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Article

13-HODE and 13-HOTrE, Present in the Traditional Chinese Medicine Herbal Extract di gu pi, Selectively Inhibit Platelet Function

by
Dylan Simpson
1,
Eliana Botta
2,
Pooja Yalavarthi
2,
Yein Ji
1,
Krista Goerger
2,
Paul Houston
1,
Sky Kareht
1,
Drewv Desai
1,
Daniela Bolaños
1,
Theodore R. Holman
1,* and
Michael Holinstat
2,3,*
1
Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, CA 95064, USA
2
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
3
Department of Internal Medicine, Division of Cardiovascular Medicine, University of Michigan, Ann Arbor, MI 48109, USA
*
Authors to whom correspondence should be addressed.
Pharmaceuticals 2026, 19(2), 263; https://doi.org/10.3390/ph19020263
Submission received: 17 January 2026 / Revised: 29 January 2026 / Accepted: 30 January 2026 / Published: 3 February 2026
(This article belongs to the Section Natural Products)
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Abstract

Background: Platelet hyperreactivity contributes to occlusive thrombus formation in vessels, precipitating acute cardiovascular events such as myocardial infarction and stroke. Traditional Chinese Medicine (TCM) has been used for centuries, and numerous TCM herbs have been reported to exert anti-inflammatory and anticoagulant effects. Objectives: We sought to identify key compounds within the TCM-derived herbal extracts that regulate platelet activity. Methods: Crude and fractioned herbal extracts were screened for their ability to inhibit platelet activation in response to multiple agonists. Platelet aggregation and flow cytometry were used to assess the potency and selectivity of the compounds within the extracts. Results: Three extracts, di gu pi (DGP), san qi (SQ), and zi cao (ZC), demonstrated inhibitory activity and were subsequently fractionated. Fractions derived from DGP, the root bark of Lycium chinense, inhibited platelet aggregation and suppressed integrin activation and granule secretion downstream of collagen receptor signaling. Further analysis identified the oxidized lipids 9(S)-hydroxy-9Z,11E-octadecadienoic acid (9-HODE), 13(S)-HODE, and 13(S)-hydroxy-9Z,11E,15Z-octadecatrienoic acid (13-HOTrE) as constituents of the bioactive fractions. Both 13-HODE and 13-HOTrE selectively inhibited collagen-mediated platelet aggregation without affecting thrombin-induced activation. Conclusions: Collectively, these findings identify oxylipins in TCM as promising candidates for the development of antiplatelet therapies targeting platelet activity and thrombosis. These oxylipins may represent novel approaches for thrombosis and have high therapeutic potential for development as next-generation antiplatelet drugs.

1. Introduction

Cardiovascular disease (CVD) remains the leading cause of death worldwide and represents a major burden on the healthcare system [1]. Platelet activation is a central contributor to the development and progression of CVD, driving the formation of occlusive thrombi that underlie acute events such as myocardial infarction and stroke [2,3,4,5]. Thus, modulation of platelet activity has proven to be clinically effective in reducing CVD risk [6,7,8,9]. Current antiplatelet therapies are limited to targeting cyclooxygenase-1 (COX-1), integrin αIIbβ3, and the purinergic P2Y12 G-protein coupled receptor (GPCR) [10,11,12]. Novel biologics or therapeutic strategies that attenuate pathological platelet activation are needed to better target the thrombotic risk observed in patients with cardiovascular disease.
Traditional Chinese Medicine (TCM) offers a rich and largely untapped resource for natural product discovery, rooted in centuries of documented clinical observation and therapeutic use [11,12,13,14,15,16]. The systematic categorization of herbs within TCM’s framework offers a structured approach for scientific investigation. Within this framework, herbs are grouped based on their observed physiological effects, offering a rational approach for targeted screening. Two such categories, “Invigorate the Blood” and “Cool the Blood”, are of particular interest due to their associations with modulation of coagulation or inflammatory pathways and platelets [13,14,15,16,17,18].
Herbs classified as “Invigorate the Blood” have traditionally been used to treat pain, physical trauma, and the recovery following injury. Emerging evidence suggests that these herbs may contain bioactive compounds capable of influencing platelet function or vascular health [15,16,19]. In contrast, herbs categorized as The “Cool the Blood” have been historically utilized as antipyretic, anxiolytic, and anti-inflammatory agents. Their relevance to coagulation is supported by the loose interplay between inflammation, platelet activation, and thrombus formation [16,19,20,21]. Due to the potential biological relevance of these two categories of Chinese herbs, we focused on commonly used herbs in these categories.
Based on these historical and mechanistic considerations, we sought to investigate herbal extracts from both the “Invigorate the Blood” and “Cool the Blood” categories to elucidate their potential cardiovascular protective effects through platelet inhibition. In an initial screen, we evaluated 48 crude herbal extracts for their ability to modulate platelet activation in response to four distinct agonists that simulate vascular injury. Extracts were selected for further purification based on their ability to inhibit at least 70 percent of collagen-induced activation, an arbitrary cut-off that facilitated separation between extracts with inhibitory versus non-inhibitory effects on platelet activation. From these, Di gu pi (DGP), the root bark of Lycium chinense, was chosen for detailed study due to its potent bioactivity [21,22,23]. Inhibitory compounds within DGP were identified using mass spectrometry, and their effects on platelet activation were subsequently characterized to understand the mechanisms underlying DGP’s cardiovascular protective properties.

