C-24), 16.6 (C-25), 17.9 (C-26), 26.4 (C-27), 178.1 (C-28), 33.3 (C-29), 24.0 (C-30), 104.6 (3-O-sugar moiety, 3-O-Ara-1′), 76.0 (3-O-sugar moiety, 3-O-Ara-2′), 74.1 (3-O-sugar moiety, 3-O-Ara-3′), 69.5 (3-O-sugar moiety, 3-O-Ara-4′), 65.7 (3-O-sugar moiety, 3-O-Ara-5′), 101.5 (3-O-sugar moiety, 2′-Rha-1″), 71.5 (3-O-sugar moiety, 2′-Rha-2″), 82.9 (3-O-sugar moiety, 2′-Rha-3″), 72.6 (3-O-sugar moiety, 2′-Rha-4″), 70.0 (3-O-sugar moiety, 2′-Rha-5″), 17.9 (3-O-sugar moiety, 2′-Rha-6″), 105.7 (3-O-sugar moiety, 3″-Glc-1‴), 75.0 (3-O-sugar moiety, 3″-Glc-2‴), 78.0 (3-O-sugar moiety, 3″-Glc-3‴), 78.9 (3-O-sugar moiety, 3″-Glc-4‴), 76.6 (3-O-sugar moiety, 3″-Glc-5‴), 60.9 (3-O-sugar moiety, 3″-Glc-6‴), 101.6 (3-O-sugar moiety, 3‴-Rha-1‴′), 72.0 (3-O-sugar moiety, 3‴-Rha-2‴′), 72.4 (3-O-sugar moiety, 3‴-Rha-3‴′), 74.3 (3-O-sugar moiety, 3‴-Rha-4‴′), 69.1 (3-O-sugar moiety, 3‴-Rha-5‴′), 18.1 (3-O-sugar moiety, 3‴-Rha-6‴′), 103.8 (3-O-sugar moiety, 4‴′-Glc-1^), 75.1 (3-O-sugar moiety, 4‴′-Glc-2^), 76.9 (3-O-sugar moiety, 4‴′-Glc-3^), 80.1 (3-O-sugar moiety, 4‴′-Glc-4^), 76.5 (3-O-sugar moiety, 4‴′-Glc-5^), 61.3 (3-O-sugar moiety, 4‴′-Glc-6^), 104.9 (3-O-sugar moiety, 4^-Xyl-1^^), 75.0 (3-O-sugar moiety, 4^-Xyl-2^^), 77.8 (3-O-sugar moiety, 4^-Xyl-3^^), 71.0 (3-O-sugar moiety, 4^-Xyl-4^^), 67.1 (3-O-sugar moiety, 4^-Xyl-5^^), 95.7 (28-O-sugar moiety, Inner-Glc-1′), 73.8 (28-O-sugar moiety, Inner-Glc-2′), 78.2 (28-O-sugar moiety, Inner-Glc-3′), 70.9 (28-O-sugar moiety, Inner-Glc-4′), 77.8 (28-O-sugar moiety, Inner-Glc-5′), 69.7 (28-O-sugar moiety, Inner-Glc-6′), 105.3 (28-O-sugar moiety, Outer-Glc-1″), 75.1 (28-O-sugar moiety, Outer-Glc-2″), 77.8 (28-O-sugar moiety, Outer-Glc-3″), 71.1 (28-O-sugar moiety, Outer-Glc-4″), 78.0 (28-O-sugar moiety, Outer-Glc-5″), 62.7 (28-O-sugar moiety, Outer-Glc-6″).

