Kratom leaf extracts exert hypolipidaemic effects via the modulation of PCSK9 and LDLR pathways in HepG2 cells

Abstract

Kratom (Mitragyna speciosa (Korth.) Havil.) has been reported to reduce serum lipids. However, the molecular mechanism underlying hypolipidaemic effect of kratom is still unclear. This study aimed to investigate the effects of kratom leaf extracts on hypolipidaemia via the expression of LDLR and PCSK9 in HepG2 cells. Real-time RT-PCR, Western blotting, and flow cytometry analyses revealed that kratom leaf extracts from red-vein and white-vein strains increased LDLR protein expression but decreased that of PCSK9 via downregulation of SREBP-2 and HNF-1α. Furthermore, a confocal laser scanning microscope revealed that kratom leaf extracts from both strains increased LDL uptake into HepG2 cells. The bioactive compounds, e.g., mitragynine, quercetin, and rutin, in kratom leaf extracts from both strains were characterized by LC-MS/MS analysis. Mitragynine also significantly increased LDLR protein expression but decreased that of PCSK9. Molecular docking studies demonstrated that mitragynine had the strongest binding affinity for EGF-A domain of LDLR (– 7.57 kcal/mol), whereas quercetin had the strongest binding affinity for PCSK9 (– 8.45 kcal/mol). In conclusion, kratom leaf extracts from red-vein and white-vein strains possessed hypolipidaemic effects by decreased PCSK9 expression and increased LDLR expression through the modulation of SREBP2 and HNF-1α. Therefore, kratom could serve as a potential supplement for ameliorating hypercholesterolemia.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-00711-1.

Introduction

Hypercholesterolemia is characterized by elevated serum levels of plasma cholesterol and low-density lipoprotein cholesterol (LDL-C) and is a major risk factor for the development of cardiovascular diseases (CVDs)^1,2. Statins, also known as hydroxymethylglutaryl coenzyme A reductase (HMGCR) inhibitors, are widely used to treat patients with elevated plasma LDL-C levels. Statins lead to reduced intracellular cholesterol levels and increased synthesis of proprotein convertase subtilisin/kexin type 9 (PCSK9) via the stimulation of nuclear sterol regulatory element-binding protein 2 (SREBP-2) and hepatocyte nuclear-factor 1 alpha (HNF-1α)³. However, increased PCSK9 levels after statin treatment could reduce the lipid-lowering efficacy⁴. Currently, the PCSK9 inhibitors alirocumab, evolocumab, and inclisiran, which have been approved by the United States Food and Drug Administration (FDA), are used as lipid-lowering drugs for the treatment of patients with primary hypercholesterolemia or severe hypercholesterolemia^5–7. However, PCSK9 inhibitors are expensive and may lead to an injection site reaction⁸. Several bioactive compounds and medicinal plants, such as berberine, kaempferol, quercetin, p-coumaric acid, pinostrobin, epigallocatechin-3-gallate (EGCG), allicin, capsaicin, and extracts of pigeon pea (Cajanus cajan (L.) Millsp.) leaves, Welsh onion, Penthorum chinense, and black raspberry, are natural PCSK9 inhibitors^9–11 and might be used as alternative lipid-lowering agents used alone or in combination with statins to alleviate hypercholesterolemia.

Mitragyna speciosa (Korth.) Havil., commonly known as “kratom” in Thailand, is a tropical tree native to Southeast Asia. People in Thailand and Malaysia chew kratom leaves to reduce fatigue and increase their tolerance to long, hard work under heat and sunlight¹². However, kratom has been reported to have psychoactive and addictive effects¹³. In addition, overdose and coingestion of kratom with other substances, e.g., diphenhydramine and O-desmethyltramadol, can cause adverse effects^14,15. Nevertheless, kratom also has pharmacological properties and has been widely used for treating opium withdrawal and for pain relief¹⁶. Moreover, kratom has several therapeutic properties, including analgesic, antinociceptive, anti-inflammatory, antidiarrhoeal, antidepressant, antioxidant, antibacterial, hypoglycaemic, and hypolipidaemic effects^17–19. Thus, the dose, duration, and ingestion of kratom in combination with other drugs should be controlled for safety. A previous study revealed that kratom users had significantly lower total cholesterol (TC) and LDL-C levels than did healthy subjects¹⁸. Similarly, other studies have demonstrated that kratom use is associated with a smaller waist circumference, low triglyceride (TG) levels, and elevated high-density lipoprotein cholesterol (HDL-C) levels^20,21. Moreover, an in vivo study demonstrated that TC, TG, and LDL-C levels were significantly decreased in kratom-treated diabetic rats, whereas HDL-C levels were significantly increased²². Kratom leaves consist of a major active compound, namely, mitragynine, as well as flavonoids (e.g., quercetin and rutin), tannins, triterpenoids, and other phenolic compounds²³. Previous studies have shown that quercetin downregulates PCSK9 expression²⁴. In addition, quercetin and rutin upregulate LDLR expression^24–26. It could be hypothesized that kratom has a hypolipidaemic effect as a result of the bioactive compounds in its leaves. Although evidence and hypotheses support the hypolipidaemic effect of kratom, the underlying molecular mechanism remains unknown. Therefore, this study aimed to investigate the effects of leaf extracts from red-vein and white-vein (Pho Thong) strains of kratom on hypolipidaemia in HepG2 cells.

