The Isolation and Preparation of Samwinol from Dracocephalum heterophyllum and Prevention on Aβ25–35-Induced Neuroinflammation in PC-12 Cells

Dracocephalum heterophyllum (D. heterophyllum) is a traditional Chinese Tibetan medicine that has been used for the treatment of lymphitis, hepatitis, and bronchitis. However, only a few selected chemical components are currently obtained from D. heterophyllum, which limits its further pharmacological applications. In this study, we have obtained samwinol from D. heterophyllum by medium- and high-pressure liquid chromatography separation for the first time. Thereafter, we investigated the protective actions of samwinol against amyloid beta protein fragment 25–35 (Aβ25–35) induced neurotoxicity in cultured rat pheochromocytoma PC-12 cells and explored its underlying mechanisms of action. The results indicated that samwinol could increase cell viability and inhibit the production of reactive oxygen species (ROS) and mitochondria-derived ROS, as assessed by MTT assay, Giemsa staining, and flow cytometry assay. Through Western blot analysis, it was found that samwinol substantially inhibited the phosphorylation of ERK(1/2) and promoted the expression of HO-1 and Nrf2. The data obtained from molecular docking were also consistent with the above conclusions. All of these results showed that samwinol from D. heterophyllum can display significant anti-neuroinflammatory and antioxidant activities in vitro, which are associated with the suppression of ERK/AKT phosphorylation and the activation of the Nrf2/HO-1 signaling pathway. In the future, additional in-depth mechanism studies will be carried out to provide more evidence for the potential of samwinol in the treatment of Alzheimer’s disease.


Introduction
Dracocephalum heterophyllum (D. heterophyllum) is a traditional Tibetan medicine (TTM) called "Ao-Ga" or "Ji-Mei-Qing-Bao", which is widely distributed in Xinjiang, Tibet, Qinghai, Gansu, and other provinces of China. It has been used in Tibetan medicine for the treatment of various diseases such as jaundice, liver disease, lymphangitis, mouth ulcers, and dental disease [1]. A number of prior reports have indicated that it also has antioxidant activity [2,3], anti-diabetic activity [4], inhibits hepatitis, and improves autoimmune diseases [5]. In addition, previous studies related to the analyses of phytochemical constituents have revealed that different flavonoids, alkaloids, triterpenes, and phenylpropanoids could be isolated from D. heterophyllum, but the chemical constituents currently obtained from D. heterophyllum are limited, which are not enough to support the subsequently reported pharmacological activities and pharmacodynamics mechanism research. Therefore, it is necessary to use a fast and efficient method to isolate pure compounds from D. heterophyllum to establish its potential in the management of various diseases.
The high-performance liquid chromatography-1,1-diphenyl-2-picrylhydrazine (HPLC-DPPH) screening system enables rapid analytical evaluation of target-specific potential antioxidants [6]. In this method, the ability of the compound to scavenge free radicals 2 of 14 is measured by the decrease in absorbance at 517 nm after the addition of 1,1-diphenyl-2-picrylhydrazine (DPPH) to the HPLC pathway. This method has been widely used to detect the presence of various antioxidants in many plants and foods, and it has screened several new active compounds with antioxidant activity [7,8]. In addition, one of the best chromatographic methods currently available for the extraction of single compounds from complex samples is preparative HPLC, which has the advantages of good separation, real-time monitoring, and high reproducibility [9]. This can also be used for the separation and purification of various chemical components of D. heterophyllum.
Alzheimer's disease (AD) is a prevalent chronic neurological condition linked to a progressive decrease in cognition and memory [10]. Progressive memory loss, poor executive function, and difficulties performing daily activities are clinical symptoms of AD, and early signals of AD development include changes in thinking or unconscious behavior, memory deficiencies for new knowledge, as well as dysfunctional alterations in both language and speech [11]. The main pathological characteristics associated with AD include Aβ plaques, neurogenic fiber tangles, and neuronal loss [12], with cerebrovascular amyloidosis, inflammation [13], and synaptic lesions of nerves [14]. There are several hypotheses proposed to explain the cause of AD, including the cholinergic hypothesis, the β-amyloid hypothesis, and the Tau protein hypothesis [15]. According to the β-amyloid hypothesis, the main component of amyloid beta (Aβ) is senile plaques, which are primarily composed of 36-43 amino acid residues [16]. A large body of medical evidence suggests that Aβ can play a significant role in the pathogenesis of AD [17][18][19].
Microglia are the only resident macrophage-type cells present in the central nervous system. Activated microglia can take up soluble or fibrillar Aβ through the process of phagocytosis and dissolve these peptides through the proteasomal pathway [20]. Aβ induces significant neurodegeneration by activating microglia, which trigger neurotoxicity through the release of inflammatory mediators such as different cytokines and reactive oxygen species (ROS) to promote the development of AD [21]. A growing number of investigations have described various pharmacological strategies to alleviate AD by treating neuroinflammation and oxidative stress [22][23][24].
To the best of our knowledge, there have been no prior reports describing the pharmacological effects of various components from D. heterophyllum in AD. The aim of this work was to extract the different monomeric compounds with antioxidant activity from D. Heterophyllum and to evaluate their possible effects on neuroinflammation by using PC12 cells.