Sweroside (DR4)

Sweroside²⁶: C₁₆H₂₂O₉; Colorless, amorphous powder; ESI/LTQ-Orbitrap-HRMS m/z: 357.117 [M-H]⁻; ¹H-NMR (600 MHz, CD₃OD-d₄) δ: 7.6 (1H, s, H-3), 4.67 (1H, d, J = 8.4 Hz, H-10b), 3.13-3.10 (1H, m, H-5), 2.71-2.67 (1H, m, H-9), 1.78-1.62 (2H, m, H-6a, b); ¹³C-NMR (150 MHz, CD₃OD-d₄) δ: 97.8 (C-1), 153.9 (C-3), 105.9 (C-4), 28.2 (C-5), 25.7 (C-6), 69.6 (C-7), 133.2 (C-8), 43.6 (C-9), 120.9 (C-10), 168.4 (C-11), 99.5 (C-1′), 74.5 (C-2′), 77.6 (C-3′), 71.3 (C-4′), 78.1 (C-5′), 62.5 (C-6′).

Cauloside A (DR5)

Cauloside A²⁷: C₃₅H₅₆O₈; ESI/LTQ-Orbitrap-HRMS m/z: 603.389 [M-H]⁻; ¹H- NMR (600 MHz, CD₃OD-d₄) δ:0.71, 0.86, 0.89, 0.95, 0.97, 1.16(3H each, s, CH₃), 4.32(1H, d, J = 6.6 Hz, H-1′ of 3-O-ara), 5.23(1H, brs, H-12); ¹³C-NMR (150 MHz, CD₃OD-d₄) δ: 39.5 (C-1), 26.3 (C-2), 83.3 (C-3), 43.9 (C-4), 48.1 (C-5), 18.6 (C-6), 34.0 (C-7), 40.5 (C-8), 49.5 (C-9), 37.7 (C-10), 24.0 (C-11), 123.4 (C-12), 145.5 (C-13), 43.0 (C-14), 28.9 (C-15), 24.5 (C-16), 47.8 (C-17), 42.8 (C-18), 47.4 (C-19), 31.6 (C-20), 35.0 (C-21), 33.5 (C-22), 64.8 (C-23), 13.4 (C-24), 16.4 (C-25), 17.9 (C-26), 26.5 (C-27), 33.6 (C-29), 24.1 (C-30), 106.3 (3-O-sugar moiety, 3-O-Ara-1′), 72.9 (3-O-sugar moiety, 3-O-Ara-2′), 74.5 (3-O-sugar moiety, 3-O-Ara-3′), 69.8 (3-O-sugar moiety, 3-O-Ara-4′), 66.8 (3-O-sugar moiety, 3-O-Ara-5′).

Dipsacus Saponin A (DR6)

Dipsacus Saponin A²⁸: C₄₂H₆₈O₁₄; White needles; ESI/LTQ-Orbitrap-HRMS m/z: 795.453 [M-H]⁻; ¹H- NMR (600 MHz, CD₃OD-d₄) δ:0.70, 0.80, 0.91, 0.94, 0.98, 1.16 (3H each, s, CH₃), 4.34 (1H, d, J = 7.8 Hz, H-1″ of 28-O-Glc), 5.26 (1H, brs, H-12), 5.35 (1H, d, J = 7.8 Hz, H-1′ of 28-O-Glc); ¹³C-NMR (150 MHz, CD₃OD-d₄) δ: 39.5 (C-1), 27.4 (C-2), 73.8 (C-3), 43.3 (C-4), 49.8 (C-5), 19.1 (C-6), 33.4 (C-7), 40.6 (C-8), 48.8 (C-9), 37.9 (C-10), 24.5 (C-11), 123.7 (C-12), 144.9 (C-13), 43.0 (C-14), 28.9 (C-15), 24.0 (C-16), 48.0 (C-17), 42.5 (C-18), 47.2 (C-19), 31.5 (C-20), 34.9 (C-21), 33.2 (C-22), 67.3 (C-23), 12.8 (C-24), 16.4 (C-25), 17.8 (C-26), 26.3 (C-27), 178.1 (C-28), 33.5 (C-29), 24.0 (C-30), 95.7 (28-O-sugar moiety, Inner-Glc-1′), 73.9 (28-O-sugar moiety, Inner-Glc-2′), 78.0 (28-O-sugar moiety, Inner-Glc-3′), 70.9 (28-O-sugar moiety, Inner-Glc-4′), 77.8 (28-O-sugar moiety, Inner-Glc-5′), 69.5 (28-O-sugar moiety, Inner-Glc-6′), 104.6 (28-O-sugar moiety, Outer-Glc-1″), 75.1 (28-O-sugar moiety, Outer-Glc-2″), 78.1 (28-O-sugar moiety, Outer-Glc-3″), 71.5 (28-O-sugar moiety, Outer-Glc-4″), 78.0 (28-O-sugar moiety, Outer-Glc-5″), 62.7 (28-O-sugar moiety, Outer-Glc-6″).