Materials and methods

Materials

HepG2 cells were purchased from American Type Culture Collection (ATCC, VA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), 1% penicillin-streptomycin, cell dissociation solution (CDS), and phosphate-buffer saline (PBS) were purchased from Gibco (Grand Island, NY, USA). Mitragynine (Lipomed, Inc., Switzerland), quercetin (ChemFaces, Wuhan, China), and rutin (Acros Organics, NJ, USA) were purchased. Formic acid, acetonitrile, methanol, dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), bovine serum albumin (BSA), and atorvastatin were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Total RNA Mini Kit was obtained from Geneaid (Taipei, Taiwan). The iScript™ Reverse Transcription Supermix for RT-qPCR, Bradford reagent, nitrocellulose membrane, and Clarity Western ECL Substrate were purchased from Bio-Rad (Hercules, CA, USA). The Luna^® Universal qPCR Master Mix was purchased from New England Biolabs Inc. (Ipswich, MA, USA). Alexa Fluor 488-conjugated goat anti-mouse IgG, BODIPY1-FL-LDL, DRAQ5, and anti-HNF-1α (MA5-17267) were purchased from Thermo Fisher Scientific (Rockford, IL, USA). Anti-LDLR (ab204941), anti-PCSK9 (ab181142), anti-SREBP-2 (ab194667), anti-β-actin (ab8226), and HRP-conjugated goat anti-mouse IgG (ab205719) were purchased from Abcam (Cambridge, UK). HRP-conjugated goat anti-rabbit IgG (7074P2), cell lysis buffer, and phenylmethylsulfonyl fluoride (PMSF) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Plant materials and extraction

Leaves from the red-vein strain Mitragyna speciosa (Korth.) Havil. were harvested from Tha Sae district, Chumphon Province, Thailand. Leaves from the white-vein (Pho Thong) strain were harvested from Thasala district, Nakhon Si Thammarat Province, Thailand. The plants were identified by Assoc. Prof. Dr. Sukanya Dej-adisai from the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand. Voucher specimens of the red-vein (SKP 165 13 19 01) and white-vein (Pho Thong) (SKP 165 13 19 02) strains were deposited at the School of Allied Health Sciences, Walailak University, Thailand. The plants were dried at 50 °C and ground into a fine powder using a mixer. Subsequently, 20 g of kratom powder was soaked with 200 ml of methanol in bottles for 2 days at room temperature, and this process was repeated three times. Each sample was subsequently filtered through gauze cloth, and the filtered liquid was concentrated using a rotary evaporator (N-1200 Series, Eyela, USA) at 50 °C in a water bath at 37 °C for 1 week. The obtained concentrates of the red-vein and white-vein (Pho Thong) strains were stored at 4 °C.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis

The bioactive compounds in kratom leaf extracts were identified using LC-MS/MS (Agilent Technologies 6490 Triple Quad LC/MS, CA, USA)²⁷. The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The composition of the mobile phase at different time intervals was set as 0–10 min, 5% B; 10–15 min, 95% B; and 15–20 min, 5% B, with a flow rate of 0.5 ml/min. The analysis was performed using the ZORBAX 300Extend-C18 (4.6 × 150 mm, 3.55 μm; Agilent) at 35 °C. Standards and samples were injected with a volume of 10 µl. The standards mitragynine, quercetin, and rutin were analysed. The precursor (m/z) and product (m/z) ions of mitragynine, quercetin, and rutin were 399.20 → 174.10, 303.05 → 153.00, and 611.10 → 303.10, respectively.