Sample Pretreatment with Medium-Pressure Liquid Chromatography
Considering the environment-friendly characteristics of methanol, it was selected as a solvent to extract the whole plant. Finally, a crude sample of about 1.3 kg was obtained from 10.0 kg of dried D. heterophyllum whole plant at about a 13.4% extraction rate. Thereafter, the crude sample was subjected to the first pretreatment by medium-pressure liquid chromatography on the silica gel. The main purpose was to remove the various polymers and sugars, which are not our target substances. The removal of these components can simplify the subsequent separation steps. At the same time, visual separation was achieved on the preparative liquid chromatography, and the crude sample was separated into two fractions, Fr1 and Fr2, as shown in Figure 1A. Baseline separation can be achieved between the two fractions. Finally, all the crude samples were processed, and two different fractions were obtained, of which the target fraction Fr1 weighed 205.5 g (15.8% recovery). On the contrary, the separated Fr1, Fr2, and crude samples were analyzed using the same conditions, and the analytical conditions were gradient elution on a ReproSil-Pur C18 AQ analytical column. The analytical chromatograms are shown in Figure 1B  The Fr1 at this stage still contained a large amount of chlorophyll. Hence, if Fr1 was prepared directly in the next step, it was possible that chlorophyll might adsorb on the stationary phase of the preparation column and cause damage to the preparation column, so it was necessary to remove the chlorophyll first. The MCI GEL ® CHP20P column was selected for the second pretreatment of Fr1 to remove the chlorophyll, whereas Fr1 was segmented to refine the fractions. Figure 1C shows a separation chromatogram. Five fractions (Fr11, Fr12, Fr13, Fr14, and Fr15) were collected after nine repetitions of the MCI GEL ® CHP20P pretreatment. Among them, Fr14 was 44.2 g, which was of high quality. We selected Fr14 as the target fraction for the subsequent separation.
In order to analyze the Fr14, 100 mg of Fr14 was solubilized in 0.1 mL MeOH and filtered through a 0.45 mm filter. Thereafter it was analyzed on an online HPLC-DPPH system using a ReproSil-Pur C18 AQ analytical column under optimal chromatographic conditions. The results have been shown in Figure 1D,E, and Fr14 had several negative peaks at 517 nm, thus indicating that there were several antioxidant peaks in Fr14. In The Fr1 at this stage still contained a large amount of chlorophyll. Hence, if Fr1 was prepared directly in the next step, it was possible that chlorophyll might adsorb on the stationary phase of the preparation column and cause damage to the preparation column, so it was necessary to remove the chlorophyll first. The MCI GEL ® CHP20P column was selected for the second pretreatment of Fr1 to remove the chlorophyll, whereas Fr1 was segmented to refine the fractions. Figure 1C shows a separation chromatogram. Five fractions (Fr11, Fr12, Fr13, Fr14, and Fr15) were collected after nine repetitions of the MCI GEL ® CHP20P pretreatment. Among them, Fr14 was 44.2 g, which was of high quality. We selected Fr14 as the target fraction for the subsequent separation.
In order to analyze the Fr14, 100 mg of Fr14 was solubilized in 0.1 mL MeOH and filtered through a 0.45 mm filter. Thereafter it was analyzed on an online HPLC-DPPH system using a ReproSil-Pur C18 AQ analytical column under optimal chromatographic conditions. The results have been shown in Figure 1D,E, and Fr14 had several negative peaks at 517 nm, thus indicating that there were several antioxidant peaks in Fr14. In addition, it was observed that the composition of Fr14 was rather complex, and it was difficult to directly separate the antioxidant peak. This requires the next step of pretreatment of Fr14 to enrich the various active components. As shown in Figure 1F, the diol mediumpressure chromatographic column divided Fr14 into five sub-fractions, and Fr145 (4.1 g) was selected as the next target fraction. The antioxidant peak of Fr145 was also recognized online on the HPLC-DPPH system using a ReproSil-Pur C18 analytical column AQ. Figure 1G,H showed a negative peak at 517 nm at around 17 min, thereby indicating that Fr145 contained an antioxidant peak (antioxidant component). Next, a Spherical C18 medium-pressure column was used for the next step of enrichment of the various active ingredients. As shown in Figure 2A, Fr145 was divided into four sub-fractions. After analysis using the online HPLC-DPPH system, it was found that the active components identified in Fr145 were enriched in Fr1451. The screening results have been shown in Figure 2B,C; there was a strong negative peak at 517nm during 45-50 min in Fr1451. To verify the reliability of these results, Fr145 and Fr1451 were compared under similar chromatographic conditions as in Figure 1G. The analysis results have been shown in Figure 2D,E. The activity peak at 17 min in Fr145 was indeed present in Fr1451. This provided direction for the subsequent separation and purification of active chromatographic peaks. difficult to directly separate the antioxidant peak. This requires the next step of pretreatment of Fr14 to enrich the various active components. As shown in Figure 1F, the diol medium-pressure chromatographic column divided Fr14 into five sub-fractions, and Fr145 (4.1 g) was selected as the next target fraction. The antioxidant peak of Fr145 was also recognized online on the HPLC-DPPH system using a ReproSil-Pur C18 analytical column AQ. Figure 1G,H showed a negative peak at 517 nm at around 17 min, thereby indicating that Fr145 contained an antioxidant peak (antioxidant component). Next, a Spherical C18 medium-pressure column was used for the next step of enrichment of the various active ingredients. As shown in Figure 2A, Fr145 was divided into four sub-fractions. After analysis using the online HPLC-DPPH system, it was found that the active components identified in Fr145 were enriched in Fr1451. The screening results have been shown in Figure 2B,C; there was a strong negative peak at 517nm during 45-50 min in Fr1451. To verify the reliability of these results, Fr145 and Fr1451 were compared under similar chromatographic conditions as in Figure 1G. The analysis results have been shown in Figure 2D,E. The activity peak at 17 min in Fr145 was indeed present in Fr1451. This provided direction for the subsequent separation and purification of active chromatographic peaks.