2. Results

2.1. Crude Herbal Extracts Modulate Platelet Aggregation

The ability of each of the 48 crude herbal extracts (Table 1) to regulate platelet activation was assessed in response to thrombin (1 nM), collagen (1 μg/mL), ADP (20 μM), and the TxA2 mimetic, U46619 (100 nM) (Figure 1). The ability to modulate platelet reactivity was defined in this study as 70 percent or more inhibition of platelet aggregation compared to vehicle (DMSO). The extracts BMG, HB, HHL, HLC, HZ, LLT, POM, SD, XD, XS, ZC, and ZJC did not inhibit agonist-induced platelet aggregation, whereas GJ and JXT inhibited aggregation mediated by all four agonists. BB, DG, DS, HHP, HQ, KS, MBC, PH, QYL, RG, SS, WB, and WZY inhibited aggregation induced by ADP, collagen, and U46619. BLG, CX, DGP, HH, HL, and YHS modulated only ADP and U46619-induced platelet aggregation. HJ attenuated ADP, collagen, and U46619-induced platelet aggregation, and MD, MY, and QP inhibited ADP and collagen-induced aggregation. Some herbal extracts attenuated aggregation induced by only one agonist. BTW, CS, DQ, LCD, SM, WLX, ZL, and ZS inhibited only ADP-mediated aggregation, while DH, RX, and ZZ inhibited collagen-mediated aggregation (Figure 1E). Following this preliminary screen, three herbal extracts were selected to further purify and analyze: DGP (Lycium chinense), which inhibited ADP and U46619 induced platelet aggregation; SQ (Panax notoginseng), which inhibited ADP induced aggregation; ZC (Lithospermum erythrorhizon), which moderately attenuated aggregation by all four agonists.

2.2. Fractions from DGP, SQ, and ZC Fractions Inhibit Platelet Activation

Fractions DGP, SQ and ZC (Table 1 and Figure 1) were subjected to chromatographic separation, with four large fractions obtained for each crude extract encompassing the entire run (Figure 2A). The fractions were then tested to determine their effects on platelet aggregation following stimulation with the four agonists—ADP, collagen U46619, and thrombin. DGP1 demonstrated no significant effects, while DGP2 only inhibited stimulation with ADP (Figure 2B–E). DGP3 inhibited ADP- and U46619-mediated aggregation, similar to DGP4, which also inhibited stimulation with collagen. All of the SQ fractions only inhibited ADP-mediated platelet aggregation. The fraction ZC2 is the only one to inhibit all four platelet agonists. Lastly, platelet aggregation induced by ADP and U46619 was inhibited by ZC3, and ZC4 was inhibited when stimulated with ADP and collagen.
The effects of the fractions from herbal extracts DGP, SQ, and ZC on platelet activation were further assessed by analyzing specific markers of platelet activation. Integrin αIIbβ3 activation, P-selectin expression, and dense granule secretion were quantified via flow cytometry on platelets pre-treated with herbal extract fractions and stimulated with the GPVI agonist, convulxin. DGP3 and DGP4 were the only fractions to significantly inhibit platelet activation, demonstrated in integrin αIIbβ3 activation, P-selectin expression, and dense granule secretion (Figure 3). The only SQ fraction with inhibitory effects was SQ4, which was observed in all three markers. All of the ZC fractions inhibited all three markers, except the fraction ZC2, which did not inhibit α-granule secretion.

2.3. DGP Active Fraction 46 Inhibits Platelet Aggregation

DGP underwent a more precise chromatography separation with 90 fractions obtained as opposed to the 4 fractions obtained previously. The 90 fractions were tested for their effects on platelet aggregation. Briefly, platelets were incubated with the active fraction prior to stimulation with the EC80 concentration of collagen (0.125–0.375 μg/mL) and thrombin (0.25–0.375 nM) determined for each individual donor to account for variations in donor sensitivity to the agonists. Fraction 46 was the only active fraction to inhibit collagen-induced platelet aggregation (Figure 4A, p < 0.001). None of the fractions tested inhibited thrombin-mediated platelet aggregation (Figure 4A and Figure S1).

2.4. 13-HOTrE and 13-HODE Oxylipins, Identified in DGP Active Fraction 46, Inhibit Collagen-Mediated Platelet Aggregation

The active fraction 46 from the DGP herbal extract was then analyzed via mass spectrometry, and three oxylipins were identified via GNPS (9-HODE, 13-HODE and 13-HOTrE), along with other unidentified compounds (Figure 4A). Upon identification of the three oxylipins, the oxylipins were enzymatically generated, purified via HPLC and confirmed with MS/MS. The purified oxylipins were subsequently incubated with washed human platelets before stimulation with EC80 concentrations of collagen or thrombin to analyze their effects on platelet aggregation. Both 13-HODE and 13-HOTrE inhibited platelet aggregation when stimulated with collagen with the inhibitory effects observed starting at 5 µM and 40 µM, respectively (Figure 5A,B), with 9-HODE having no effect. None of the three compounds inhibited thrombin-induced platelet aggregation (Figure 5C,D).