Cell preparation and compound treatment

The cell line was maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin‒streptomycin at 37 °C in a humidified atmosphere containing 5% CO₂. HepG2 cells were cultured for 24 h in serum-starved medium containing 0.5% FBS. Then, cells were pretreated for 24 h with the following substances: vehicle (0.1% DMSO), leaf extracts from the red-vein strain of kratom (0.125 and 0.250 mg/ml), leaf extracts from the white-vein strain of kratom (Pho Thong) (0.125 and 0.250 mg/ml), and atorvastatin (10 µM). Subsequently, real-time RT-qPCR, Western blotting, and flow cytometric analyses, as well as BODIPY‑LDL uptake assays, were conducted.

Cell viability assay

Cell viability was measured via the MTT assay. HepG2 cells were cultured in 96-well plates at a density of 1 × 10⁴ cells/well to reach 80–90% confluence. The cells were treated with leaf extracts from the red-vein strain of kratom (0, 0.0625, 0.125, 0.250, 0.50, and 1 mg/ml), leaf extracts from the white-vein strain of kratom (Pho Thong) (0, 0.0625, 0.125, 0.250, 0.50, and 1 mg/ml), and mitragynine (0.0625, 1.25, 2.5, 5, and 10 µM) at 37 °C and 5% CO₂ for 24 h. After treatment, MTT (1 mg/ml) was added to the cells, which were subsequently incubated at 37 °C for 4 hours. Insoluble formazan crystals were dissolved using DMSO. The absorbance was measured at 560 nm using a microplate reader (Multiskan FC Microplate Photometer, Thermo Scientific, USA).

Real-time reverse transcription quantitative PCR (RT-qPCR) analysis

HepG2 cells were grown at 7 × 10⁵ cells/well to reach 80–90% confluence in 6-well plates. Total RNA was extracted from the cells using the Total RNA Mini Kit. RNA was converted to cDNA using iScript™ Reverse Transcription Supermix for RT-qPCR. The quantitative real-time PCR analysis of LDLR, PCSK9, SREBP-2, and HNF-1α mRNA expression was performed with an Applied Biosystems QuantStudio 5 Real-Time PCR System (Applied Biosystems, USA). The reaction mixtures contained cDNA, primers (Table 1)^28–30, and Luna^® Universal qPCR Master Mix. The relative mRNA expression levels were analysed using the 2^ΔΔCt method and normalized to the level of GAPDH.

Table 1.

Forward and reverse primers for real-time RT-qPCR.

Gene name	Forward primer	Reverse primer
LDLR	5’-AGTTGGCTGCGTTAATGTGA-3’	5’-TGATGGGTTCATCTGACCAGT-3’
PCSK9	5’-GCTGAGCTGCTCCAGTTTCT-3’	5’-AATGGCGTAGACACCCTCAC-3’
SREBP-2	5’-CTCTGACCAGCACCCACACT-3’	5’-CACACCATTTACCAGCCATAAG-3’
HNF-1α	5’-TGGCGCAGCAGTTCACCCAT-3’	5’-TGAAACGGTTCCTCCGCCCC-3’
GAPDH	5’-CATGAGAAGTATGACAACAGCCT-3’	5’-AGTCCTTCCACGATACCAAAGT-3’

Western blotting analysis

HepG2 cells were grown at 1 × 10⁶ cells/well to reach 70–80% confluence in petri dishes. The cells were collected and lysed using cell lysis buffer supplemented with 1 mM PMSF. The protein concentration was determined using the Bradford protein assay. Protein samples were separated using 8% SDS-PAGE and then transferred onto a nitrocellulose membrane. These membranes were incubated with specific antibodies, anti-LDLR (1:1,000), anti-PCSK9 (1:1,000), anti-SREBP-2 (1:500) anti-β-actin (1:1,000), and anti-HNF-1α (1:2,000), overnight at 4 °C. After incubation, the membranes were washed and incubated with HRP-conjugated goat anti-mouse IgG secondary antibody (1:10,000) or HRP-conjugated goat anti-rabbit IgG secondary antibody (1:2,000). The Clarity Western ECL Substrate was applied to the membrane to visualize protein bands, which were subsequently detected and quantified using the ChemiDoc Imaging System (Bio-Rad) to capture the chemiluminescent signal. ImageJ software was used for protein band quantification, and protein expression levels were normalized to those of β-actin.

