Anti-Inflammatory Effects of Alphitolic Acid Isolated from Agrimonia coreana Nakai Extracts Are Mediated via the Inhibition of ICRAC Activity in T Cells

Agrimonia pilosa Ledeb., an important medicinal herb in traditional East Asian medicine, is primarily used to treat abdominal pain, dysentery, and hemostasis. There are ten other reported species of Agrimonia plants, including Agrimonia coreana Nakai—a naturally growing species in South Korea—and Agrimonia eupatoria Linn. Although recent studies have isolated numerous active constituents and investigated their effects, the medicinal utility of this herb is not yet fully explored. Through patch-clamp recording, a previous study reported that Agrimonia plant extracts inhibit the function of Ca2+ release-activated Ca2+ channels (CRACs). Herein, we aimed to identify and isolate the main compounds in A. coreana responsible for CRAC inhibition while assessing the anti-inflammatory effects mediated by this inhibition. We demonstrated for the first time that alphitolic acid isolated from A. coreana has a dose-dependent inhibitory effect on CRAC activity and, thus, an inhibitory effect on intracellular calcium increase. Furthermore, analysis of human CD4+ T cell proliferation via the carboxyfluorescein diacetate succinimidyl ester method revealed that alphitolic acid inhibited T cell proliferation in a concentration-dependent manner. Our findings provide a theoretical basis for the potential therapeutic use of alphitolic acid in the treatment of inflammatory diseases.


Introduction
Inflammation is a defense mechanism against tissue damage caused by numerous factors in response to stimulants arising from the internal and external environments, including invading pathogens, such as viruses, bacteria, and fungi.Most direct causal effectors of inflammation are produced by immune cells.Further, although immune cells, such as T cells, protect the host by neutralizing diverse external pathogens, their excessive activity can cause disease development.For example, autoimmune diseases, such as rheumatism, are caused by the excessive activity of helper T type 1 cells, whereas the excessive activity of helper T type 2 cells leads to allergic diseases [1,2].The T cell-mediated immune responses are initiated by increased intracellular calcium concentrations via calcium entry through ion channels, resulting in T cell proliferation and differentiation and cytokine secretion.Ca 2+ release activated Ca 2+ channels (CRACs) have a crucial role in calcium influx into T cells [3].In particular, store-operated calcium entry (SOCE) is a specific type of calcium influx through CRACs.SOCE is initiated following T cell receptor (TCR) and co-receptor stimulation, leading to phospholipase C-γ1 (PLCγ1) activation, which triggers the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol-1,4,5-triphosphate (IP3) and diacylglycerol.Subsequently, IP3 binds to the IP3 receptor in the endoplasmic reticulum (ER)-an intracellular calcium storage compartment-causing a decrease in ER calcium levels.This depletion triggers the binding between stromal interaction molecule 1 (STIM1) in the ER and ORAI1 ion channels in the cell membrane, effectively opening the ion channels and enabling the influx of calcium into the cell.SOCE is pivotal in regulating intracellular calcium signaling and activation of downstream pathways, including the Ca 2+ -dependent kinase-calmodulin (CaMK) and calcineurin (CaN) pathways and the nuclear translocation of the transcriptional regulator nuclear factor of activated T cell (NFAT).These events ultimately lead to the upregulation of target genes critical for innate and adaptive immunity [3,4].
CRACs also play a key role in most immune cells and, thus, participate in various immune mechanisms from phagocytosis and B cell activation to degranulation [3,5,6].Abnormal functioning of CRACs has been linked to severe combined immunodeficiency and various other medical conditions, including allergies, atherosclerosis, and inflammatory bowel disease.Additionally, CRACs reportedly influence cancer cell proliferation and metastasis [7][8][9][10][11].Accordingly, increased attention has been focused on substances that can modulate the activity of these channels [12].In particular, curcumin, vitexin, alphamangostin, and fargesin exhibit CRAC inhibitory and anti-inflammatory effects [13][14][15][16][17].The ongoing research related to these natural substances holds promise for developing novel compounds capable of inhibiting CRACs, contributing to the treatment and prevention of myriad diseases [12,18].
Agrimonia coreana Nakai is a perennial plant of the Rosaceae family and a species of the same genus as Agrimonia pilosa.Although these species have slight morphological variations, their active compounds are relatively conserved [19].Nevertheless, few studies have examined the anti-inflammatory effects or the constituent compounds of A. coreana, a traditional medicinal herb used in East Asia, including Korea, Japan, China, and India, and listed in the Korean Herbal Pharmacopoeia under the name Yong-A-Cho.Records show that A. coreana has mainly been used for detoxification, alleviating fever, and treating enteritis, malaria, and hemostatic disorders.Moreover, it is used in traditional Chinese medicine for hemostatic, anti-malarial, detoxifying, and anti-tumor effects [20,21].However, other medicinal effects of A. coreana are being investigated, with reports on anti-oxidant, anti-inflammatory, and anti-viral properties [22][23][24][25].Furthermore, A. coreana contains myriad active compounds from flavonoids to triterpenes and their glycosides, isoflavones, isocoumarins, phloroglucinol derivatives, tannins, and organic acids, comprising 252 constituents, with numerous associated ongoing studies [20].
Alphitolic acid (ALA) is a pentacyclic triterpene constituent commonly isolated from Alphitonia petriei (Rhamnaceae), a tropical tree that naturally grows in areas from the north of New South Wales on the east of Australia to the Torres Straight in Queensland.However, ALA has been isolated from various other plants, including Ziziphus jujuba [26,27].The numerous reported therapeutic effects of ALA range from anti-inflammatory and antioxidant to NO inhibition and anti-cancer activities [28][29][30].Meanwhile, few studies have evaluated the anti-inflammatory effect within the context of ion channels.
Herein, we evaluated the anti-inflammatory effects of A. coreana via CRAC inhibition.To this end, we identified the main active constituent in A. coreana responsible for inhibiting CRAC activity and verified whether it elicited anti-inflammatory effects.The results of this study provide evidence for the potential application of A. coreana-derived ALA in the treatment of inflammatory diseases.