3. Discussion

While existing biologically derived and synthetically developed antiplatelet therapies have reduced cardiovascular morbidity and mortality, their use is accompanied by significant bleeding risk [3,6,7,8]. Therefore, identifying new biologics and developing therapies that limit pathological platelet activation without compromising hemostasis remains a critical unmet need. To this end, the regulation of platelet activity through oxylipins or analogues of oxylipins has recently been described [24,25,26,27].
TCM has been used for centuries and has well-documented therapeutic use and physiological effects across diverse clinical contexts. The vast array of bioactive compounds within TCM herbs has often been overlooked in the context of discovering new, promising therapeutics. Our group sought to investigate the cardiovascular protective effects observed from commonly utilized herbs in TCM through evaluating their effects on platelet activation. We selected 48 herbal extracts, which are categorized as either “invigorating” or “cooling” the blood. Each extract was tested against multiple platelet agonists to identify agents that selectively inhibited specific activation pathways in the platelet. From our initial screen, we identified three herbal extracts that exhibited inhibitory effects on a variety of platelet agonists, DGP, SQ and ZC (Figure 1).
The three herbal extracts were further purified (Figure 2A) and analyzed to determine which of the purified fractions retained the inhibitory effects on platelet activation. Both DGP3 and DGP4 inhibited aggregation when stimulated with ADP and U46619, similar to the crude DGP herbal extract (Figure 1), suggesting that the antiplatelet effects of DGP are due to compounds in fractions 3 and 4. Interestingly, while the crude extract did not inhibit collagen-mediated aggregation, both DGP3 and DGP4 significantly inhibited integrin activation and granule secretion following GPVI stimulation (Figure 3). It is possible that the inhibitory effects of the purified fractions may not have been observed in the crude extract due to competing effects from other compounds in the extract.
Similar to the crude extract, all four of the SQ fractions significantly inhibited ADP-mediated aggregation and had no significant effect on the other agonists. However, when looking at the specific markers, SQ4 did inhibit integrin activation and granule secretion and thus was not investigated further. Lastly, despite the ZC crude extract having inhibitory effects on aggregation, the fractions demonstrated variability in their inhibitory effects and thus were also not studied further.
As both DGP3 and DGP4 demonstrated inhibitory effects on platelet activation and previous studies indicated that the root bark of Lycium chinense also exerts significant anti-inflammatory effects in the blood, we narrowed our focus to DGP [19,20,21,28,29]. The DGP root bark has been shown to enhance cytokine (TNF-α, IL-6) and chemokine (RANTES, MIP-1α) production in macrophages, indicating a regulatory effect on immune responses [30,31,32,33,34,35]; however, no studies to date have examined whether the documented anti-inflammatory effects were also due to inhibition of platelet activation. To further determine the compounds in the DGP extract that resulted in platelet inhibition, DGP underwent a more precise chromatography separation with 90 fractions.
Among the compounds isolated from the DGP extract, fraction 46 of the 90 was the only fraction that significantly modulated collagen-induced platelet aggregation (Figure 4), suggesting that the concentrated molecules within this fraction were primarily responsible for the observed antiplatelet effects. Subsequent mass spectrometry analysis identified 13-HODE, 13-HOTrE, and 9-HODE as the most abundant constituents of fraction 46. Both 13-HODE and 13-HOTrE inhibited collagen-induced platelet aggregation, with 13-HODE demonstrating greater potency, achieving complete inhibition at 5 µM (Figure 5). 9-HODE had no detectable effect, suggesting that it is not responsible for the observed antiplatelet effects of the DGP extract. Similar to fraction 46, 13-HODE and 13-HOTrE demonstrated selectivity to the collagen signaling pathway and had no inhibitory effect on thrombin-induced platelet aggregation. Since the inhibition of thrombin signaling through the protease-activated receptor (PAR)1 pathway is associated with an increased bleeding risk, the attenuation of platelet activation selectively through the GPVI pathway may result in protection from thrombosis with minimal impact on hemostasis [8].
9-HODE and13-HODE are the oxidized derivatives of linoleic acid and 13-HOTrE is the derivative of gamma-linolenic acid, with all being generated by 15-LOX-1 present in macrophages. 13-HODE has previously shown anti-inflammatory effects and reduced platelet adhesion to endothelial cells in vitro [35,36]. Our data demonstrating the inhibition of collagen-induced platelet activation provides further insight into the mechanism by which it inhibits platelet adhesion and thrombus formation. The role of 13-HOTrE in biology is also relevant, due to its presence in human plasma [37]. Increased plasma levels of 13-HOTrE correlate with systemic inflammation in postpartum cows [38], with worsening hot flashes in breast cancer patients [39], and with aortic dissection [40]. We demonstrate that both 13-HODE and 13-HOTrE inhibit collagen-mediated aggregation, but 13-HODE is more potent (complete inhibition at 5 µM) than 13-HOTrE (partial inhibition at 40 µM) (Figure 5).
Compared with other oxylipins with established antiplatelet activity, such as 12-HETrE and 12-HEPE, 13-HODE exhibits comparable potency despite differences in carbon chain length, degree of unsaturation, and oxygenation site [24,41]. Notably, whereas 12-HETrE and 12-HEPE inhibit platelet aggregation in response to both collagen and thrombin, 13-HOTrE and 13-HODE selectively inhibit collagen-induced aggregation. This selectivity suggests that these molecules may serve as promising scaffolds for the development of antiplatelet therapies that spare thrombin-mediated hemostatic signaling. The distinct structural features and agonist-selective antiplatelet effects of 13-HODE and 13-HOTrE further imply engagement of a unique mechanistic pathway. Accordingly, additional studies are warranted to elucidate their mechanisms of action, particularly with respect to modulation of the GPVI–immunoreceptor tyrosine-based activation motif (ITAM) signaling pathway [42,43,44].
Although the precise mechanisms of action require further investigation, this study provides new insight into the cardiovascular protective properties of Lycium chinense root bark, a widely used herb in TCM. Through systematic screening and purification, we identified 13-HODE and 13-HOTrE as a bioactive oxylipin present in the extract that selectively inhibits collagen-induced platelet aggregation while sparing thrombin-dependent activation. While the micromolar potency of 13-HODE alone may not fully account for the inhibitory activity of the crude extract due to its relatively low abundance in the extract, our findings suggest that 13-HODE likely contributes to a broader, additive effect of bioactive compounds within the herb. Collectively, this work establishes TCM herbs, including Lycium chinense, as a source of pathway-selective antiplatelet agents and underscores the value of TCM as a platform for natural product discovery by identifying novel molecular scaffolds with the potential for the development of novel therapeutics.

4. Materials and Methods

4.1. Chemicals

Fatty acids used in this study were purchased from Nu Chek Prep, Inc. (Elysian, MN, USA) and the oxylipins were isolated in-house (vide infra). Soybean Lipoxygenase-1 (SLO-1) was purchased from Cayman Chemicals (Ann Arbor, MI, USA). All other solvents and chemicals were reagent grade or better and were used as purchased without further purification from Sigma-Aldrich (St. Louis, MO, USA).