Flow cytometric analysis of cell-surface LDLR

HepG2 cells were seeded at a density of 1 × 10⁶ cells/well to reach 80–90% confluence in a 6-well plate. The cells were detached using cell dissociation solution (CDS) at 1 ml/well. The cells were washed twice with PBS and incubated with PBS containing 1% bovine serum albumin at room temperature (RT) for 30 min. The cells were blocked and incubated with an anti-LDLR antibody (1:400) at RT for 1 h and then incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1,000) at RT for 1 h. The cells were resuspended in PBS and analysed by flow cytometry (FACScan, BD Biosciences, San Jose, CA, USA). The relative fluorescence intensity was quantified by recording the fluorescence of 10,000 events.

BODIPY‑LDL uptake assay

HepG2 cells were grown at 5 × 10⁵ cells/well to reach 50–60% confluence on cover slips in 6-well plates. The cells were washed twice with PBS. The cells on cover slips were subsequently incubated with serum-free media containing 5 µg/ml BODIPY1-FL-LDL at 37 °C for 4 h, followed by nuclear staining with 5 µM DRAQ5 at 37 °C for 15 min in the dark. The cells were fixed in 4% paraformaldehyde at RT for 15 min. BODIPY-LDL uptake was visualized using a confocal laser scanning microscope (Leica SP5 II Laser Scanning Confocal Microscope). ImageJ software was used to quantify the relative fluorescence intensity.

Molecular docking studies

The 3D structures of LDLR EGF-A (PDB: 3BPS) and PCSK9 (PDB: 2P4E) were downloaded from the Protein Data Bank (http://www.rcsb.org) and processed using BIOVIA Discovery Studio (Version 2021 Client) to remove water and cofactors. Proteins were optimized using the AutoDock 1.5.6 program by adding hydrogen atoms, assigning Kollman charges, and converting to PDBQT format. The ligands, including mitragynine, quercetin, rutin, and atorvastatin, were obtained from the PubChem database in SDF format and converted to PDB and PDBQT formats using BIOVIA Discovery Studio and AutoDock 1.5.6. Protein-ligand interactions were simulated using AutoDock 1.5.6. The grid box sizes for the X, Y, and Z dimensions and spacing were set as shown in Table 2. The results revealed that lower binding energy values were correlated with stronger protein-ligand interactions, indicating favourable binding.

Table 2.

The grid box size and spacing of the targeted proteins.

Protein name	PDB code	X axis	Y axis	Z axis	Spacing (Å)
EGF-A domain of LDLR	3BPS	98	120	122	0.653
PCSK9	2P4E	96	62	100	0.836

Statistical analyses

Statistical analyses were performed using SPSS 23.0 software (SPSS Inc., Chicago, IL, USA). Graphs were created using GraphPad Prism version 9.0 (GraphPad Software LLC, San Diego, CA, USA). The Shapiro-Wilk test was used to assess the normality of the data. The data were analysed by one-way ANOVA using Bonferroni post hoc correction for parametric factors or the Kruskal-Wallis and Mann-Whitney U tests for nonparametric factors. A P value of < 0.05 was considered to indicate statistical significance. All experiments were performed in triplicate.

Results

LC-MS/MS analysis of Mitragynine, Quercetin, and Rutin in Kratom leaf extracts

LC-MS/MS analysis was performed to determine the bioactive compounds in leaf extracts from red-vein and white-vein (Pho Thong) strains of kratom. The retention times of mitragynine, quercetin, and rutin in kratom leaf extracts were 7.97, 7.49, and 5.82 min, respectively (Fig. 1). The concentrations of mitragynine, quercetin, and rutin in leaf extracts from red-vein and white-vein (Pho Thong) strains of kratom are shown in Table 3.

Fig. 1.

Chromatograms of reference standards, including mitragynine (A), quercetin (D) and rutin (G), as well as leaf extracts from red-vein (B, E, H) and white-vein (Pho Thong) (C, F, I) strains of kratom. The retention times of mitragynine (A), quercetin (D), and rutin (G) were 7.97, 7.49, and 5.82 min, respectively.

Table 3.

Concentrations of Mitragynine, Quercetin, and Rutin in leaf extracts from red-vein and white-vein (Pho Thong) strains of Kratom.

Kratom leaf extracts	Mitragynine	Quercetin	Rutin
Red vein strain	38.61 ± 0.55 (3.86%w/w dry leaf weight)	5.57 ± 0.08 (0.56%w/w dry leaf weight)	21.04 ± 0.29 (2.10%w/w dry leaf weight)
White vein (Pho Thong) strain	39.79 ± 1.81 (3.98%w/w dry leaf weight)	0.48 ± 0.04 (0.05%w/w dry leaf weight)	35.78 ± 0.43 (3.58%w/w dry leaf weight)

The results are presented as the means ± SEMs of three independent experiments (N = 3); mg/g (% w/w dry leaf weight).