Preparation of A. coreana Extracts and Isolation of Active Fractions
We prepared A. coreana extracts using 70% ethanol (EtOH) and 95% EtOH extracts (AC 70ext and AC 95ext , respectively) from the collected A. coreana samples to determine the active constituent of A. coreana with inhibitory effects on CRAC activity.The yields of the two extracts were 10.85% and 4.25%, respectively, with AC 70ext showing a higher yield than AC 95ext .Importantly, 95% ethanol and 70% ethanol have different polarities; hence, the carbohydrates, the flavonoid glucuronide (or glycoside), and tannin contained in A. coreana were relatively well soluble in 70% ethanol, whereas the lipids and terpenes were primarily extracted with 95% ethanol.As A. coreana is rich in flavonoids, the yield was expected to be higher with a 70% ethanol solvent.Analysis of the inhibition of CRAC (I CRAC ) current using the two extracts revealed a stronger inhibitory effect with AC 70ext (81.27 ± 3.880%) than with AC 95ext (73.23 ± 8.790%; Figure 1a,b).This extract was separated into n-hexane (HEX), chloroform (CHCl 3 ), ethyl acetate (EtOAc), butanol (BuOH), and water fractions.

Preparation of A. coreana Extracts and Isolation of Active Fractions
We prepared A. coreana extracts using 70% ethanol (EtOH) and 95% EtOH extracts (AC70ext and AC95ext, respectively) from the collected A. coreana samples to determine the active constituent of A. coreana with inhibitory effects on CRAC activity.The yields of the two extracts were 10.85% and 4.25%, respectively, with AC70ext showing a higher yield than AC95ext.Importantly, 95% ethanol and 70% ethanol have different polarities; hence, the carbohydrates, the flavonoid glucuronide (or glycoside), and tannin contained in A. coreana were relatively well soluble in 70% ethanol, whereas the lipids and terpenes were primarily extracted with 95% ethanol.As A. coreana is rich in flavonoids, the yield was expected to be higher with a 70% ethanol solvent.Analysis of the inhibition of CRAC (ICRAC) current using the two extracts revealed a stronger inhibitory effect with AC70ext (81.27 ± 3.880%) than with AC95ext (73.23 ± 8.790%; Figure 1a,b).This extract was separated into nhexane (HEX), chloroform (CHCl3), ethyl acetate (EtOAc), butanol (BuOH), and water fractions.Each fraction was evaluated for its inhibitory effect on ICRAC; a stronger effect was observed with HEX (76.02 ± 5.654%) and CHCl3 fractions (92.84 ± 1.901%) than with EtOAc fractions (43.31 ± 3.439%; Figure 1c-e).It was thus postulated that, in A. coreana, the main active constituent responsible for the anti-inflammatory effect via ICRAC inhibition was a nonpolar compound in the HEX and CHCl3 fractions.
Therefore, to isolate and purify the active constituent in the CHCl3 fraction with the highest activity, silica column chromatography was performed on the CHCl3 layer to obtain seven fractions (AC-C-1 to AC-C-7).AC-C-3 (50.96 ± 7.190%), AC-C-6 (53.98 ± 9.776%), and AC-C-7 (63.91 ± 9.654%) exhibited inhibitory effects on ICRAC (Figure 2a).We then assessed the fraction with the highest activity (AC-C-7) to identify the active ingredients.However, sufficient yields were not obtained to isolate the ingredients.Therefore, we Each fraction was evaluated for its inhibitory effect on I CRAC ; a stronger effect was observed with HEX (76.02 ± 5.654%) and CHCl 3 fractions (92.84 ± 1.901%) than with EtOAc fractions (43.31 ± 3.439%; Figure 1c-e).It was thus postulated that, in A. coreana, the main active constituent responsible for the anti-inflammatory effect via I CRAC inhibition was a nonpolar compound in the HEX and CHCl 3 fractions.