Target Preparation of Samwinol of Fr1451 with High-Pressure Liquid Chromatography
The analytical conditions in Figure 2B can effectively separate the active peaks from the impure peaks that exist next to them. We performed the preparation of Fr1451 on a ReproSil-Pur C18 AQ preparative column with linear amplification of the above conditions, and Figure 2F depicted the preparative chromatogram of the Fr142 fraction. The retention times of the active peaks observed on the ReproSil-Pur C18 AQ preparative column ( Figure 2F) and the ReproSil-Pur C18 AQ analytical column ( Figure 2B) were found

Target Preparation of Samwinol of Fr1451 with High-Pressure Liquid Chromatography
The analytical conditions in Figure 2B can effectively separate the active peaks from the impure peaks that exist next to them. We performed the preparation of Fr1451 on a ReproSil-Pur C18 AQ preparative column with linear amplification of the above conditions, and Figure 2F depicted the preparative chromatogram of the Fr142 fraction. The retention times of the active peaks observed on the ReproSil-Pur C18 AQ preparative column ( Figure 2F) and the ReproSil-Pur C18 AQ analytical column ( Figure 2B) were found to be similar in comparison to Figure 2B. After preparation, 16.1 mg of Fr14511 was finally obtained with a recovery of 4.3%.
Next, both the purity analysis and activity verification of Fr14511 were carried out using an online HPLC-DPPH system. As shown in Figure 3A,B, there was a strong negative activity peak at 517 nm. However, from Figure 3A, this chromatographic peak was found to be impure. It appears that the two chromatographic peaks were merged, and there were some small magazine peaks behind the main peak, so Fr14511 needs to be further purified. ther purified.
The chromatographic conditions were further optimized on the ReproSil-Pur C18 AQ column. When 40% acetonitrile was used for isocratic elution, two distinct chromatographic peaks were successfully separated in the range of 90-110 min ( Figure 3C). After the linear amplification of the chromatographic conditions, Fr14511 was purified. The main peak was Fr145112, the final collected fraction solution was concentrated and dried to obtain 3.74 mg of Fr145112, and the recovery rate was 23.2%.

Purity, Activity, and Structure of Fr145112
Isolated Fr145112 was re-evaluated for purity and activity using the online HPLC-DPPH system with the ReproSil-Pur C18 AQ analytical column. It was observed to possess purities greater than 95%, as illustrated in Figure 3D,E. To elucidate the structure of the target compound, Fr145112 were identified by comparing their ESI-MS and NMR spectrum data with the previously reported data. The supporting information includes the full spectra generated in this study. Fr145112 had NMR and MS data that matched with the data for samwinol. The chemical structure is shown in Figure 3D.  The chromatographic conditions were further optimized on the ReproSil-Pur C18 AQ column. When 40% acetonitrile was used for isocratic elution, two distinct chromatographic peaks were successfully separated in the range of 90-110 min ( Figure 3C). After the linear amplification of the chromatographic conditions, Fr14511 was purified. The main peak was Fr145112, the final collected fraction solution was concentrated and dried to obtain 3.74 mg of Fr145112, and the recovery rate was 23.2%.