4.2. Plant Materials

Forty-eight medicinal herbs were procured from the herbal pharmacy at Five Branches University (Santa Cruz, CA, USA). The herbs were provided as dried material and used as received, with identities confirmed by the pharmacy’s labeling.

4.3. Initial Extraction Procedure

Each herb (30 g) was subjected to extraction by refluxing with 100 mL of HPLC-grade methanol (Sigma-Aldrich, St. Louis, MO, USA) three times, each for 30 min. Reflux was conducted using a heating mantle and condenser at approximately 65 °C. After each reflux, the mixture was filtered through Whatman No. 1 filter paper (Whatman, Maidstone, UK) under vacuum to separate the liquid extract from solid residues. The filtrates from the three extractions were combined for each herb. The combined methanol extracts were concentrated under reduced pressure at 40 °C using a rotary evaporator (Buchi Rotavapor RE-111, Buchi Corporation, New Castle, DE, USA) to yield crude extracts. The crude extracts were stored at −20 °C until further processing for bioactivity-guided fractionation.

4.4. Large-Scale Lycium Chinense Methanol Extraction

Dried Lycium chinense herb (2.5 kg split into two 1.25 kg batches) was ground to a coarse powder using a laboratory blender. The ground material was transferred to a 10 L high-density polyethylene carboy, and enough methanol was added to fully cover the plant material. The mixture was agitated manually twice daily for 7 days at room temperature and stored in the dark. The methanolic extract was filtered through Whatman No. 1 filter paper (Whatman, Maidstone, UK) under vacuum, and the residue was washed with additional methanol. The combined filtrate was concentrated under reduced pressure at 40 °C using a rotary evaporator (Buchi Rotavapor R-300, Buchi Corporation, New Castle, DE, USA) to yield a concentrated extract at 1 g/mL The process was repeated two additional times.

4.5. Buchi Flash Purification

Purification of the extracted material was performed using a Buchi Pure C-815 Flash Chromatography System (Buchi Corporation, New Castle County, DE, USA) equipped with integrated UV (200–400 nm) and evaporative light scattering detection (ELSD) equipped with a 40 g FlashPure EcoFlex C18 reverse-phase cartridge (50 µm particle size; Buchi Corporation, New Castle County, DE, USA). The concentrated extract (2 mL), adsorbed onto Celite 545, was loaded via a solid-phase loading cartridge. Elution was carried out with the following program at a flow rate of 45 mL/min: 2.5 min at 95:5 ACN:H 2 O, a linear gradient from 5 to 100 ACN over 27.5 min, and 100 ACN for 10 min. General characteristics between runs were similar, as was their biological activity.

4.6. Fractionation Procedure

Fractions were broken into the following groups with the following designations on the first fractionation: 2–15 (XX1); 16–35 (XX2); 36–60 (XX3); and 61–90 (XX4). Fractions were then separated into the following groups on the second fractionation: 2–5 (XX1A); 6–9 (XX1B); 10–12 (XX1C); 13–15 (XX1D); 16–20 (XX2A); 21–25 (XX2B); 26–30 (XX2C); 31–35 (XX2D); 36–40 (XX3A); 41–45 (XX3B); 46–52 (XX3C); 53–60 (XX3D); 61–67 (XX4A); 68–74 (XX4B); 75–83 (XX4C); 83–90 (XX4D). The two-letter term is specific to each herbal extract. For example purposes, XX has been used as the generic term.

4.7. Liquid Chromatography–Tandem Mass Spectrometry

Ultra-high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-MS/MS) was performed using a Thermo Scientific Orbitrap Velos Pro mass spectrometer interfaced with a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA). Chromatographic separation was achieved on a Hypersil GOLD C18 column (50 × 2.1 mm, 1.9 µm particle size; Thermo Fisher Scientific, Waltham, MA, USA) maintained at 30 °C. The mobile phase consisted of (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid, delivered at a flow rate of 0.5 mL/min. The gradient program was as follows: 33–95% B (0–8 min), 95% B (8–12 min), followed by re-equilibration at 33% B (12–14 min). The injection volume was 2 µL. Mass spectrometry was conducted in two separate trials: one in positive electrospray ionization (ESI+) mode and one in negative electrospray ionization (ESI−) mode. The ion source parameters were as follows: spray voltage, 3.5 kV; spray current, 1 µA; source temperature, 54 °C; sheath gas flow, 20 arbitrary units; sweep gas flow, 3 arbitrary units; capillary temperature, 240 °C. Full-scan MS data were acquired at a resolution of 60,000 (FWHM at m/z 400) over a mass range of 100–1000 m/z. Data were processed using Xcalibur software (version 2.2 sp1.48, Thermo Fisher Scientific, Waltham, MA, USA). Targeted MS/MS was performed for specific precursor ions in the negative mode: d4-13-hydroxyoctadecadienoic acid (HODE) (m/z 299.2), 13-HODE (m/z 295.2), 9-HODE (m/z 295.2), 13-hydroxy-9Z,11E,15Z-octadecatrienoic acid (HOTrE) (m/z 293.2), and 9-HOTrE (m/z 293.2). Quantitation was based on the area under the curve of characteristic fragment ions: parent m/z of 299.2, and fragment m/z of 198 for d4-13- HODE; parent m/z of 295.2, and fragment m/z of 195 for 13-HODE; parent m/z of 295.2 and fragment m/z of 171 for 9-HODE; parent m/z of 293.2, and fragment m/z of 193 for 13-HOTrE; and, parent m/z of 293.2 and fragment m/z of 169 for 9-HOTrE.