Dipsacus Saponin IV (DR7)

Dipsacus Saponin IV²⁹: C₄₉H₇₈O₁₉; White powder; ESI/LTQ-Orbitrap-HRMS m/z: 969.506 [M-H]⁻; ¹H- NMR (600 MHz, CD₃OD-d₄) δ: 0.72, 0.80, 0.91, 0.94, 0.99, 1.17 (3H each, s, CH₃), 2.11 (3H, s, -COCH₃), 4.41 (1H, d, J = 7.2 Hz, H-1″ of 28-O-Glc), 4.34 (1H, d, J = 7.2 Hz, H-1′ of 3-O-ara), 5.25 (1H, brs, H-12), 5.35 (1H, d, J = 8.4 Hz, H-1′ of 28-O-Glc); ¹³C-NMR (150 MHz, CD₃OD-d₄) δ: 39.5 (C-1), 26.3 (C-2), 83.7 (C-3), 43.9 (C-4), 48.1 (C-5), 18.9 (C-6), 33.5 (C-7), 40.7 (C-8), 48.2 (C-9), 37.7 (C-10), 24.6 (C-11), 123.7 (C-12), 145.0 (C-13), 43.0 (C-14), 28.9 (C-15), 24.0 (C-16), 48.1 (C-17), 42.6 (C-18), 47.2 (C-19), 31.5 (C-20), 34.9 (C-21), 33.2 (C-22), 64.8 (C-23), 13.4 (C-24), 16.6 (C-25), 17.9 (C-26), 26.3 (C-27), 178.1 (C-28), 33.4 (C-29), 24.0 (C-30), 106.5 (3-O-sugar moiety, 3-O-Ara-1′), 73.3 (3-O-sugar moiety, 3-O-Ara-2′), 72.8 (3-O-sugar moiety, 3-O-Ara-3′), 72.8 (3-O-sugar moiety, 3-O-Ara-4′), 64.7 (3-O-sugar moiety, 3-O-Ara-5′), 172.5 (CH₃CO-CO), 21.0 (CH3), 95.8 (28-O-sugar moiety, Inner-Glc-1′), 73.8 (28-O-sugar moiety, Inner-Glc-2′), 78.2 (28-O-sugar moiety, Inner-Glc-3′), 70.9 (28-O-sugar moiety, Inner-Glc-4′), 77.8 (28-O-sugar moiety, Inner-Glc-5′), 69.5 (28-O-sugar moiety, Inner-Glc-6′), 104.6 (28-O-sugar moiety, Outer-Glc-1″), 78.0 (28-O-sugar moiety, Outer-Glc-2″), 71.5 (28-O-sugar moiety, Outer-Glc-3″), 78.0 (28-O-sugar moiety, Outer-Glc-4″), 77.7 (28-O-sugar moiety, Outer-Glc-5″), 62.7 (28-O-sugar moiety, Outer-Glc-6″).

Dipsacus Saponin V (DR8)