Purity, Activity, and Structure of Fr145112
Isolated Fr145112 was re-evaluated for purity and activity using the online HPLC-DPPH system with the ReproSil-Pur C18 AQ analytical column. It was observed to possess purities greater than 95%, as illustrated in Figure 3D,E. To elucidate the structure of the target compound, Fr145112 were identified by comparing their ESI-MS and NMR spectrum data with the previously reported data. The supporting information includes the full spectra generated in this study. Fr145112 had NMR and MS data that matched with the data for samwinol. The chemical structure is shown in Figure 3D. . These data were consistent with the previously reported data for samwinol [25]. 6 of 14 To further evaluate the DPPH scavenging activity of samwinol, the protocol was performed by using a slight modification [26][27][28]. As shown in Figure 3F, samwinol exhibited significant antioxidant activity, and the IC 50 value was 211 ± 20.43 µM. The presence of the hydroxyl groups on the branched chains might have enhanced the antioxidant activity of the analyzed compounds. Both chromatographic and chemical methods have confirmed that the compound has good antioxidant activity, and substantial antioxidant activity could be the basis for the treatment of several other diseases. Using the existing compounds, other pharmacological activities can be further explored.

Binding Ability of Samwinol to Antioxidant Proteins
Since the above-ground part of D. heterophyllumand has been reported to treat lymphadenitis, hepatitis, and bronchitis, it was speculated that samwinol might also possess substantial antioxidant effects. Molecular docking analyses were performed to evaluate the potential interaction between samwinol and antioxidative protein Nrf2, HO-1, and NQO 1. The protein conformations of Nrf2, HO-1, and NQO1 and the possible amino acid sites of these antioxidant proteins bound to samwinol are depicted in Figure 4. The binding energy and ligand interaction have been shown in Table 1. Samwinol possessed more potential binding sites for Nrf2 and HO-1 and lowered the binding energy more than NQO1, which suggested that samwinol might influence the antioxidant pathway from Nrf2 to HO-1.

The Effects of Samwinol on PC12 Cells
To detect the potential effects of samwinol on the viability of PC12 cells, an MTT a was carried out with various concentrations of samwinol. The results in Figure 5A sho that the cell viability exhibited no significant changes when the concentration was less 10 μM. Thus, 5 μM and 10 μM of samwinol were selected in the following experiments Aβ25-35 has been commonly used to build the AD model. The PC12 cells were sta with Giemsa staining solution to observe the possible morphological changes. The re in Figure 6 showed that cell death, leakage of material from the nucleus, shortening o synapses, and cell crumpling were observed in the Aβ25-35 group. After the samwinol t ment, the cell density was found to increase, cell synapses were restored, and the gradually became round in appearance following the treatment.

The Effects of Samwinol on PC12 Cells
To detect the potential effects of samwinol on the viability of PC12 cells, an MTT assay was carried out with various concentrations of samwinol. The results in Figure 5A showed that the cell viability exhibited no significant changes when the concentration was less than 10 µM. Thus, 5 µM and 10 µM of samwinol were selected in the following experiments.

The Effects of Samwinol on PC12 Cells
To detect the potential effects of samwinol on the viability of PC12 cells, an MTT assay was carried out with various concentrations of samwinol. The results in Figure 5A showed that the cell viability exhibited no significant changes when the concentration was less than 10 μM. Thus, 5 μM and 10 μM of samwinol were selected in the following experiments. Aβ25-35 has been commonly used to build the AD model. The PC12 cells were stained with Giemsa staining solution to observe the possible morphological changes. The results in Figure 6 showed that cell death, leakage of material from the nucleus, shortening of cell synapses, and cell crumpling were observed in the Aβ25-35 group. After the samwinol treatment, the cell density was found to increase, cell synapses were restored, and the cells gradually became round in appearance following the treatment. The mitochondrial respiratory chain is one of the major sources of cellular ROS, which consists of a group of unstable molecules, including hydrogen peroxide (H2O2), hydroxyl radicals (OH − ), singlet oxygen (O 2 ), and superoxide (O 2− ) [29]. Under normal physiological conditions, the balance between ROS production and ROS clearance is tightly controlled [30]. When the body is in a state of oxidative stress, high levels of ROS Aβ [25][26][27][28][29][30][31][32][33][34][35] has been commonly used to build the AD model. The PC12 cells were stained with Giemsa staining solution to observe the possible morphological changes. The results in Figure 6 showed that cell death, leakage of material from the nucleus, shortening of cell synapses, and cell crumpling were observed in the Aβ 25-35 group. After the samwinol treatment, the cell density was found to increase, cell synapses were restored, and the cells gradually became round in appearance following the treatment.