Effects of leaf extracts from red-vein and white-vein (Pho Thong) strains of Kratom and Mitragynine on the viability of HepG2 cells

To investigate the cytotoxic effects of leaf extracts from red-vein and white-vein (Pho Thong) strains of kratom, as well as mitragynine, HepG2 cells were treated with a vehicle (0.1% DMSO), leaf extracts from the red-vein strain of kratom (0, 0.0625, 0.125, 0.250, 0.50, and 1 mg/ml), leaf extracts from the white-vein strain of kratom (Pho Thong) (0, 0.0625, 0.125, 0.250, 0.50, and 1 mg/ml), and mitragynine (0.0625, 1.25, 2.5, 5, and 10 µM), and cell viability was measured via the MTT assay. No compounds had cytotoxic effects on HepG2 cells (cell viability > 80% at all concentrations) (Fig. 2).

Fig. 2.

Effects of leaf extracts from red-vein (A) and white-vein (Pho Thong) (B) strains of kratom, as well as mitragynine (C), on the viability of HepG2 cells. The assessment of cell viability was conducted using the MTT assay. The results are presented as the means ± SEMs of three independent experiments (N = 3).

Effects of leaf extracts from red-vein and white-vein (Pho Thong) strains of Kratom on the expression of LDLR and PCSK9 and the uptake of LDL in HepG2 cells

To investigate the effects of kratom leaf extracts on LDLR mRNA and protein expression, RT-qPCR and Western blotting analyses were performed, respectively. The results revealed that leaf extracts from the white-vein (Pho Thong) strain of kratom (0.25 mg/ml) but not the red-vein strain of kratom significantly elevated LDLR mRNA expression (Fig. 3A). In contrast, leaf extracts from both red-vein (0.25 mg/ml) and white-vein (Pho Thong) (0.25 mg/ml) strains of kratom demonstrated a significantly increased mature LDLR protein expression of approximately 2.19-fold (p < 0.0001) and 2.12-fold (p < 0.0001), respectively, in comparison with that in the control group (Figs. 4A, S1). The effects of leaf extracts from red-vein and white-vein (Pho Thong) strains of kratom on the cell-surface LDLR level were further investigated by flow cytometric analysis. Leaf extracts from both red-vein (0.25 mg/ml) and white-vein (Pho Thong) (0.25 mg/ml) strains of kratom significantly increased the cell-surface LDLR by 229.9% (p < 0.0001) and 175.3% (p < 0.005), respectively, compared with that of the control group (Fig. 5). The effects of kratom leaf extracts on the activity of LDLR and LDL uptake were also observed by a confocal laser scanning microscope (CLSM) (Fig. 6A). The leaf extracts from red-vein (0.25 mg/ml) and white-vein (Pho Thong) (0.25 mg/ml) strains of kratom significantly increased LDL uptake by approximately 2.86-fold (p < 0.0005) and 2.61-fold (p < 0.005), respectively, compared with the control group (Fig. 6B). These findings suggest that leaf extracts from red-vein and white-vein (Pho Thong) strains of kratom contribute to an increased expression of cell-surface LDLR protein and enhanced LDL uptake into HepG2 cells.

Fig. 3.

Effects of leaf extracts from red-vein and white-vein (Pho Thong) strains of kratom on mRNA expression in HepG2 cells. The mRNA expression levels of LDLR (A), PCSK9 (B), SREBP-2 (C), and HNF-1α (D) were analysed by real-time RT-qPCR. The results are presented as the means ± SEMs of three independent experiments (N = 3). * p < 0.05 indicates a significant difference compared with the vehicle control (0.1% DMSO). ATV, atorvastatin.

Fig. 4.

Effects of leaf extracts from red-vein and white-vein (Pho Thong) strains of kratom on protein expression in HepG2 cells. The protein expression levels of LDLR (A), PCSK9 (B), SREBP-2 (C), and HNF-1α (D) were analysed by Western blotting analysis. The results are presented as the means ± SEMs of three independent experiments (N = 3). * p < 0.05, ** p < 0.005, *** p < 0.0005, and **** p < 0.0001 indicate significant differences compared with the vehicle control (0.1% DMSO). ATV, atorvastatin.