Dipsacus Saponin V²⁹: C₄₇H₇₆O₁₇; White powder; ESI/LTQ-Orbitrap-HRMS m/z: 911.500 [M-H]⁻; ¹H- NMR (600 MHz, CD₃OD-d₄) δ: 0.80, 0.85, 0.91, 0.94, 0.96, 1.04, 1.16 (3H each, s, CH₃), 4.27 (1H, d, J = 7.2Hz, H-1″ of 28-O-Glc), 4.34 (1H, d, J = 7.2 Hz, H-1′ of 3-O-ara), 5.25 (1H, brs, H-12), 5.35 (1H, d, J = 7.8 Hz, H-1′ of 28-O-Glc); ¹³C-NMR (150 MHz, CD₃OD-d₄) δ: 39.7 (C-1), 26.9 (C-2), 90.6 (C-3), 40.1 (C-4), 56.9 (C-5), 19.2 (C-6), 33.8 (C-7), 40.6 (C-8), 48.9 (C-9), 37.8 (C-10), 24.4 (C-11), 123.6 (C-12), 144.7 (C-13), 42.8 (C-14), 28.8 (C-15), 23.9 (C-16), 47.9 (C-17), 42.4 (C-18), 47.1 (C-19), 31.4 (C-20), 34.7 (C-21), 33.4 (C-22), 28.4 (C-23), 16.9 (C-24), 16.0 (C-25), 17.7 (C-26), 26.2 (C-27), 177.9 (C-28), 33.1 (C-29), 23.9 (C-30), 107.1 (3-O-sugar moiety, 3-O-Ara-1′), 72.7 (3-O-sugar moiety, 3-O-Ara-2′), 74.2 (3-O-sugar moiety, 3-O-Ara-3′), 69.3 (3-O-sugar moiety, 3-O-Ara-4′), 66.3 (3-O-sugar moiety, 3-O-Ara-5′), 95.6 (28-O-sugar moiety, Inner-Glc-1′), 73.7 (28-O-sugar moiety, Inner-Glc-2′), 78.0 (28-O-sugar moiety, Inner-Glc-3′), 70.8 (28-O-sugar moiety, Inner-Glc-4′), 77.7 (28-O-sugar moiety, Inner-Glc-5′), 69.4 (28-O-sugar moiety, Inner-Glc-6′), 104.5 (28-O-sugar moiety, Outer-Glc-1″), 75.0 (28-O-sugar moiety, Outer-Glc-2″), 77.9 (28-O-sugar moiety, Outer-Glc-3″), 71.4 (28-O-sugar moiety, Outer-Glc-4″), 77.9 (28-O-sugar moiety, Outer-Glc-5″), 62.6 (28-O-sugar moiety, Outer-Glc-6″).

HN Saponin F (DR9)

HN Saponin F³⁰: C₄₁H₆₆O₁₃; White powder; ESI/LTQ-Orbitrap-HRMS m/z: 765.442 [M-H]⁻; ¹H- NMR (600 MHz, CD₃OD-d₄) δ:0.71, 0.80, 0.91, 0.93, 0.98, 1.17 (3H each, s, CH₃), 4.31(1H, d, J = 7.2 Hz, H-1′ of 3-O-ara), 5.30(1H, brs, H-12), 5.38(1H, d, J = 7.8 Hz, H-1′ of 28-O-Glc); ¹³C-NMR (150 MHz, CD₃OD-d₄) δ: 39.6 (C-1), 26.3 (C-2), 83.3 (C-3), 43.9 (C-4), 48.2 (C-5), 18.9 (C-6), 33.2 (C-7), 40.7 (C-8), 49.9 (C-9), 37.7 (C-10), 24.6 (C-11), 123.8 (C-12), 144.9 (C-13), 43.0 (C-14), 28.9 (C-15), 24.0 (C-16), 48.0 (C-17), 42.6 (C-18), 47.2 (C-19), 31.5 (C-20), 34.9 (C-21), 33.4 (C-22), 64.9 (C-23), 13.4 (C-24), 16.5 (C-25), 17.8 (C-26), 26.3 (C-27), 178.1 (C-28), 33.5 (C-29), 24.0 (C-30), 106.3 (3-O-sugar moiety, 3-O-Ara-1′), 73.0 (3-O-sugar moiety, 3-O-Ara-2′), 74.5 (3-O-sugar moiety, 3-O-Ara-3′), 69.8 (3-O-sugar moiety, 3-O-Ara-4′), 66.8 (3-O-sugar moiety, 3-O-Ara-5′), 95.7 (28-O-sugar moiety, Glc-1′), 73.9 (28-O-sugar moiety, Glc-2′), 78.7 (28-O-sugar moiety, Glc-3′), 71.1 (28-O-sugar moiety, Glc-4′), 78.3 (28-O-sugar moiety, Glc-5′), 62.4 (28-O-sugar moiety, Glc-6′).