The Effects of Samwinol on PC12 Cells
To detect the potential effects of samwinol on the viability of PC12 cells, an MTT assay was carried out with various concentrations of samwinol. The results in Figure 5A showed that the cell viability exhibited no significant changes when the concentration was less than 10 μM. Thus, 5 μM and 10 μM of samwinol were selected in the following experiments. Aβ25-35 has been commonly used to build the AD model. The PC12 cells were stained with Giemsa staining solution to observe the possible morphological changes. The results in Figure 6 showed that cell death, leakage of material from the nucleus, shortening of cell synapses, and cell crumpling were observed in the Aβ25-35 group. After the samwinol treatment, the cell density was found to increase, cell synapses were restored, and the cells gradually became round in appearance following the treatment. The mitochondrial respiratory chain is one of the major sources of cellular ROS, which consists of a group of unstable molecules, including hydrogen peroxide (H2O2), hydroxyl radicals (OH − ), singlet oxygen (O 2 ), and superoxide (O 2− ) [29]. Under normal physiological conditions, the balance between ROS production and ROS clearance is tightly controlled [30]. When the body is in a state of oxidative stress, high levels of ROS The mitochondrial respiratory chain is one of the major sources of cellular ROS, which consists of a group of unstable molecules, including hydrogen peroxide (H 2 O 2 ), hydroxyl radicals (OH − ), singlet oxygen (O 2 ), and superoxide (O 2− ) [29]. Under normal physiological conditions, the balance between ROS production and ROS clearance is tightly controlled [30]. When the body is in a state of oxidative stress, high levels of ROS can lead to oxidative damage. The production of ROS is central to the progression of several inflammatory diseases. ROS produced by cells is involved in the host defense response [31]. Therefore, the changes in ROS can be used to demonstrate the anti-inflammation ability of samwinol.
To verify the previous speculation, the ROS and Mito-ROS levels in PC12 were detected by flow cytometry. The results in Figure 5 show that the trend of the production of ROS (B) and Mito-ROS (C) results were consistent. The production of ROS and Mito-ROS in the Aβ 25-35 group were obviously higher than that of the control group, and 10 µM of samwinol could obviously reverse the situation. These findings suggested that Aβ [25][26][27][28][29][30][31][32][33][34][35] causes an increase in ROS production in PC12 cells, which was primarily derived from mitochondria, and the treatment of samwinol can effectively alleviate oxidative stress.

Western Blot Assay for Protein Expression in PC12 Cells
Mitogen-activated protein kinase (MAPKs) are a family of serine/threonine protein kinases, which include members of the extracellular receptor-activated kinase (ERK) 1/2, p38, and c-Jun N-terminal kinase (JNK) [32]. It is widely believed that MAPKs can play critical roles in the regulation of inflammation and induce the expression of various inflammatory mediators and pro-inflammatory cytokines [33]. The experimental results showed that the expression of p-ERK (1/2) was upregulated in the model group compared with the control group, and 10 µM of samwinol demonstrated superior anti-inflammatory activity than that of 5 µM. Samwinol might exert anti-inflammatory effects by inhibiting the activation of p-ERK (1/2). In addition, samwinol also suppressed the activation of p-AKT induced by Aβ [25][26][27][28][29][30][31][32][33][34][35] in a concentration-dependent manner.
In mammals, nuclear-associated factor 2 (Nrf2) plays a pivotal role in counteracting oxidative stress mechanisms, and Nrf2 is a defense system used to maintain cellular redox homeostasis [34]. During this process, oxidative stress can activate Nrf2 gene expression and transcriptional activity in the body. Next, Nrf2 can regulate the expression of antioxidant proteins such as heme oxygenase 1 (HO-1), and NQO1 can be activated in response to ROS to protect the cells from inflammation-induced oxidative stress [35]. The results in Figure 7 showed that samwinol markedly increased the expression of Nrf2 and HO-1. Since Aβ [25][26][27][28][29][30][31][32][33][34][35] caused oxidative stress in the cells, it led to the inhibition of Nrf2 expression. However, samwinol reversed this change, which could augment the expression of Nrf2 and promote the translocation of Nrf2 into the nucleus. Nrf2 can then increase the expression of antioxidant proteins HO-1 to counteract oxidative stress in PC12 cells. The protein expression of NQO1 displayed no significant difference (p > 0.05) between the Aβ 25-35 group and the samwinol-treated group. Thus, these results showed that samwinol exhibited minimal effects on the expression of NQO1 protein.
causes an increase in ROS production in PC12 cells, which was primarily derived from mitochondria, and the treatment of samwinol can effectively alleviate oxidative stress.

Western Blot Assay for Protein Expression in PC12 Cells
Mitogen-activated protein kinase (MAPKs) are a family of serine/threonine protein kinases, which include members of the extracellular receptor-activated kinase (ERK) 1/2, p38, and c-Jun N-terminal kinase (JNK) [32]. It is widely believed that MAPKs can play critical roles in the regulation of inflammation and induce the expression of various inflammatory mediators and pro-inflammatory cytokines [33]. The experimental results showed that the expression of p-ERK (1/2) was upregulated in the model group compared with the control group, and 10 μM of samwinol demonstrated superior anti-inflammatory activity than that of 5 μM. Samwinol might exert anti-inflammatory effects by inhibiting the activation of p-ERK (1/2). In addition, samwinol also suppressed the activation of p-AKT induced by Aβ25-35 in a concentration-dependent manner.
In mammals, nuclear-associated factor 2 (Nrf2) plays a pivotal role in counteracting oxidative stress mechanisms, and Nrf2 is a defense system used to maintain cellular redox homeostasis [34]. During this process, oxidative stress can activate Nrf2 gene expression and transcriptional activity in the body. Next, Nrf2 can regulate the expression of antioxidant proteins such as heme oxygenase 1 (HO-1), and NQO1 can be activated in response to ROS to protect the cells from inflammation-induced oxidative stress [35]. The results in Figure 7 showed that samwinol markedly increased the expression of Nrf2 and HO-1. Since Aβ25-35 caused oxidative stress in the cells, it led to the inhibition of Nrf2 expression. However, samwinol reversed this change, which could augment the expression of Nrf2 and promote the translocation of Nrf2 into the nucleus. Nrf2 can then increase the expression of antioxidant proteins HO-1 to counteract oxidative stress in PC12 cells. The protein expression of NQO1 displayed no significant difference (p > 0.05) between the Aβ25-35 group and the samwinol-treated group. Thus, these results showed that samwinol exhibited minimal effects on the expression of NQO1 protein.