4.8. Data Analysis in Global Natural Products Social Molecular Networking

Raw data were converted into an mzML file using MS Convert from Proteowizard, (3.0.21229)with a molecular network being created from the MS/MS spectra on the Global Natural Products Social Molecular Networking (GNPS) 2 website. The GNPS website allows one to search known natural products based on their MS features, thus allowing for easy identification. The precursor ion mass tolerance was set to 0.05 Da, and an MS/MS fragment ion tolerance of 0.05 Da. A network was created where edges were filtered to have a cosine score above 0.65 and more than 6 matched peaks. Further, edges between two nodes were kept in the network if and only if each of the nodes appeared in each other’s respective top 10 most similar nodes. The maximum size of the molecular family was set to 100, and the lowest-scoring edges were removed from molecular families until the molecular family size was below this threshold. The maximum precursor ion mass difference between two MS/MS was set to 100 Da. The spectra in the network were then searched against the GNPS2 spectral library. The library spectra were filtered in the same manner as the input data. The GNPS Job is available under this link: https://gnps2.org/status?task=07dd79075d574a05acbcb2b3f1d24710 (accessed on 22 May 2025).

4.9. Oxylipin Isolation and Characterization by Mass Spectrometry

To synthesize the 13-oxylipins, SLO-1 (0.1 μM, pH 9.2) was reacted with either linoleic acid (LA) or gamma-linolenic acid (GLA) in 500 mL of 50 mM Borate at room temperature and ambient oxygen. The enzymatic turnover was monitored by absorbance at 234 nm, with 20 μM fatty acid reacted until 90% turnover occurred. To synthesize the 9-oxylipin, SLO-1 (0.3 μM, pH 6.5) was reacted with either LA in 500 mL of 50 mM HEPES at room temperature and ambient oxygen. The enzymatic turnover was monitored by absorbance at 234 nm, with the end of the reaction corresponding to 90% of 20 μM of the fatty acid being turned over. The reactions were quenched with 1% glacial acetic acid and extracted three times with dichloromethane (DCM). The products were then reduced with trimethyl phosphite and evaporated under a stream of nitrogen gas. The reaction products were reconstituted in methanol and analyzed via liquid chromatography–tandem mass spectrometry (LC-MS/MS). Chromatographic separation was performed using a C18 column (Phenomenex Kinetex, Torrance, CA, USA, 4 μm, 150 × 2.0 mm). Mobile phase solvent A consisted of 99.9% water and 0.1% formic acid, and solvent B consisted of 99.9% acetonitrile and 0.1% formic acid. Analysis was carried out over 60 min using isocratic 50:50 A:B for 0–30 min followed by a gradient from 50:50 A:B to 75:25 A:B from 30 to 60 min. The chromatography system was coupled to a Thermo-Electron LTQ LC-MS/MS instrument for mass analysis (Thermo Fischer Scientific, Waltham, MA, USA). All analyses were performed in negative ionization mode at the normal resolution setting. MS/MS was performed in a targeted manner with a mass list containing the following m/z ratios containing the following m/z ratios of 295.2 ± 0.5 (13-HODE), 293.2 ± 0.5 (13-HOTrE), 295.2 ± 0.5 (9-HODE), 293.2 ± 0.5 (9-HOTrE) were used. Matching retention times, UV spectra, and fragmentation patterns for four common fragments were used to identify products with comparison to known standards. The commercially obtained standards that were used were: d4-13-HODE and 13-HODE. The enzyme products coeluted with the known standards. Along with the catalytic nature of lipoxygenase (LOX) isozymes, we were able to assume the products were of the S-configuration. The same protocol was used for purifying the oxylipins, except for the high-performance liquid chromatography (HPLC) method and the amounts of product injected. The products were purified isocratically via HPLC on a Higgins Haisil Semi- preparative (5 μm, 250 mm × 10 mm) C18 column with 45:55 acetonitrile:water, and 0.1% acetic acid. Oxylipins were isolated from collected fractions and confirmed with MS/MS (Higgins Analytical, San Jose, CA, USA).

4.10. Oxylipin Quantification

The concentrations of four oxylipins—13-HODE, 9-HODE, 13-HOTrE, and 9-HOTrE—were quantified in a crude extract derived from 2.5 kg of dried herb, using d4-13-HODE as an internal standard. The extract, prepared at a concentration of 1 mg/mL, yielded 10 mg of crude extract, from which the oxylipin content was determined via LC-MS across three trials. The average mass of each oxylipin in the 10 mg crude extract, along with the standard deviation, was calculated based on molar quantities normalized to 0.5 ng of d4-13-HODE (with 0.01 ngs being the limit of detection). The results are as follows: 13-HODE, 19.1 ± 0.5 µg; 9-HODE, 55 ± 3 µg; 13-HOTrE, 1.84 ± 0.02 µg; and 9-HOTrE, 0.056 ± 0.005 µg. Corresponding concentrations in the dried herb were determined by scaling these masses to the 2.5 kg of starting material, yielding 7.6 ± 0.2 µg/kg for 13-HODE, 22 ± 1 µg/kg for 9-HODE, 0.736 ± 0.006 µg/kg for 13-HOTrE, and 0.023 ± 0.002 µg/kg for 9-HOTrE. It should be noted that the internal standard, d4-13-HODE, and the enzyme generated oxylipins have the S-configuration. We have assumed the natural products from the root are also in the S-configuration, but this was not confirmed due to their low levels.

4.11. Preparation of Washed Human Platelets

Human subjects research was carried out and approved by the University of Michigan Institutional Board (approval number: HUM00100677) and in accordance with the Declaration of Helsinki. Subjects provided written informed consent prior to drawing. Blood was collected from self-reported healthy donors via venipuncture into 3.2% sodium citrate vacutainer tubes (Greiner Bio-One, Monroe, NC, USA). Platelet-rich plasma (PRP) was isolated by centrifuging whole blood at 200 g for 15 min. Acid citrate dextrose (2.5% sodium citrate, 1.5% citric acid, 2% D glucose) and apyrase (0.02 U/mL) were added to PRP to prevent platelet activation by chelating Ca2+ ions and inactivating ADP/ATP. Plasma was then centrifuged at 2000 g for 10 min before resuspension of the subsequent platelet pellet in Tyrode’s buffer (10 mM HEPES, 10 mM, 12 mM NaHCO3, 127 mM NaCl, 5 mM KCl, 0.5 mM NaH2PO4, 1 mM MgCl2, 5 mM D-glucose, pH 7.4). Platelet concentration was analyzed using a Hemavet (950FS, Drew Scientific, Miami Lakes, FL, USA) before dilution to a physiological concentration of 3 × 108 platelets/mL.