Therefore, to isolate and purify the active constituent in the CHCl 3 fraction with the highest activity, silica column chromatography was performed on the CHCl 3 layer to obtain seven fractions (AC-C-1 to AC-C-7).AC-C-3 (50.96 ± 7.190%), AC-C-6 (53.98 ± 9.776%), and AC-C-7 (63.91 ± 9.654%) exhibited inhibitory effects on I CRAC (Figure 2a).We then assessed the fraction with the highest activity (AC-C-7) to identify the active ingredients.However, sufficient yields were not obtained to isolate the ingredients.Therefore, we selected AC-C-3, which had the second strongest activity, and confirmed the effect.Through Sephadex LH-20 column chromatography, the AC-C-3 fraction was further separated into five fractions (AC-C-3-1 to AC-C-3-5).The observed inhibitory effects of these five fractions on the I CRAC were negligible compared with those of the previously obtained fractions.Nevertheless, the fraction with the most robust effect was AC-C-3-3 (25.40 ± 1.414%), which was used to perform preparative ODS medium-pressure liquid chromatography (MPLC) and for further fractionation into five groups (Figure 2b).Among these groups, No. 4 elicited the strongest effect (81.86 ± 8.631%; Figure 2c).The details of this fractionation are presented in the schematic diagram of Figure 3, and the I CRAC inhibition results for each fraction are presented in Table 1.
Int. J. Mol.Sci.2023, 24, x FOR PEER REVIEW 4 of 13 selected AC-C-3, which had the second strongest activity, and confirmed the effect.Through Sephadex LH-20 column chromatography, the AC-C-3 fraction was further separated into five fractions (AC-C-3-1 to AC-C-3-5).The observed inhibitory effects of these five fractions on the ICRAC were negligible compared with those of the previously obtained fractions.Nevertheless, the fraction with the most robust effect was AC-C-3-3 (25.40 ± 1.414%), which was used to perform preparative ODS medium-pressure liquid chromatography (MPLC) and for further fractionation into five groups (Figure 2b).Among these groups, No. 4 elicited the strongest effect (81.86 ± 8.631%; Figure 2c).The details of this fractionation are presented in the schematic diagram of Figure 3, and the ICRAC inhibition results for each fraction are presented in Table 1.

Inhibitory Effects of Active Constituents on ICRAC and Ca 2+ Influx
Next, we analyzed the inhibitory effect of a single-type compound isolated fro coreana, i.e., ACC-311, on ICRAC.The results revealed concentration-dependent ICRAC in itory activities of ACC-311, leading to inhibition by 6.17 ± 1.057% at 1.0 µM, 34.46 ± 6.0 at 10.0 µM, and 75.22 ± 2.956% at 100 µM, whereas the calculated half maximal inhib concentration (IC50) was 17.52 ± 2.458 µM (Figure 5a,b).Considering that intracellular cium influx through ICRAC plays a major role in T cell activation and proliferation, w duced calcium influx through CRAC in Jurkat T cells to determine whether the ICRA hibitory effect of ACC-311 reduces calcium influx.ER depletion was achieved thapsigargin treatment while maintaining 0 mM calcium in the extracellular solution.sequently, 2 mM calcium was added to induce intracellular calcium influx.When the cium influx was constant, 30 µM ACC-311 was added.Results confirmed suppressio calcium influx (Figure 5c,d).