Fig. 5.

Effects of leaf extracts from red-vein (0.125 and 0.250 mg/ml) and white-vein (Pho Thong) (0.125 and 0.250 mg/ml) strains of kratom on cell-surface LDLR levels in HepG2 cells. The mean fluorescence intensity (MFI) of cell-surface LDLR was observed by flow cytometric analysis. The data represent the means ± SEMs of three independent experiments (N = 3). ** p < 0.005, **** p < 0.0001 represent significant differences compared with the vehicle control (0.1% DMSO). ATV, atorvastatin.

Fig. 6.

Effects of leaf extracts from red-vein (0.125 and 0.250 mg/ml) and white-vein (Pho Thong) (0.125 and 0.250 mg/ml) strains of kratom on LDL uptake in HepG2 cells measured using a confocal laser scanning microscope (A). The image presents BODIPY-LDL (green), DRAQ5 (red), and the overlay. The relative fluorescence intensity was analysed using ImageJ software. (B) The data represent the means ± SEMs of a minimum of three independent experiments (N = 3). ** p < 0.005, *** p < 0.0005, **** p < 0.0001 represent significant differences compared to vehicle control (0.1% DMSO). ATV, atorvastatin.

Moreover, leaf extracts from the white-vein (Pho Thong) but not the red-vein strain of kratom presented a significant decrease in PCSK9 mRNA expression compared with the control group (Fig. 3B). In addition, leaf extracts from both red-vein (0.25 mg/ml) and white-vein (Pho Thong) (0.25 mg/ml) strains of kratom significantly decreased PCSK9 protein expression by approximately 0.30-fold (p < 0.0005) and 0.23-fold (p < 0.0001), respectively, compared with that in the control group (Figs. 4B, S1). In contrast, a positive control, atorvastatin (10 µM), significantly increased PCSK9 mRNA and protein expression (Figs. 3B and 4B).

The effects of mitragynine on LDLR and PCSK9 expression in HepG2 cells were also investigated. Compared with the control, mitragynine (2.5 µM) significantly increased LDLR protein expression by 1.65-fold (p < 0.005) (Figs. 7A, S2). However, compared with the control, mitragynine (2.5 µM) significantly decreased PCSK9 protein expression by approximately 0.53-fold (p < 0.05) (Figs. 7B, S2).

Fig. 7.

Effects of mitragynine on protein expression in HepG2 cells. The protein expression levels of LDLR (A), and PCSK9 (B) were analysed by Western blotting analysis. The results are presented as the means ± SEMs of three independent experiments (N = 3). * p < 0.05, and ** p < 0.005 indicate significant differences compared with the vehicle control (0.1% DMSO).

Effects of leaf extracts from red-vein and white-vein (Pho Thong) strains of Kratom on SREBP-2 and HNF-1α expression in HepG2 cells

The expression of the LDLR and PCSK9 genes is regulated by SREBP-2^3,31. Moreover, PCSK9 gene expression is strongly induced by HNF-1α³². To investigate the potential induction of LDLR and PCSK9 expression by these transcription factors, the effects of kratom leaf extracts on the expression of SREBP-2 and HNF-1α were further investigated. The leaf extracts from red-vein and white-vein (Pho Thong) strains of kratom did not affect SREBP-2 or HNF-1α mRNA expression (Fig. 3C and D). However, leaf extracts from red-vein and white-vein (Pho Thong) strains of kratom showed significantly decreased protein expression levels of SREBP-2 (0.57-fold and 0.67-fold, respectively) (Fig. 4C, S1) and HNF-1α (0.29-fold and 0.59-fold, respectively) (Figs. 4D, S1) compared with those in the control group. Moreover, atorvastatin (10 µM), a positive control, significantly increased SREBP-2 and HNF-1α mRNA and protein expression (Figs. 3C and D and 4C and D).

Molecular docking results

The binding energy, inhibition constant (K_i), and residues involved in hydrogen bonding and hydrophobic formation are presented in Fig. 8; Table 4. The results revealed that mitragynine had the strongest binding affinity with the EGF-A domain of LDLR (– 7.57 kcal/mol). Moreover, quercetin had the highest binding affinity for PCSK9 (– 8.45 kcal/mol). In contrast, atorvastatin had a lower binding affinity for the EGF-A domain of LDLR (– 5.37 kcal/mol) and PCSK9 (– 5.34 kcal/mol). In addition, rutin had the lowest binding affinity for the EGF-A domain of LDLR (– 4.42 kcal/mol) and PCSK9 (– 4.76 kcal/mol).