4.12. Platelet Aggregation

Washed platelets at physiological concentration (3 × 108 platelets/mL) incubated with herbal extract (0.2 mg/mL), active compound (1–4 ng/mL), or bioactive lipid (13-HOTrE, 9 HODE, 13-HODE; 1–4 ng/mL), or the equivalent volume of vehicle (DMSO) for 10 min. Platelets were stimulated with adenosine 5′-diphosphate (ADP) (20 μM; Millipore Sigma, Burlington, MA, USA), type I collagen (Chrono-log, Havertown, PA, USA), U46619 (100 nM; Cayman Chemical, Ann Arbor, MI, USA), a thromboxane (Tx) A2 mimetic, or human α thrombin (Enzyme Research Labs, South Bend, IN, USA). For the initial screens, the concentration of the agonist was based on a known concentration response range for the ligand–receptor response in platelets (20 μM ADP, 1 μg/mL collagen, 100 nM U46619, 1 nM thrombin). Subsequent studies with purified compounds used the lowest effective concentration reaching at least 80% platelet aggregation (EC80) of collagen (0.125–0.375 μg/mL) and thrombin (0.25–0.375 nM) for each individual donor to account for variations in agonist sensitivity. Platelet aggregation was measured via light transmission for 10 min at 37 °C under stirring conditions (1200 rpm) in a lumi-aggregometer (Model 700D; Chrono-log). While all assays were repeated in multiple donors, the variability of platelet activation by donor is a limitation of the study.

4.13. Integrin Activation and Granule Secretion via Flow Cytometry

Washed platelets at physiological concentration (3 × 108 platelets/mL) were incubated with the selected herbal extract or vehicle (DMSO) for 10 min at 37 °C. Platelets were then stained with a FITC-conjugated antibody recognizing the active form of integrin αIIbβ3 (PAC1; BD Bioscience, Franklin Lakes, NJ, USA), an APC/Cyanine 7-conjugated antibody recognizing P-selectin, a protein released from ɑ-granules and expressed on the surface of the platelet upon activation (CD62P; BioLegend, San Diego, CA, USA), and a PE/Dazzle conjugated antibody recognizing dense granule secretion upon platelet activation (CD63; BioLegend, San Diego, CA, USA) and stimulated with convulxin (50 ng/mL) before incubation at 37 °C for 10 min in the dark. Due to the static nature of flow cytometry, convulxin, a GPVI agonist, was used instead of collagen, which requires a dynamic environment. Platelets were then fixed with 2% paraformaldehyde. Mean fluorescence intensity was quantified via flow cytometry (Cyto-FLEX; Beckman Coulter, Brea, CA, USA) to analyze integrin αIIbβ3 activation, ɑ-granule secretion, and dense granule secretion.

4.14. Data Analysis

GraphPad Prism 10 (GraphPad Software, La Jolla, CA, USA) was utilized for statistical analysis. Multiple statistical analyses were used in this study; the statistical test used in each assay is noted in the figure legend. Data are represented as mean ± standard error of the mean (SEM).

Supplementary Materials

The following information can be downloaded at https://www.mdpi.com/article/10.3390/ph19020263/s1, Supplemental Figure S1: Washed platelets were incubated with DGP active compound fractions 43, 44, 45, 47, or the equivalent volume of vehicle (DMSO) for 10 min prior to stimulation with the EC80 concentration of collagen (A) or thrombin (B) and subsequent analysis of platelet aggregation via light transmission. Data are presented as representative aggregation curves over 10 min.

Author Contributions

Conceptualization, D.S., T.R.H. and M.H.; Methodology, D.S., T.R.H. and M.H.; Validation, K.G. and T.R.H.; Formal analysis, D.S., E.B., P.Y., K.G., T.R.H. and M.H.; Investigation, D.S., E.B., P.Y., Y.J., K.G., P.H., S.K., D.D. and D.B.; Resources, D.S., T.R.H. and M.H.; Writing—original draft, D.S., T.R.H. and M.H.; Writing—review & editing, D.S., E.B., P.Y., K.G., T.R.H. and M.H.; Supervision, T.R.H. and M.H.; Funding acquisition, M.H. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by US National Institutes of Health grants R35GM131835 (M.H.).

Institutional Review Board Statement

This study was conducted in accordance with the Declaration of Helsinki and approved by the University of Michigan Institutional Review Board (HUM# HUM00100677) on 21 May 2015.

Informed Consent Statement

Informed consent was obtained from all subjects involved in this study.

Data Availability Statement

The original contributions presented in this study are included in the article and Supplementary Material. Further inquiries can be directed to the corresponding authors.