Inhibitory Effects of Active Constituents on CD4 + T Cell Proliferation
To determine whether ACC-311 could suppress T cell proliferation via ICRAC in tion, CD4 + T cells were produced by stimulating human peripheral blood mononu cells (PBMCs) with anti-CD3 and anti-CD28 antibodies.To confirm T cell prolifera

Discussion
The anti-inflammatory, antioxidant, and anti-viral effects of A. coreana have be demonstrated previously.Many studies have also investigated the diversity of consti ents in this plant species [20].Herein, a constituent of A. coreana exhibiting an anti-infla matory effect was identified, and the anti-inflammatory effect was evaluated in terms ion channel inhibition.According to a previous study, A. pilosa methanol extract and fractions could inhibit ORAI1 [33].In contrast, the current study obtained A. coreana tracts using 70% and 95% ethanol rather than methanol; the extraction yields and inhi tory effects were compared based on the EtOH content.Next, to identify the active co ponent that exhibited an ICRAC inhibitory effect, the extracts of A. coreana were fractionat with HEX, CHCl3, EtOAc, BuOH, and water.The CHCl3 fraction exhibited ICRAC inhibiti and was further separated via several columns.Ultimately, one constituent, i.e., ACC-3 was identified as ALA in the NMR analysis.To the best of our knowledge, among all stu ies reporting on the constituents of A. coreana, the present study is the first to report t isolation and identification of ALA.
We further assessed whether ACC-311 exerts an anti-inflammatory effect by regul ing intracellular calcium signals through ICRAC inhibition.The estimated IC50 for ACC-3 to inhibit ICRAC was 17.52 ± 2.458 µM, and ACC-311 inhibited intracellular calcium infl through CRAC in Jurkat T cells.We thus posited that inhibition of intracellular calciu

Discussion
The anti-inflammatory, antioxidant, and anti-viral effects of A. coreana have been demonstrated previously.Many studies have also investigated the diversity of constituents in this plant species [20].Herein, a constituent of A. coreana exhibiting an anti-inflammatory effect was identified, and the anti-inflammatory effect was evaluated in terms of ion channel inhibition.According to a previous study, A. pilosa methanol extract and its fractions could inhibit ORAI1 [33].In contrast, the current study obtained A. coreana extracts using 70% and 95% ethanol rather than methanol; the extraction yields and inhibitory effects were compared based on the EtOH content.Next, to identify the active component that exhibited an I CRAC inhibitory effect, the extracts of A. coreana were fractionated with HEX, CHCl 3 , EtOAc, BuOH, and water.The CHCl 3 fraction exhibited I CRAC inhibition and was further separated via several columns.Ultimately, one constituent, i.e., ACC-311, was identified as ALA in the NMR analysis.To the best of our knowledge, among all studies reporting on the constituents of A. coreana, the present study is the first to report the isolation and identification of ALA.
We further assessed whether ACC-311 exerts an anti-inflammatory effect by regulating intracellular calcium signals through I CRAC inhibition.The estimated IC50 for ACC-311 to inhibit I CRAC was 17.52 ± 2.458 µM, and ACC-311 inhibited intracellular calcium influx through CRAC in Jurkat T cells.We thus posited that inhibition of intracellular calcium signaling would suppress T cell proliferation.To test this hypothesis, we evaluated the inhibitory effect of ACC-311 on human CD4+ T cell proliferation following stimulation with anti-CD3 and anti-CD28.Apparent inhibition was observed on cell proliferation.The I CRAC inhibitor, BTP2, also effectively inhibited T cell proliferation.The inhibitory activity of ACC-311 on CRAC may be caused by STIM1 and ORAI1 regulation, two major proteins involved in CRAC activity.However, this study did not further elucidate whether the associated mechanism involves the ORAI1 pathway or STIM1, or interference in the formation of the ORAI1 and STIM1 complex.This will be a topic of a future study [34].The role of CRAC in T cell activation and the CaMK/CaN/NFAT signaling pathway is well known [3,4].Immunosuppressive drugs like cyclosporine A and tacrolimus, which have been clinically proven to be efficacious, target the same pathway by regulating the NFAT pathway through CaN inhibition [35,36].CRACs play a crucial role in this NFAT pathway and have been suggested as a target for CaN/NFAT regulation.It can be assumed that ACC-311 inhibits T cell activity by suppressing NFAT activity, as intracellular calcium concentration decreases through CRAC inhibition.Indeed, altered intracellular calcium concentration can act as a crucial signal for T cell proliferation and activation [37][38][39][40].In a previous study, ALA was shown to inhibit the nuclear factor-kappa B (NF-κB) pathway [41].As with intracellular calcium signaling, the NF-κB pathway is associated with immune cell activity, proliferation, and differentiation, whereas the pathway proteins are engaged in activities such as epithelial cell differentiation or apoptosis [42].Regulation of the NF-κB pathway in immune cells affects the production of various cytokines that control cellular proliferation and apoptotic differentiation.Hence, the inhibitory effect on T cells in the present study could have led to a simultaneous anti-inflammatory effect via inhibition of the NF-κB pathway.
The inhibitory effects of ALA on immune cell proliferation were mediated by inhibiting a CRAC.However, it remains uncertain whether ALA is the sole contributor to the antiinflammatory effect of A. coreana as not all constituents of all fractions with a CRAC inhibitory effect were investigated.Hence, further investigation is needed to verify or expand our findings.
In summary, we isolated and purified a previously unreported ALA from A. coreana extract.Additionally, we demonstrated its inhibitory effect on I CRAC and the associated suppression of intracellular calcium influx, resulting in inhibition of CD4 + T cell proliferation.CRAC inhibition is a mechanism that has not yet been targeted to achieve immunosuppression because it modulates upstream signaling pathways rather than inhibiting CaN or regulating the activity of NFAT.The ALA we discovered is a drug with these effects and can be effective in treating inflammation and allergic diseases caused by T cell activation.