Dipsacus Saponin C (DR10)

Dipsacus Saponin C³¹: C₆₄H₁₀₄O₃₀; White powder; ESI/LTQ-Orbitrap-HRMS m/z: 1351.653 [M-H]⁻; ¹H- NMR (600 MHz, CD₃OD-d₄) δ: 0.70, 0.83, 0.90, 0.94, 0.97, 1.17 (3H each, s, CH₃), 1.22 (3H, d, J = 6.6 Hz, Rha-CH₃), 1.25 (3H, d, J = 6.0 Hz, Rha-CH₃), 4.37 (1H, d, J = 7.8 Hz, H-1′ of Ara), 4.47 (1H, d, J = 8.4 Hz, 4″″of Xyl), 4.50 (2H, dd, J = 8.4, 7.8 Hz, H-1″′,H-1^ of Glc), 5.34 (1H, brs, H-12); ¹³C-NMR (150 MHz, CD₃OD-d₄) δ: 39.7 (C-1), 26.6 (C-2), 82.4 (C-3), 44.0 (C-4), 49.6 (C-5), 18.8 (C-6), 33.4 (C-7), 40.5 (C-8), 48.1 (C-9), 37.6 (C-10), 24.5 (C-11), 123.3 (C-12), 145.6 (C-13), 43.0 (C-14), 28.9 (C-15), 24.1 (C-16), 47.9 (C-17), 42.9 (C-18), 47.4 (C-19), 31.7 (C-20), 35.0 (C-21), 34.0 (C-22), 64.6 (C-23), 13.8 (C-24), 16.4 (C-25), 17.9 (C-26), 26.5 (C-27), 33.7 (C-29), 24.2 (C-30), 105.0 (3-O-sugar moiety, 3-O-Ara-1′), 75.9 (3-O-sugar moiety, 3-O-Ara-2′), 74.9 (3-O-sugar moiety, 3-O-Ara-3′), 69.8 (3-O-sugar moiety, 3-O-Ara-4′), 65.8 (3-O-sugar moiety, 3-O-Ara-5′), 101.6 (3-O-sugar moiety, 2′-Rha-1″), 71.0 (3-O-sugar moiety, 2′-Rha-2″), 82.9 (3-O-sugar moiety, 2′-Rha-3″), 77.8 (3-O-sugar moiety, 2′-Rha-4″), 69.1 (3-O-sugar moiety, 2′-Rha-5″), 18.1 (3-O-sugar moiety, 2′-Rha-6″), 103.8 (3-O-sugar moiety, 3″-Glc-1‴), 75.0 (3-O-sugar moiety, 3″-Glc-2‴), 76.6 (3-O-sugar moiety, 3″-Glc-3‴), 80.0 (3-O-sugar moiety, 3″-Glc-4‴), 76.9 (3-O-sugar moiety, 3″-Glc-5‴), 61.3 (3-O-sugar moiety, 3″-Glc-6‴), 105.7 (3-O-sugar moiety, 4‴-Glc-1‴′), 75.1 (3-O-sugar moiety, 4‴-Glc -2‴′), 77.0 (3-O-sugar moiety, 4‴-Glc-3‴′), 78.8 (3-O-sugar moiety, 4‴-Glc-4‴′), 76.5 (3-O-sugar moiety, 4‴-Glc-5‴′), 60.8 (3-O-sugar moiety, 4‴-Glc-6‴′), 105.3 (3-O-sugar moiety, 4‴′-Xyl-1^), 74.1 (3-O-sugar moiety, 4‴′-Xyl-2^), 77.9 (3-O-sugar moiety, 4‴′-Xyl-3^), 71.0 (3-O-sugar moiety, 4‴′-Xyl-4^), 67.1 (3-O-sugar moiety, 4‴′-Xyl-5^), 101.6 (3-O-sugar moiety, 4″-Rha-1‴), 72.4 (3-O-sugar moiety, 4″-Rha-2‴), 72.6 (3-O-sugar moiety, 4″-Rha-3‴), 74.3 (3-O-sugar moiety, 4″-Rha-4‴), 70.0 (3-O-sugar moiety, 4″-Rha-5‴), 18.1 (3-O-sugar moiety, 4″-Rha-6‴).

