Immunomodulatory effect of Cannabis root extract on inflammatory cascades via endocannabinoid system regulation

Abstract

Background

Cannabis roots have been widely used in traditional medicine, with documented references in classical texts describing their use for the treatment of various inflammatory diseases and pain. Despite their longstanding ethnopharmacological significance, the bioactive compounds responsible for these effects and their underlying mechanisms remain unexplored. The present study was conducted to evaluate the unique anti-inflammatory mechanisms of Cannabis sativa root fractions, and moreover, to investigate its mechanism related with the endocannabinoid system (ECS).

Methods

Antioxidant activities and phenol contents of various Cannabis root fractions were determined by chemical assays. The effects of cannabis root fractions on inflammatory markers and endocannabinoid receptor (CB1, CB2) levels were evaluated in LPS-stimulated RAW 264.7 cells. Intracellular 2-arachidonoylglycerol (2-AG) levels were measured using LC-MS/MS. The fraction with the highest potential was further investigated to elucidate its mechanism using endocannabinoid receptor antagonists.

Results

Among the fractions, ethyl acetate fraction (CSREA) demonstrated the highest potential in both antioxidant and anti-inflammatory effects. However, its effect was not attributed to the inhibition of NF-κB signaling pathways. LC-MS/MS analysis showed that CSREA affected intracellular 2-AG levels, supporting its potential via the ECS. CSREA also effectively suppressed ERK phosphorylation, a critical inflammatory signaling pathway modulated by ECS. However, CSREA activity was reduced by co-treatment with a CB1 antagonist.

Conclusion

This study demonstrates that CSREA suppresses inflammatory responses and restores cellular homeostasis primarily by regulating the endocannabinoid system. However, its exclusive use of an acute in vitro inflammation model represents a limitation, and the effects of CSREA in chronic and in vivo settings require further investigation.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12906-026-05317-2.

Background

Cannabis sativa L. is an annual herbaceous dioecious plant with separate male and female flowers, belonging to the Cannabaceae family [1]. The major constituents of Cannabis (cannabinoids such as cannabidiol and 9-tetrahydrocannabinol) were legally regulated in many countries [2]. In the Republic of Korea, Cannabis is legally classified as a narcotic [3]; however, the “Narcotics Control Act” exempts the mature roots of Cannabis from this classification, citing the minimal presence of the psychoactive compounds delta-8-tetrahydrocannabinol (THC) and delta-9-THC [4].

Cannabis root (or Cannabis radix) has been documented in traditional medicine for various therapeutic properties. According to the Korean medical classic Donguibogam, Cannabis root is used to treat blood stasis, urolithiasis, dystocia and retained placenta, as well as to alleviate abdominal distension [5]. Additionally, Bencao Gangmu records its use in pain relief for bone fractures [6]. In Shennong Bencao Jing, a classical Chinese pharmacopoeia, it is described to exhibit hemostatic effects postpartum [7]. Historical records from ancient Greece and Rome indicate its application in treating gout, pain, and skin burns [8]. As such, traditional medicine recognizes Cannabis root as a valuable medicinal resource.

Previous studies utilizing network pharmacology analysis have identified the potential impact of Cannabis root on inflammatory signaling pathways such as PI3K-AKT, AMPK, MAPK, and PPAR [9]. Furthermore, the presence of anti-inflammatory triterpenoid compounds such as Friedelin and Epifriedelanol in Cannabis root has been reported [10]. A recent study demonstrated that Cannabis root extract significantly reduced IL-6 and NQO1 expression in LPS-treated THP-1 macrophages [11]. Similarly, Huang and his colleagues confirmed that Cannabis root inhibits the expression of inflammatory cytokines [12], suggesting its potential role in modulating inflammatory responses.

The endocannabinoid system (ECS) is present in various organs and regulates multiple physiological functions through two primary receptors: cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2) [13]. CB1 is predominantly distributed in the central and peripheral nervous systems [14] and plays a crucial role in pain modulation and neuroinflammatory responses [15]. In contrast, CB2 is primarily found in immune cells and directly regulates inflammation [16]. Exogenous cannabinoids, such as cannabidiol (CBD), exert anti-inflammatory effects via multiple mechanisms, including modulation of classical cannabinoid receptors (CB1 and CB2) and other receptor pathways such as 5-HT1A and TRPV1 [17, 18].