Extraction and Isolation
The dried aerial parts of A. coreana were ground; 200 g of each sample was extracted with 20-fold (4 L) ethanol solvent (70%, 95%) for 4 h and filtered at room temperature using a 5 µm filter paper.The filtered samples were concentrated under reduced pressure at 45 • C and subsequently freeze-dried.The yield from 70% ethanol was 10.85%, resulting in 21.7 g of extract, while that of the 95% ethanol extract was 4.25% (8.5 g of extract).
The dried aerial part of A. coreana (254.3 g) was extracted with 70% aqueous EtOH at room temperature (25 • C).The extract was concentrated under reduced pressure, and the resultant aqueous fraction was sequentially partitioned using hexane, chloroform, and water.The CHCl3-soluble portion was concentrated under reduced pressure, subjected to silica gel column chromatography, and eluted stepwise with hexane:EtOAc (10:1-1:1, v/v).The active fraction A was further separated by Sephadex LH-20 column chromatography and eluted with CHCl3:MeOH (1:1, v/v) to produce an active fraction.This active fraction was chromatographed on a column of Sephadex LH-20 and eluted with methanol to create an active sub-fraction A1, which was subjected to reversed-phase MPLC and eluted with a gradient using increasing MeOH in water (30% to 60% aqueous MeOH) to produce the A1-1 subfraction.This sub-fraction was subjected to preparative HPLC using a system equipped with a COSMOSIL C18 (i.d. 10 × 150 mm) column with isocratic elution using 65% aqueous acetonitrile at a flow rate of 3 mL/min to yield ACC-311.To increase the purity of ACC-311, it was subjected to preparative HPLC using a system equipped with TSKgel ODS (i.d.4.6 × 150 mm) column with a gradient elution using increasing acetonitrile in water (70% to 100% aq.acetonitrile) at a flow rate of 1 mL/min to yield ACC-311 (2.8 mg).

HPLC
The purity of ACC-311 (2.8 mg) was assessed using an HPLC system equipped with a TSKgel ODS (4.6 i.d.× 150 mm) column and photodiode array (Hitachi L-2455 diode array detector, Japan) and eluted with a gradient solvent system of 20% aq.acetonitrile to acetonitrile containing 0.04% trifluoroacetic acid at a flow rate of 1 mL/min.