Discussion
In this study, a quick and efficient approach of the medium-and high-pressure liquid chromatography combined with an online HPLC-DPPH system was used to recognize, separate, and purify antioxidative lignan from D. heterophyllum. The isolated compound was identified as samwinol. Samwinol is a lignin that was originally isolated from Sambucus williamsii [25]. Natural polyphenols can serve as potent antioxidants with the advantages of being non-toxic, highly safe, naturally available, and found in abundance in nature. The phenolic hydroxyl groups of lignin can contribute to its antioxidant activity [36]. The above-ground parts of Dracocephalum heterophyllum have been reported to be used for the treatment of hypertension, lymphadenitis, hepatitis, and bronchitis in Tibetan medicine [37]. The antioxidant capacity of samwinol isolated from D. heterophyllum was also established by molecular docking and DPPH assays for the first time.
In our study, the activity of the compounds was initially corroborated using the compounds obtained by isolation for the first time around the antioxidant system in the inflammatory process of AD. However, the development process of AD is complicated, and oxidative stress is also only one aspect of the interaction. This study only used the PC12 cells model to conduct preliminary activity evaluation and study in vitro. Combined with the molecular docking antioxidant, the results were in good agreement with the cell experiment, suggesting that our compound may have better findings in antioxidants. More studies are needed in the future to reveal the detailed mechanism of action of samwinol in the treatment of AD.

Instrumentation and Reagents
The preparative liquid chromatography used in this study included two NP7000 prep-HPLC pumps, an NU3000 ultraviolet-visible (UV-Vis) detector, and an LC workstation (Hanbon Science & Technology Co., Huaian, China). The online HPLC-DPPH system was constructed with Essentia LC-16 (Shimadzu Instruments, Co., Shanghai, China) and LC-10AD HPLC equipment (Shimadzu Instruments, Co., Kyoto, Japan). The device was equipped with two binary gradient pumps, a UV-Vis detector, a column oven, and an LC workstation. The two HPLCs were connected by a triple valve and a polyether ether ketone reaction coil (18.0 m × 0.25 mm i.d.). LC-16 was used for HPLC analysis, and LC-10AD was used to obtain DPPH screening chromatograms. ESI-MS analysis was performed by a Waters QDa Electrospray Ionization Mass Spectrometer (ESI-MS) (Waters Instruments Co., Milford, MA, USA). The 1H and 13C NMR spectra were acquired on a 600 MHz Bruker Avance spectrometer (Bruker Instruments Co., Berlin, Germany). The solvent for NMR was DMSO-d 6 . UV absorbance values were obtained with a Readmax 1900 microplate reader (Flash, Co., Shanghai, China).
DPPH was purchased from Sigma-Aldrich (Steinheim, Germany). Analytical and HPLC grade ethanol (MeOH), analytical grade dichloromethane (CH 2 Cl 2 ), analytical grade, and HPLC grade ACN, analytical grade n-hexane, and analytical grade ethyl acetate were purchased from the Kelon Chemical Reagent Factory (Chengdu, China). HPLC grade H 2 O was prepared using a water purifier from Moore (Chongqing, China).