Dipsacus Saponin XI (DR11)

Dipsacus Saponin XI³²: C₅₉H₉₆O₂₆; White powder; ESI/LTQ-Orbitrap-HRMS m/z: 1219.610 [M-H]⁻; ¹H- NMR (600 MHz, CD₃OD-d₄) δ: 0.70, 0.83, 0.90, 0.94, 0.97, 1.17 (3H each, s, CH₃), 1.22 (3H, d, J = 6.0 Hz, Rha-CH₃), 1.26 (3H, d, J = 6.0 Hz, Rha-CH₃), 4.37 (1H, d, J = 7.2 Hz, H-1′ of Ara), 4.46 (2H, dd, J = 8.4, 7.8 Hz, H-1″′,H-1^ of Glc), 5.33 (1H, brs, H-12); ¹³C-NMR (150 MHz, CD₃OD-d₄) δ: 39.7 (C-1), 26.6 (C-2), 82.4 (C-3), 44.0 (C-4), 49.7 (C-5), 18.8 (C-6), 33.1 (C-7), 40.5 (C-8), 49.6 (C-9), 37.7 (C-10), 23.7 (C-11), 123.3 (C-12), 145.6 (C-13), 43.0 (C-14), 29.0 (C-15), 24.1 (C-16), 47.5 (C-17), 42.9 (C-18), 48.1 (C-19), 31.7 (C-20), 34.1 (C-21), 33.5 (C-22), 64.6 (C-23), 13.8 (C-24), 16.4 (C-25), 17.9 (C-26), 26.5 (C-27), 33.7 (C-29), 24.5 (C-30), 104.9 (3-O-sugar moiety, 3-O-Ara-1′), 76.6 (3-O-sugar moiety, 3-O-Ara-2′), 74.1 (3-O-sugar moiety, 3-O-Ara-3′), 70.0 (3-O-sugar moiety, 3-O-Ara-4′), 65.7 (3-O-sugar moiety, 3-O-Ara-5′), 101.6 (3-O-sugar moiety, 2′-Rha-1″), 71.1 (3-O-sugar moiety, 2′-Rha-2″), 82.9 (3-O-sugar moiety, 2′-Rha-3″), 72.6 (3-O-sugar moiety, 2′-Rha-4″), 70.0 (3-O-sugar moiety, 2′-Rha-5″), 18.0 (3-O-sugar moiety, 2′-Rha-6″), 105.7 (3-O-sugar moiety, 3″-Glc-1‴), 74.7 (3-O-sugar moiety, 3″-Glc-2‴), 77.1 (3-O-sugar moiety, 3″-Glc-3‴), 79.1 (3-O-sugar moiety, 3″-Glc-4‴), 76.8 (3-O-sugar moiety, 3″-Glc-5‴), 60.9 (3-O-sugar moiety, 3″-Glc-6‴), 101.7 (3-O-sugar moiety, 3‴-Rha-1‴′), 72.0 (3-O-sugar moiety, 3‴-Rha-2‴′), 72.4 (3-O-sugar moiety, 3‴-Rha-3‴′), 74.3 (3-O-sugar moiety, 3‴-Rha-4‴′), 69.2 (3-O-sugar moiety, 3‴-Rha-5‴′), 18.1 (3-O-sugar moiety, 3‴-Rha-6‴′), 104.0 (3-O-sugar moiety, 4‴-Glc-1^), 75.4 (3-O-sugar moiety, 4‴-Glc-2^), 77.9 (3-O-sugar moiety, 4‴-Glc-3^), 70.7 (3-O-sugar moiety, 4‴-Glc-4^), 78.0 (3-O-sugar moiety, 4‴-Glc-5^), 62.0 (3-O-sugar moiety, 4‴-Glc-6^).