Fig. 8.

Two-dimensional (2D) view of the molecular docking analysis of interactions between compounds: mitragynine (A), quercetin (B), rutin (C), and atorvastatin (D) in kratom leaf extracts against target proteins; the EGF-A domain of LDLR; and PCSK9.

Table 4.

Binding affinity, Inhibition constant, and interactive residues of the compounds and protein targets determined via molecular Docking analysis.

Protein targets	Ligand	Binding affinity (Kcal/mol)	Inhibition constant (µM)	Hydrogen bonds	Pi-Pi/Pi-alkyl/Pi-cation/ Pi-anion/ Pi-sigma/ Hydrophobic interactions
EGF-A domain of LDLR	Mitragynine	− 7.57	1.74	LUE:529, GLN:531	LEU:528, GLU:410
	Quercetin	− 7.48	2.66	ARG:434, VAL:435, ASP:651, THR:459, ASP:422	PRO:438, VAL:650
	Rutin	− 4.42	573.03	ASN:207, VAL:208, GLU:211, PRO:209	LEU:112, LYS:110
	Atorvastatin	− 5.37	116.09	THR: 61, LYS:83, LEU:135	ALA:62, LYS:136; PRO:138, GLU:85, LEU:137, HIS:139, VAL:140
PCSK9	Mitragynine	− 6.19	1.98	SER:642, VAL:644	TRP:566, LEU:571, HIS:591, ASP:570
	Quercetin	− 8.45	0.64	ALA:649, TRP:461, VAL:435, ASP:651, ASN:439, ARG:525, GLU:426	THR:459, VAL:650
	Rutin	− 4.76	325.65	ARG:476, ILE:474, ALA:463, ASP:360, ALA:330, ARG:357, TYR:293	CYS:358
	Atorvastatin	− 5.34	122.18	–	PHE:150, PHE:318, GLN:152, LEU:351, ASP:320, TYR:325, ALA:68, LYS:69, ASP:70, PRO:71, TRP:72, LYS:258, GLN:256

Discussion

This is the first study to demonstrate the effects of leaf extracts from red-vein, and white-vein (Pho Thong) strains of kratom on hypolipidaemia via an increase in LDLR expression as well as a decrease in PCSK9 protein expression via the downregulation of the transcription factors SREBP-2 and HNF-1α in HepG2 cells. Leaf extracts from both red-vein, and white-vein (Pho Thong) strains of kratom were also associated with increased LDL uptake into HepG2 cells. This may further lead to a reduction in LDL-C levels. Similarly, other medicinal plants and bioactive compounds, including berberine³³, pterostilbene³⁴, aloe-emodin³⁵, piceatannol³⁶, and butein³⁷, have also been found to decrease PCSK9 expression via the downregulation of SREBP-2 and HNF-1α.

PCSK9 plays a critical role as a posttranslational regulator of cell-surface LDLR. It binds to the epidermal growth factor repeat-A (EGF-A) of LDLR and leads to the degradation of LDLR in lysosomes, resulting in increased levels of LDL-C in the circulation³⁸. In this study, PCSK9 expression was significantly decreased after treatment with kratom leaf extracts. The decreased PCSK9 expression may be due to the downregulation of SREBP-2 and HNF-1α. SREBP-2 and HNF-1α are transcription factors that regulate PCSK9 gene expression by binding to the SRE and HNF1 sites in the PCSK9 promoter, respectively³. SREBP-2 also regulates LDLR gene expression by binding SRE sites in the promoters of these genes³¹.

In this study, although SREBP-2 was downregulated, LDLR expression was still increased. It could be speculated that kratom leaf extracts may increase LDLR mRNA or protein stability. Furthermore, a synergistic reduction in HNF-1α and SREBP-2 may strongly repress PCSK9 expression and prevent the lysosomal degradation of LDLR, thereby increasing LDLR levels in hepatocytes. A previous study showed that berberine increased LDLR expression through increased LDLR mRNA stability³⁹. In addition, LDLR degradation can be accelerated by an inducible degrader of LDLR (IDOL), an E3 ubiquitin ligase⁴⁰. Thus, the effects of kratom leaf extracts on hypolipidaemia in relation to LDLR mRNA stability and the inhibition of IDOL should be further clarified.