Acknowledgments

We thank Joe Zeng for contributions to analysis of the extracts (University of Michigan). We thank Amand Prieur for recruiting subjects and performing blood draws for human platelet studies. We thank Laura Rodriguez-Velandia and Laura M. Sanchez for thoughtful discussions regarding MS interpretations.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Initial screening of herbal extracts for modulatory effects on platelet aggregation. Crude herbal extracts (0.2 mg/mL) or the equivalent volume of vehicle (DMSO) incubated with washed human platelets for 10 min prior to stimulation with ADP (20 μM; (A)), collagen (1 μg/mL; (B)), thromboxane A2 mimetic U46619 (100 nM; (C)), or thrombin (1 nM; (D)). Extracts selected for further investigation are denoted: DGP (blue), DQ (red), and ZC (green). The red line marks the cutoff for defined as 70 percent or more inhibition of platelet aggregation. Data are presented as percent of vehicle ± SEM (n = 3). The ability of each extract to inhibit agonist-induced platelet aggregation by more than 70% was mapped (E).
Figure 1. Initial screening of herbal extracts for modulatory effects on platelet aggregation. Crude herbal extracts (0.2 mg/mL) or the equivalent volume of vehicle (DMSO) incubated with washed human platelets for 10 min prior to stimulation with ADP (20 μM; (A)), collagen (1 μg/mL; (B)), thromboxane A2 mimetic U46619 (100 nM; (C)), or thrombin (1 nM; (D)). Extracts selected for further investigation are denoted: DGP (blue), DQ (red), and ZC (green). The red line marks the cutoff for defined as 70 percent or more inhibition of platelet aggregation. Data are presented as percent of vehicle ± SEM (n = 3). The ability of each extract to inhibit agonist-induced platelet aggregation by more than 70% was mapped (E).
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Figure 2. Fractions of DGP, SQ, and ZC crude extracts inhibit platelet aggregation. Flow chart diagram of separation and purification of crude herbal extracts (A). Washed human platelets incubated with the four flash LC fractions from DGP, SQ, and ZC or vehicle (DMSO) for 10 min were stimulated with 20 μM ADP (B), 1 μg/mL collagen (C), 100 nM U46619 (D), or 1 nM thrombin (E). Light transmission aggregometry was analyzed for 10 min following stimulation. Data are presented as the average percent of maximum aggregation ± SEM (left panels; n = 3), and representative aggregation curves over time with fractions DGP3, DGP4, ZC2, ZC4 (right panels). One-way ANOVA with Dunnet’s multiple comparisons; asterisks indicate statistically significant differences compared with the vehicle-treated group. * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 2. Fractions of DGP, SQ, and ZC crude extracts inhibit platelet aggregation. Flow chart diagram of separation and purification of crude herbal extracts (A). Washed human platelets incubated with the four flash LC fractions from DGP, SQ, and ZC or vehicle (DMSO) for 10 min were stimulated with 20 μM ADP (B), 1 μg/mL collagen (C), 100 nM U46619 (D), or 1 nM thrombin (E). Light transmission aggregometry was analyzed for 10 min following stimulation. Data are presented as the average percent of maximum aggregation ± SEM (left panels; n = 3), and representative aggregation curves over time with fractions DGP3, DGP4, ZC2, ZC4 (right panels). One-way ANOVA with Dunnet’s multiple comparisons; asterisks indicate statistically significant differences compared with the vehicle-treated group. * p < 0.05; ** p < 0.01; *** p < 0.001.
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Figure 3. Fraction DGP3 inhibits platelet integrin activation and granule secretion. Washed human platelets incubated with the four flash LC fractions from DGP, SQ, and ZC or vehicle (DMSO) for 10 min were stimulated with convulxin (50 ng/mL). Integrin αIIbβ3 activation (A,B), α-granule secretion (C,D), and dense granule secretion (E,F) were detected via PAC1, CD62P, and CD63 fluorescently conjugated antibodies and analyzed via flow cytometry. Data are presented as average mean fluorescent intensity (MFI) (left panels; n = 6) and representative histograms (right panels). One-way ANOVA with Dunnet’s multiple comparisons; asterisks indicate statistically significant differences compared with the vehicle-treated group. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
Figure 3. Fraction DGP3 inhibits platelet integrin activation and granule secretion. Washed human platelets incubated with the four flash LC fractions from DGP, SQ, and ZC or vehicle (DMSO) for 10 min were stimulated with convulxin (50 ng/mL). Integrin αIIbβ3 activation (A,B), α-granule secretion (C,D), and dense granule secretion (E,F) were detected via PAC1, CD62P, and CD63 fluorescently conjugated antibodies and analyzed via flow cytometry. Data are presented as average mean fluorescent intensity (MFI) (left panels; n = 6) and representative histograms (right panels). One-way ANOVA with Dunnet’s multiple comparisons; asterisks indicate statistically significant differences compared with the vehicle-treated group. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
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Figure 4. DGP active fraction 46 inhibits collagen-mediated platelet aggregation but does not inhibit thrombin-mediated platelet aggregation. Washed platelets were incubated with DGP active compound fractions 43–47 or the equivalent volume of vehicle (DMSO) for 10 min prior to stimulation with the EC80 concentration of collagen (A) or thrombin (B) and subsequent analysis of platelet aggregation via light transmission. Data are presented as the average percent or maximum aggregation ± SEM (left panels; n = 4), and active fraction 46 representative aggregation curves over 10 min (right panels). One-way ANOVA with Dunnet’s multiple comparisons with all groups compared to the vehicle. *** p < 0.001.
Figure 4. DGP active fraction 46 inhibits collagen-mediated platelet aggregation but does not inhibit thrombin-mediated platelet aggregation. Washed platelets were incubated with DGP active compound fractions 43–47 or the equivalent volume of vehicle (DMSO) for 10 min prior to stimulation with the EC80 concentration of collagen (A) or thrombin (B) and subsequent analysis of platelet aggregation via light transmission. Data are presented as the average percent or maximum aggregation ± SEM (left panels; n = 4), and active fraction 46 representative aggregation curves over 10 min (right panels). One-way ANOVA with Dunnet’s multiple comparisons with all groups compared to the vehicle. *** p < 0.001.
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Figure 5. 13-HODE and 13-HOTrE significantly inhibit collagen-induced platelet aggregation. DGP active compound was assessed via mass spectrometry to highlight 13-HOTrE (A). Washed platelets were incubated with 9-HODE, 13-HODE, 13-HOTrE, or equivalent volume of vehicle (DMSO) for 10 min prior to stimulation with the EC80 concentration of collagen (A,B) or thrombin (C,D). Platelet aggregation was analyzed via light transmission aggregometry for 10 min. Data are presented as the average of the maximum percent aggregation ± SEM ((A,C); n = 4) and the representative aggregation curves (B,D). One-way ANOVA with Dunnet’s multiple comparisons with all groups compared to the vehicle. * p < 0.05; ** p < 0.01.
Figure 5. 13-HODE and 13-HOTrE significantly inhibit collagen-induced platelet aggregation. DGP active compound was assessed via mass spectrometry to highlight 13-HOTrE (A). Washed platelets were incubated with 9-HODE, 13-HODE, 13-HOTrE, or equivalent volume of vehicle (DMSO) for 10 min prior to stimulation with the EC80 concentration of collagen (A,B) or thrombin (C,D). Platelet aggregation was analyzed via light transmission aggregometry for 10 min. Data are presented as the average of the maximum percent aggregation ± SEM ((A,C); n = 4) and the representative aggregation curves (B,D). One-way ANOVA with Dunnet’s multiple comparisons with all groups compared to the vehicle. * p < 0.05; ** p < 0.01.
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Table 1. Identification of acronyms used according to Latin and Pinyin designations.
Table 1. Identification of acronyms used according to Latin and Pinyin designations.
AbbreviationPinyinLatin BinomialBotanical Part Used
BBBi baPiper longumFructus Piperis
BLGBan lan genIsatis tinctoriaRadix Isatidis
BMGBai mao genImperata cylindricaRhizoma Imperatae
BTWBai tou wengPulsatilla chinensisRadix Pulsatillae
CSChi shaoPaeonia lactifloraRadix Paeoniae
CXChuan xiongLigusticum chuanxiongRhizoma Chuanxiong
DGDang guiAngelica sinensisRadix Angelicae
DGPDi gu piLycium chinenseCortex Lycii
DHDa huangRheum palmatumRadix et Rhizoma Rhei
DSDan shenSalvia miltiorrhizaRadix Salviae
HBHuang baiPhellodendron amurenseCortex Phellodendri
HHHong huaCarthamus tinctoriusFlos Carthami
HHLHe huang lianCoptis chinensisRhizoma Coptidis
HHPHe huan piAlbizia julibrissinCortex Albiziae
HLHuang lianCoptis chinensisRhizoma Coptidis
HLCHan lian caoEclipta prostrataHerba Ecliptae
HQHuang qinScutellaria baicalensisRadix Scutellariae
HZHu zhangReynoutria japonicaRhizoma Reynoutria
KSKu shenSophora flavescensRadix Sophorae
LDCLong dan caoGentiana manshuricaRadix et Rhizoma Gentianae
LLTLu lu tongLiquidambar taiwanianaFructus Liquidambaris
MBCMa bian caoVerbena officinalisHerba Verbenae
PHNot TCMPeganum harmalaSemen Pegani
POMNot TCMPunica granatumSemen Granati
QYLQi ye lianTricyrtis macropoda.Rhizoma Tricyrtidis
RGRou guiCinnamomum cassiaCortex Cinnamomi
RXRu xiangBoswellia carteriResina Olibani
SDSheng diRehmannia glutinosaRadix Rehmanniae
SMSu muCaesalpinia sappanLignum Sappan
SQSan qiPanax notoginsengRadix et Rhizoma Notoginseng
SSSang shenMorus albaFructus Mori
WBWang bu liu xingVaccaria segetalisSemen Vaccariae
WLXWei ling xianClematis chinensisRadix et Rhizoma Clematidis
WZYWu zhu yuEvodia rutaecarpa.Fructus Evodiae
XDXu duanDipsacus asperRadix Dipsaci
XSXuan shenScrophularia ningpoensisRadix Scrophulariae
YHSYan hu suoCorydalis yanhusuoRhizoma Corydalis
ZCZi caoLithospermum erythrorhizonRadix Lithospermi
ZLZe lanLycopus lucidusHerba Lycopi
ZSShan zhaCrataegus pinnatifidaFructus Crataegi
ZZZhi ziGardenia jasminoidesFructus Gardeniae
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Simpson, D.; Botta, E.; Yalavarthi, P.; Ji, Y.; Goerger, K.; Houston, P.; Kareht, S.; Desai, D.; Bolaños, D.; Holman, T.R.; et al. 13-HODE and 13-HOTrE, Present in the Traditional Chinese Medicine Herbal Extract di gu pi, Selectively Inhibit Platelet Function. Pharmaceuticals 2026, 19, 263. https://doi.org/10.3390/ph19020263