Inflammation is a defense mechanism against pathogen infections, tissue damage, and autoimmune responses. Acute inflammation is essential for tissue healing; however, chronic inflammation is implicated in the pathogenesis of various diseases, including cardiovascular diseases, cancer, diabetes, and arthritis [19]. Nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroids, which are commonly used for inflammation management, exhibit strong anti-inflammatory effects. However, long-term use of these drugs can lead to adverse effects such as gastrointestinal damage, immunosuppression, and drug resistance [20]. Consequently, there is growing interest in developing alternative therapeutics derived from natural compounds.

Cannabis root is known to contain minimal levels of cannabinoids that directly interact with the ECS [21]. However, it includes various bioactive compounds such as hexanoyl-CoA, a precursor of cannabinoid substances, which have been reported to exhibit anti-inflammatory and antioxidant effects [16]. This study investigated and compared the efficacy of different fractions of Cannabis root on inflammatory responses by modulating the ECS. Furthermore, the potent EA fraction was further investigated to identify its unique mechanism using CB1 and CB2 antagonist. Therefore, we provide scientific evidence of Cannabis root for usage as novel drugs in treating chronic inflammatory diseases.

Methods

Chemicals and reagents

Lipopolysaccharide (LPS), Stigmasterol, Dexamethasone, DPPH, 2-Arachidonoylglycerol (2-AG), gallic acid, catechin, tannic acid, ammonium formate, and formic acid were procured from Sigma Chemicals (St. Louis, MO, USA). Hexane and chloroform were obtained from Junsei Chemical (Tokyo, Japan), while ethyl acetate and butanol were sourced from Daejung Chemical (Gyeonggi-do, Republic of Korea). Friedelin and Epifriedelanol were purchased from ChemFaces (Wuhan, China). HPLC-grade water and acetonitrile were acquired from J.T. Baker (Deventer, Holland). Cannabinoid receptor 1 antagonist SR141716A and cannabinoid receptor 2 antagonist AM630 were obtained from Tocris Bioscience (Bristol, UK).

Extraction and solvent fractionation of Cannabis root

The plant names of Cannabis root (Cannabis sativa Linnaeus) were authenticated via the online databases The Plant List and World Flora Online. Dried and pulverized Cannabis roots (200 g) were extracted at room temperature for three days using 3 L of 70% ethanol. The extracts were then sequentially fractionated over two-day periods using hexane, ethyl acetate, butanol, and water. The resulting fractions were concentrated, freeze-dried, and processed into powder form. The fractions were designated as Cannabis Sativa Root Hexane fraction (CSRH), Cannabis Sativa Root Ethyl Acetate fraction (CSREA), Cannabis Sativa Root Butanol fraction (CSRB), and Cannabis Sativa Root Water fraction (CSRW). The obtained yields were CSRH (0.55 g, 0.275%), CSREA (0.67 g, 0.335%), CSRB (0.6 g, 0.3%), and CSRW (5.41 g, 2.705%). The ethanol-extracted Cannabis root fractions were dissolved in dimethyl sulfoxide (DMSO) and filtered using a 20 μm syringe filter before use.

Thin-Layer chromatography (TLC) analysis

Cannabis roots (Cannabis sativa cultivar ‘Cheungsam’) cultivated in various locations, including open fields in Imha-myeon (IF, Andong, Gyeongsangbuk-do), greenhouses in Imha-myeon (IG), open fields in Pungcheon-myeon (PF, Andong, Gyeongsangbuk-do), and open fields in Okcheon-myeon (OF, Yangpyeong, Gyeonggi-do), were analyzed by thin-layer chromatography (TLC) to detect the presence of Epifriedelanol, Friedelin, and Stigmasterol. The roots were dissolved in chloroform at a concentration of 100 mg/mL and applied onto a TLC silica gel plate (TLC silica gel 60 aluminum sheet, Merck, Darmstadt, Germany). The mobile phase consisted of toluene/chloroform (9:1), and the developed plates were sprayed with vanillin-sulfuric acid reagent, dried, and heated at 105 °C for 5 min for visualization. The fractions were dissolved in methanol at a concentration of 100 mg/mL and applied to a TLC silica gel plate. Standard compounds were prepared at 1 mg/mL in methanol and applied under identical conditions. The mobile phase used was chloroform/methanol (98.5:1.5) and the total migration distance was 14 cm. After development, the TLC plates were sprayed with a vanillin-sulfuric acid reagent (0.05 g vanillin, 2 mL sulfuric acid, 8 mL ethanol), dried, and heated at 105 °C for 5 min to visualize the spots.