HPLC Condition

The Waters Sunfire™ C₁₈ (4.600 × 250 mm, 5 μm) column was used to analyze the target compounds isolated from water fractions. The mobile phase system consisted of 0.05% phosphoric acid in water (solvent A) and acetonitrile (solvent B) at a flow rate of 1 mL/min. The linear gradient elution was implemented with the following elution program: 0 to 5 min, 10%-15% B; 5-10 min, 15%-20% B; 10-30 min, 20%-30% B; 30-40 min, 30%-35% B; 40-60 min, 35%-45% B; 60-65 min, 45%-49% B. All eluents were filtered in a 0.200 µm PVDF syringe filter. The column temperature was set to 25°C. The sample injection volume was 10 µL, and UV 212 nm was selected as the optimal wavelength for detecting the compounds. For the preparation of extract solutions, samples were sonicated with 70% MeOH for 60 min. Prior to injection, all analyzed solutions were strained using 0.200 µm PVDF syringe filters. All extraction methods were performed with 1 g of sample powder and 10 mL of extraction solvent.

The Development of Inhibition Experiment for AD

AChE and BChE Inhibition

The process of AChE and BChE inhibition experiment was developed from a previous study. The AChE and BChE activities were reflected by the formation of generated the yellow 5-thio-2-nitrobenzoate anion due to the ChE-mediated hydrolysis of DTNB and the release of thiocholine. 0.1 M sodium phosphate buffer (pH 7.8) was used for dissolving berberine (positive control) and samples.

In the AChE inhibition experiment, 20 µL of sample solution (concentration gradient: 0.001-0.600 mg/mL for extract and fraction, 0.003-2.500 mM for compounds and positive control), 50 µL of 3.996 U/mL AChE, 50 µL of 0.600 mM ATCh, 50 µL of 2 mM DTNB were added to the sample group one by one. 20 µL of sodium phosphate buffer, 50 µL of 3.996 U/mL AChE, 50 µL of 0.600 mM ATCh, 50 µL of 2 mM DTNB were added to the blank group one by one. 20 µL of sample solution, 50 µL of sodium phosphate buffer, 50 µL of 0.600 mM ATCh, 50 µL of 2 mM DTNB were added to the control group one by one. The reaction was performed in the 96-well plates for 10 min. All processes were performed in triplicate. 10 min later, the absorbance of each well was detected at 412 nm and the inhibition ratio (%) was calculated with the following formula: (1−[S-C]/B) × 100, where C was the absorbance of the control group, S was the absorbance of sample group and B was the absorbance of the blank group. The results were shown by IC₅₀, calculated from the log-dose inhibition curve.

In the BChE inhibition experiment, 20 µL of sample solution (concentration gradient: 0.010-0.300 mg/mL for extract and fraction, 0.010-4.00 mM for compounds and positive control), 20 µL of 0.360 U/mL BChE, 50 µL of 0.600 mM BTCh, 50 µL of 1 mM DTNB were added to the sample group one by one. 20 µL of sodium phosphate buffer, 20 µL of 0.360 U/mL BChE, 50 µL of 0.600 mM BTCh, 50 µL of 1 mM DTNB were added to the control group one by one. 20 µL of sample solution, 20 µL of sodium phosphate buffer, 50 µL of 0.600 mM BTCh, 50 µL of 1 mM DTNB were added to the blank group one by one. The reaction was performed in the 96-well plates for 24 h. All processes were performed in triplicate. 24 h later, the absorbance of each well was detected at 412 nm and the inhibition ratio (%) was calculated with the following formula: ([C-B] − [S-B]/[C-B]) × 100, where C was the absorbance of the control group, S was the absorbance of sample group and B was the absorbance of the blank group. The results were shown by IC₅₀, calculated from the log-dose inhibition curve.