Nuclear Magnetic Resonance Spectrometry
All NMR spectra were recorded on a JEOL JNM ECA500 FT-NMR spectrometer.The NMR measurements, including those from 1 H, 13 C, HMQC, HMBC, and 1 H-1 H COSY analyses, were carried out using 5 mm probe tubes at a temperature of 30 • C in CD 3 OD solutions.The 1 H NMR and 13 C NMR chemical shifts were referenced to the residual solvent peak of CD 3 OD at [δ H 3.31, δ C 49.0] ppm for 1 H nucleus and 13 C nucleus, respectively.

Figure 1 .
Figure 1.Column fractions for evaluating the ICRAC inhibition effects, and isolating the active constituents, of A. coreana.The inhibitory effects of A. coreana extracts prepared using 70% and 95% ethanol (AC70ext and AC95ext, respectively) on ICRAC.(a) Representative chart trace of the ICRAC activity inhibition caused by AC70ext; the concentration at treatment was 100 mg/mL.(b) Current-voltage (IV) relationship curve of the inhibitory effects of AC70ext and AC95ext; ICRAC inhibition was detected at 100 mg/mL in each case.(c) Representative chart trace of the ICRAC inhibition caused by the CHCl3 fraction of A. coreana extract (ACCHCl3); the concentration at treatment was 100 µg/mL.BTP2 was used as the control for comparing the inhibition rates, and its concentration at treatment was 10.0 µM.(d) IV curve of the inhibitory effects of ACCHCl3; the concentration at treatment was 100 µg/mL.(e) ICRAC inhibitory effects of various A. coreana fractions.*p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 1 .
Figure 1.Column fractions for evaluating the I CRAC inhibition effects, and isolating the active constituents, of A. coreana.The inhibitory effects of A. coreana extracts prepared using 70% and 95% ethanol (AC 70ext and AC 95ext , respectively) on I CRAC .(a) Representative chart trace of the I CRAC activity inhibition caused by AC 70ext ; the concentration at treatment was 100 mg/mL.(b) Currentvoltage (IV) relationship curve of the inhibitory effects of AC 70ext and AC 95ext ; I CRAC inhibition was detected at 100 mg/mL in each case.(c) Representative chart trace of the I CRAC inhibition caused by the CHCl 3 fraction of A. coreana extract (AC CHCl3 ); the concentration at treatment was 100 µg/mL.BTP2 was used as the control for comparing the inhibition rates, and its concentration at treatment was 10.0 µM.(d) IV curve of the inhibitory effects of AC CHCl3 ; the concentration at treatment was 100 µg/mL.(e) I CRAC inhibitory effects of various A. coreana fractions.* p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 3 .
Figure 3. Schematic diagram of the isolation of purified compounds using different columns from AC70ext.

Figure 3 .
Figure 3. Schematic diagram of the isolation of purified compounds using diff AC70ext.

Figure 3 .
Figure 3. Schematic diagram of the isolation of purified compounds using different columns from AC 70ext .

Figure 6 .
Figure 6.ICRAC inhibition induced by ACC-311 causes inhibitory effects on human primary T proliferation.(a) Cell proliferation of human CD4 + T cells stained with CFSE and cultured for 3 d after stimulation with anti-CD3 and anti-CD28.Flow cytometry results of T cell proliferation bef and after stimulation.BTP2, a CRAC inhibitor at 10 µM, served as the negative control.(b) Gra showing the proliferation rate of CD4+ T cells.(c) Inhibition of human primary CD4 + T cell prolif ation by ACC-311.The control reagent used was an ICRAC inhibitor, 10 µM BTP2.(d) Proliferation primary CD4 + T cells based on ACC-311 concentration.***p < 0.001, ****p < 0.0001.

Figure 6 .
Figure 6.I CRAC inhibition induced by ACC-311 causes inhibitory effects on human primary T cell proliferation.(a) Cell proliferation of human CD4 + T cells stained with CFSE and cultured for 3 days after stimulation with anti-CD3 and anti-CD28.Flow cytometry results of T cell proliferation before and after stimulation.BTP2, a CRAC inhibitor at 10 µM, served as the negative control.(b) Graph showing the proliferation rate of CD4+ T cells.(c) Inhibition of human primary CD4 + T cell proliferation by ACC-311.The control reagent used was an I CRAC inhibitor, 10 µM BTP2.(d) Proliferation of primary CD4 + T cells based on ACC-311 concentration.*** p < 0.001, **** p < 0.0001.

Table 1 .
Inhibition rates of the fractions from A. coreana.