Plant Sample Preparation and Medium Pressure Liquid Chromatography Pretreatment
Our group has previously successfully completed the isolation and identification of several new active compounds [7,8]. Based on the previous studies, the separation method of D. heterophyllum was adjusted to some extent in this study. The herb of D. heterophyllum was collected from North Mountain in Huzhu, Qinghai, and verified by Professor Lijuan Mei of Northwest Plateau Institute of Biology. The sample (nwipb-2016-10-10) was kept in the Qinghai-Tibet Plateau Biological Museum. The collected whole herbs were dried in the shade, weighed, and thereafter all the whole herbs (10.0 kg) were crushed and extracted three times with 100% methanol, each using 80.0 L of solvent, and the extraction time was 12 h. Finally, 240.0 L of extract solution was collected, filtered, and concentrated at 40 • C using a rotary evaporator. When the volume of the concentrate was reduced to 5.0 L, 1.5 kg of amorphous silica gel was added, mixed well, and dried in a 40 • C oven to obtain 2.8 kg of silica gel sample mixture. The silica gel mixture was pretreated by silica medium-pressure liquid chromatography using MeOH and CH 2 Cl 2 as mobile phases, and the elution conditions were 0-30 min, 0% methanol; 30-60 min, 0-100% methanol; and 60-90 min, 100% methanol, flow rate kept at 57.0 mL/min; the single sample loading was 65 g, and the chromatogram was recorded at 210 nm. After repeating this process 43 times, fraction Fr1 (205.5 g) was obtained as the subsequent separation material.
Fraction Fr1 (205.5 g) was dissolved in 2.0 L MeOH. Then 234.0 g of amorphous silica gel was added to the methanol solution of Fr1, mixed, and dried in an oven at 40 • C to finally obtain 439.5 g of a dry mixture. The mixture was further pretreated using MCI GEL ® CHP20P medium-pressure columns (49 × 460 mm). The mobile phase used was MeOH/H 2 O with elution conditions of 0-150 min, 20-100% methanol, and 150-210 min, 100% methanol. The flow rate and wavelength were the same as in the first step of pretreatment, and the single loading was 55.0 g. After repeating the process eight times, fractions Fr11-Fr15 were obtained and concentrated by drying. The fraction Fr14 (44.2 g) was selected for the subsequent isolation. Similarly, fraction Fr14 (44.2 g) was dissolved in 400.0 mL of MeOH, mixed with 74.0 g of amorphous silica gel, and dried in an oven at 40 • C. The dry mixture (118.2 g) was subjected to a third pretreatment with a diol mediumpressure column (50 × 500 mm) using N-hexane/ethyl acetate as a mobile phase. The linear gradient elution conditions were 0-90 min and 0-100% ethyl acetate. The flow rate was 57.0 mL/min, and the chromatogram was recorded at 280 nm. After the two replicate separations, the target fractions (Fr145) were collected, pooled, and concentrated to yield 4.1 g of enriched sample with 9.3% recovery.
Fr145 was further separated using the Spherical C18 medium-pressure column (50 × 500 mm) preparative column. HPLC grade water and ACN were used as mobile phases A and B, respectively. The gradient elution step for Fr145 was 0-90 min, 50-80% B. The equilibration time was 15 min at a flow rate of 57.0 mL/min. The chromatogram was recorded at 210 nm. Thereafter, 374 mg of Fr1451 was obtained.

High-Pressure Liquid Chromatography Separation and Purification of Samwinol from Fr1451
Fr1451 were separated by using a ReproSil-Pur C18 AQ (20 × 250 mm, 5 µm) preparative column. Mobile phase A was HPLC grade water, and mobile phase B was ACN. The isocratic elution step was performed by using 40% B for 60 min. The flow rate of the eluent was maintained at 19.0 mL/min, and the elution process was measured at 210 nm. Finally, 16 mg of Fr14511 was obtained. Then, Fr14511 (16 mg) was completely dissolved in MeOH (1.0 mL), eluted isocratically with 30% ACN/H 2 O on the ReproSil-Pur C18 AQ (20 × 250 mm, 5 µm) preparative column for 120 min, the chromatogram was recorded at 210 nm, and the second main peak (Samwinol) was collected and dried to obtain 3.45 mg of the compound.

Assessment of Purity and Activity of Samwinol
The purity and activity of samwinol were assessed by using an online HPLC-DPPH system. The analysis was performed by using a ReproSil-Pur C18 AQ (4.6 × 250 mm, 5 µm) column. The mobile phase system was ACN/H 2 O, with a linear gradient based on 40-80% ACN, an elution time 60 min, and a flow rate 1.0 mL/min. The absorbance was measured at 210 nm. The concentration of DPPH was 25 µg/mL, and the eluate flowed at a rate of 0.8 mL/min. DPPH ethanol solution chromatogram was recorded at 517 nm.
DPPH was prepared in an ethanol solution at a concentration of 25 µg/mL, and the isolated Samwinol was prepared in solutions with different concentration gradients (0.1, 1, 10, 50, 100, and 500 µg/mL). The sample solution (30 µL) and the ethanol solution (70 µL) were added to the 96-well plate, respectively. Each sample was repeated three times. The mixed solution was then incubated in the dark for 30 min. Finally, the UV absorption value of the mixture was recorded at 517 nm, denoted as A. This experiment was repeated three times. The scavenging rate of the DPPH radicals has been represented by the following formula.
where A 0 denotes the absorbance of the blank group (ethanol), A 1 is the absorbance of the control group, and A is the absorbance of the sample solution.