In this study, LC-MS/MS analysis revealed that leaf extracts from red-vein, and white-vein (Pho Thong) strains of kratom consisted of mitragynine, quercetin, and rutin. Our findings revealed that the concentration of mitragynine in kratom leaves ranged from 38.61 to 39.79 mg/g (3.86–3.97% w/w dry leaf weight). Similarly, a previous report revealed that the concentration of mitragynine was 36.70–63.60 mg/g (3.67–6.36% w/w dry leaf weight)⁴¹. Another study reported that the mitragynine concentration was 0.31–4.94% w/w dry leaf weight⁴². The variation in the concentration of mitragynine has been reported to depend on the season and geography⁴². Mitragynine is a major alkaloid (66% w/w total alkaloids) found in kratom leaves. Nevertheless, other alkaloids, e.g., paynantheine (9% w/w total alkaloids), speciogynine (7% w/w total alkaloids), and 7-hydroxymitragynine (2% w/w total alkaloids), as well as other compounds, e.g., flavonoids, terpenoid saponins, and polyphenols, are also found in kratom leaves^23,43.

In this study, we first showed that mitragynine upregulated LDLR but downregulated PCSK9 protein expression. Molecular docking studies also revealed that mitragynine has a strong binding affinity for the EGF-A domain of LDLR, suggesting its potential role in inhibiting or interrupting the binding between PCSK9 and LDLR. Moreover, quercetin also exhibited the highest binding affinity to the PCSK9 protein according to molecular docking, indicating its ability to inhibit PCSK9 function. However, atorvastatin and rutin had lower binding affinities for both the EGF-A domain of LDLR and PCSK9. These findings suggest that atorvastatin and rutin may have a lower ability to interfere with the binding between PCSK9 and LDLR. In fact, the major mechanism by which statins reduce hepatic cholesterol synthesis is the inhibition of hydroxymethylglutaryl-CoA reductase (HMGCR)⁴⁴. Taken together, these findings suggest that the hypolipidaemic effect of kratom leaf extracts may be a consequence of the bioactive constituents, particularly mitragynine, and quercetin in kratom leaves.

Mitragynine and 7-hydroxymitragynine are partial agonists of the µ-opioid receptor and antagonists of the κ- and δ-opioid receptors^45,46, which have effects similar to those of cocaine and opium^47,48. Chewing 1–5 g of raw kratom leaves daily (∼4 leaves daily) had a stimulant effect and could help workers resist fatigue. Chewing 5–15 g of raw kratom leaves daily (∼13 leaves daily) could produce opioid-like effects, e.g., sedation, analgesia, constipation and euphoria. However, chewing more than 15 g of raw kratom leaves daily (> ∼14 leaves daily) can cause adverse effects, e.g., drowsiness, respiratory distress, liver damage, confusion, stupor, and coma^49–51. Consequently, the safety or toxicity risk of kratom for medical or pharmacological purposes should be strongly considered. Although kratom has hypolipidaemic effects, the recommended amount of kratom consumed daily to reduce hyperlipidaemia is still unknown. The optimal dosage of kratom leaf consumption for the effects of hypolipidaemia should be further investigated.

In conclusion, leaf extracts from red-vein and white-vein (Pho Thong) strains of kratom, which contain several bioactive compounds, exhibited hypolipidaemic effects because of decreased PCSK9 and increased LDLR expression through the modulation of SREBP2 and HNF-1α (Fig. 9). These findings suggest that leaf extracts from red-vein and white-vein (Pho Thong) strains of kratom, as well as mitragynine, could serve as potential supplements for ameliorating hypercholesterolemia.

Fig. 9.

Prospective molecular mechanism of the hypolipidaemic effects of leaf extracts from red-vein and white-vein (Pho Thong) strains of kratom in HepG2 cells.

Electronic supplementary material

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Author contributions

“N.N., M.L., S.P., B.P., N.J.: Contributed to the conception and design of the experiments. N.N.: Performed the experiments and collected the data. N.N., S.P., B.P., N.J.: Analyzed the data and helped interpret the results. N.N., M.L., N.J.: Provided resources and funding. N.N., N.J.: Prepared and revised the manuscript. N.N., N.J.: Confirmed the authenticity of all the raw data. N.J.: Supervised, and administered the project. All authors have read and approved the final manuscript.”

Funding

This research project is supported by National Research Council of Thailand (NRCT): (Contract No. N41A670408), financially supported by Walailak University Graduate Research Fund, and partially supported by the Walailak University Graduate Scholarship.

Data availability

All data that support the findings of this study are available within the article.

Competing interests

The authors declare no competing interests.