BACE1 Inhibition

The process of the BACE1 inhibition experiment was performed based on a previous study.³³ In the BACE1 inhibition experiment, BACE1 buffer was used for dissolving quercetin (positive control) and samples. 10 µL of sample solution (concentration gradient: 0.010-1.700 mg/mL for extract and fraction, 0.003-6.100 mM for compounds and positive control), 10 µL of 1 U/mL BACE1, 10 µL of 0.750 mM BACE1 substrate were added to the sample group one by one. 10 µL of BACE1 buffer, 10 µL of 1 U/mL BACE1, 10 µL of 0.750 mM BACE1 substrate were added to the control group one by one. The reaction was performed in the 96-well plates for 12 h in the dark at room temperature. All processes were performed in triplicate. 12 h later, the absorbance of each well was detected at 540 nm (Excitation wavelength) and 594 nm (Emission wavelength). The inhibition ratio (%) was calculated with the following formula: (1−[S₁₂-S₀]/[C₁₂-C₀]) × 100, where C₀ was the absorbance of the control group before incubation, C₁₂ was the absorbance of the control group after incubation, S₀ was the absorbance of sample group before incubation and S₁₂ was the absorbance of sample group after incubation. The results were shown by IC₅₀, calculated from the log-dose inhibition curve.

AGE Formation Inhibition

The process of AGEs inhibition experiment was performed based on a previous study. In the AGEs inhibition experiment, phosphate buffer (pH7.4) was used for dissolving aminoguanidine hydrochloride (positive control) and samples. 30 µL of sample solution (concentration gradient: 0.060-0.800 mg/mL for extract and fraction, 0.090-1.800 mM for compounds and positive control), 100 µL of Enzyme (10 mg/mL bovine serum albumin), 100 µL of 0.400M AGE substrate (0.400M Fructose and Glucose mixed solution) were added to the sample group one by one. 30 µL of phosphate buffer, 100 µL of Enzyme, 100 µL of 0.400M AGE substrate were added to the control group one by one. 30 µL of sample solution, 100 µL of phosphate buffer, 100 µL of 0.400M AGE substrate were added to the blank group one by one. The reaction was performed in the 96-well plates for 48 h in the dark at 60°C. All processes were performed in triplicate. 48 h later, the absorbance of each well was detected at 360 nm (Excitation wavelength) and 465 nm (Emission wavelength). The inhibition ratio (%) was calculated with the following formula: ([C-B] − [S-B]/[C-B]) × 100, where C was the absorbance of control group, S was the absorbance of sample group and B was the absorbance of blank group. The results were shown by IC₅₀, calculated from the log-dose inhibition curve.

Statistical Analysis

All of process of experiment was performed in triplicate. The data of results were presented as Mean ± Standard Deviation (SD). Student's t-test and a one-way ANOVA were applied to statistical significance analysis and when P was <.050, the data was considered statistically significant.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X211044603 - Supplemental material for The Effect of Terpenoids of Dipsacus Asperoides Against Alzheimer's Disease and Development of Simultaneous Analysis by High Performance Liquid Chromatography

Supplemental material, sj-docx-1-npx-10.1177_1934578X211044603 for The Effect of Terpenoids of Dipsacus Asperoides Against Alzheimer's Disease and Development of Simultaneous Analysis by High Performance Liquid Chromatography by Zi Ying Wang, Xiao Dan Zhang and Wan Kyunn Whang in Natural Product Communications

Footnotes

Acknowledgments

This research was supported by the Chung-Ang University Young Scientist Scholarship in 2019.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Author's Contributions

ZYW and WKW conceived and designed the experiments; ZYW performed the extraction, isolation, bioactivities experiments, and quantitative analysis, analyzed the data, and wrote the paper. XDZ took part in designing the experiments and solving the difficulties in the experiment. All authors have read and agreed to the published version of the manuscript.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Chung-Ang University (grant number Chung-Ang University Young Scientist Scholarship i).

ORCID iDs

Zi Ying Wang

Xiao Dan Zhang

Supplemental Material

Supplemental material for this article is available online.

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