AMA Style

Simpson D, Botta E, Yalavarthi P, Ji Y, Goerger K, Houston P, Kareht S, Desai D, Bolaños D, Holman TR, et al. 13-HODE and 13-HOTrE, Present in the Traditional Chinese Medicine Herbal Extract di gu pi, Selectively Inhibit Platelet Function. Pharmaceuticals. 2026; 19(2):263. https://doi.org/10.3390/ph19020263

Chicago/Turabian Style

Simpson, Dylan, Eliana Botta, Pooja Yalavarthi, Yein Ji, Krista Goerger, Paul Houston, Sky Kareht, Drewv Desai, Daniela Bolaños, Theodore R. Holman, and et al. 2026. "13-HODE and 13-HOTrE, Present in the Traditional Chinese Medicine Herbal Extract di gu pi, Selectively Inhibit Platelet Function" Pharmaceuticals 19, no. 2: 263. https://doi.org/10.3390/ph19020263

APA Style

Simpson, D., Botta, E., Yalavarthi, P., Ji, Y., Goerger, K., Houston, P., Kareht, S., Desai, D., Bolaños, D., Holman, T. R., & Holinstat, M. (2026). 13-HODE and 13-HOTrE, Present in the Traditional Chinese Medicine Herbal Extract di gu pi, Selectively Inhibit Platelet Function. Pharmaceuticals, 19(2), 263. https://doi.org/10.3390/ph19020263

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