Predicting the Binding Capacity of Samwinol to Antioxidant Proteins by Using Molecular Docking
The binding ability of samwinol and antioxidant proteins Nrf2, HO-1, and NQO1 was predicted by using a computerized molecular docking analysis, which was used to predict the antioxidant capacity of Samwinol. The ID of Nrf2 (ID: 4IQK), HO-1 (ID: 1N3U), and NQO1 (ID:2FUG) can be found in the protein data bank (https://www.rcsb.org/, accessed on 23 August 2022). First, irons and water were eliminated from the receptor. Next, the Kollaman charges and polar hydrogen were added. The Lamarckian genetic algorithm, the pseudo-Solis, and Wets methods were used for the minimization, setting the parameters to default. According to the dock score values, a total of 100 peptide conformations were obtained. Model development predefined the minimum binding energy. These results were visualized and analyzed using Discovery Studio 2020. 4.6. Cellular Antioxidant Activity Assays 4.6.1. Culture of PC12 Cells and Aβ [25][26][27][28][29][30][31][32][33][34][35] Induced AD Model Aβ 25-35 (A107852, Aladdin, Shanghai, China) was dissolved in the ultrapure water used for reserve fluid (1 mM). The reserve fluid was stored at −20 • C and incubated at 37 • C for 72 h before use. The PC-12 cell line was purchased from the Cell Bank of the Chinese Academy of Science. The cells were maintained at 37 • C in a 5% CO 2 incubator with saturated humidity. The culture medium was DMEM (12800-082, Gibco, Beijing, China) with 1% penicillin and 10% fetal bovine serum.

Effects of Samwinol on Cell Viability
The PC12 cells were seeded in 96-well plates at a density of 5 × 10 3 cells/well and then incubated with samwinol at various concentrations (1-80 µM) for 24 h, respectively. After incubating, 10 µL of MTT solution (5 mg/mL) was added to each well for 4 h, and the medium was aspirated and shaken with 100 µL DMSO (67-68-5, Sinopharm, Beijing, China) for 15 min, and the absorbance value was measured at 490 nm using a microplate reader (Molecular Divice, Sunnyvalr, CA, USA).

Morphological Changes as Analyzed by GIEMSA Staining
The PC12 cells were inoculated in 24-well plates (5 × 10 4 cells/well), samwinol was incubated in advance for 2 h, then Aβ 25-35 was added and incubated for 24 h. The plates were washed twice with PBS when the medium was aspirated, and 4% paraformaldehyde (PH0427, Phygene, Fuzhou, China) was added to fix at 4 • C for one h. Giemsa staining solution was added for 10 min and washed with PBS. Finally, the cell morphology was observed under the microscope (Leica Microsystems CMS GmbH, Wetzlar, Germany).

Measurement of ROS and Mito-ROS Levels by Flow Cytometry
The PC12 cells were inoculated in 6-well plates (1 × 10 5 cells/well), samwinol was incubated in advance for 2 h, then Aβ 25-35 was added and incubated for 2 h. The cells were then incubated with a DCFH-DA probe (5 µM) and Mito-sox probe (10 µM) for 15 min and 30 min, respectively. Thereafter, the cells were digested for 5 min and collected for centrifugation. The supernatant was aspirated, and the cells were mixed well by adding PBS. ROS and Mito-ROS levels were assayed by flow cytometry (ACEA Biosciences, San Diego, CA, USA).

Detection of Protein Expression by Western Blot Assay
Western blot was used to analyze changes in cellular signaling. Briefly, the total proteins were extracted by lysing the cells with cell lysis solution (P0013, Beyotime, Shanghai, China) with 1%PMSF (ST506-2, Beyotime, Shanghai, China), and the protein concentration was measured by the BCA Protein Assay Kit. (P0009, Beyotime, Shanghai, China). Then, 10% SDS-PAGE was used to separate the protein samples and transfer the target protein to the PVDF membrane (ISEQ00010, Millipore, Ireland) under a 250 mA current. Then, 5% skimmed milk (G5002, Servicebio, Wuhan, China) was used to seal the membranes for 1.5 h. After removing the milk, the membranes were washed 3 times with TBST. The PVDF membranes were incubated with the primary antibody overnight, and β-actin (8H10D10, cell signal technology, Danvers, MA, USA) was used as an internal control. The membranes were then washed three times with TBST and incubated with rabbit polyclonal secondary antibody for 1 h. The membrane was exposed after adding a developer to the membrane.

Statistical Analysis
All of the experiments were performed in triplicates, and the data have been presented as the mean ± standard deviation. One-way ANOVA or Student's t-test using SPSS 18.0 (SPSS, Chicago, IL, USA) was conducted. p < 0.05 was considered statistically significant.

Conclusions
In this study, we isolated samwinol from D. heterophyllum for the first time and confirmed that samwinol could prevent the neuroinflammation induced by Aβ [25][26][27][28][29][30][31][32][33][34][35] in PC-12 cells by modulating the activation of the ERK pathway. The involved mode of action was associated with the activation of the Nrf2/HO-1 pathway, which was attributed to the suppression of oxidative stress. In the future, additional mechanistic studies will be carried out to provide complete evidence for the potential of samwinol in the treatment of AD.