High-performance liquid chromatography (HPLC) with UV-DAD detection

The fractions were dissolved in methanol (100 mg/mL) and filtered prior to injection into an Agilent 1260 Infinity HPLC system (Agilent, CA, USA) equipped with an Agilent Standard Eclipse XDB-C18 chromatographic column (250 × 4.6 mm, 5 μm pore size). The mobile phase consisted of solvent A (HPLC-grade water) and solvent B (100% acetonitrile). A gradient elution was applied, where solvent B increased from 45% to 50% over 30 min. The flow rate was maintained at 1.0 mL/min, and the column temperature was set at 24 °C. A UV detector was used to monitor the absorbance at 205 nm, and 10 µL of each sample was injected.

Determination of radical scavenging activity

The free radical scavenging activity of ethanol-extracted Cannabis root fractions was assessed using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, as described by Bondet et al. [22]. A 0.3 mM DPPH solution was prepared in ethanol and mixed with various concentrations of the fractions at a 10:1 ratio. The reaction mixture was incubated at room temperature for 30 min. After incubation, the absorbance of the reaction mixture was measured at 515 nm using a Versamax microplate reader (Molecular Devices, Sunnyvale, CA, USA) in a 96-well plate. Ascorbic acid was used as the reference standard.

Determination of antioxidant compound content

The total phenolic content (TPC) was quantified using the Folin-Ciocalteu reaction [23]. A 50 µL aliquot of the Cannabis root fractions was mixed with 1 mL of 2% Na₂CO₃ solution and incubated for 3 min. Subsequently, 0.05 mL of 50% Folin-Ciocalteu reagent was added, and the mixture was incubated in the dark for 30 min. The absorbance was measured at 765 nm using a Versamax microplate reader (Molecular Devices, USA) in a 96-well plate. Gallic acid was used as the standard reference compound.

The total flavonoid content (TFC) was determined using the aluminum chloride colorimetric assay [24]. A 250 µL aliquot of the Cannabis root fractions was mixed with 1 mL of distilled water and 75 µL of 5% NaNO₂, followed by incubation for 5 min. Then, 300 µL of AlCl₃·6 H₂O was added and allowed to react for 6 min. Subsequently, 500 µL of 1 M NaOH was added, and the absorbance was measured at 510 nm using a Versamax microplate reader (Molecular Devices, USA) in a 96-well plate. (+)-Catechin was used as the reference standard.

The total tannin content (TTC) was determined following the Folin-Denis method [25]. A 100 µL aliquot of the Cannabis root fractions was mixed with Folin-Denis reagent and diluted with 70% ethanol to a final volume of 7.5 mL. After the addition of 500 µL of Folin-Denis reagent and 1 mL of 2% Na₂CO₃, the mixture was further diluted to 10 mL with 70% ethanol. The absorbance was measured at 700 nm using a Versamax microplate reader (Molecular Devices, USA) in a 96-well plate. Tannic acid was used as the standard reference. The absorbance values obtained from the antioxidant assays were converted to total contents using the respective standard calibration curves.

Cell culture and maintenance

The murine macrophage cell line RAW264.7 was obtained from the Korean Cell Line Bank (Seoul, Korea). RAW264.7 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 1% penicillin-streptomycin (Gibco, USA) and 10% fetal bovine serum (FBS; Thermo Fisher Scientific, USA). The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ and were subcultured every two days.

Cell viability assay

Cell viability was assessed using the EZ-Cytox kit (DaeilLab, Seoul, Korea, EZ-3000). RAW264.7 cells were seeded in a 96-well cell culture plate at a density of 5 × 10⁴ cells/well and incubated at 37 °C in a 5% CO₂ humidified incubator for 24 h. The Cannabis root fractions were then applied at various concentrations, and the cells were incubated for an additional 24 h. Following treatment, the culture medium was replaced with fresh medium containing 10% EZ-Cytox reagent, and the cells were further incubated for 30 min. The absorbance was measured at 450 nm using a Versamax microplate reader (Molecular Devices, USA) to evaluate cell viability.

Nitric oxide assay

Nitric oxide (NO) production was quantified using the Griess reaction. RAW264.7 cells were seeded in a 6-well plate at a density of 3 × 10⁵ cells/well and incubated for 24 h at 37 °C in a 5% CO₂ humidified incubator. Cells were pretreated with Cannabis root fractions and antagonists for 1 h, followed by stimulation with lipopolysaccharide (LPS) at final concentrations of 1 µg/mL and 0.1 µg/mL. After an additional 24-hour incubation, culture supernatants were collected. The griess reagent was prepared by mixing Griess A (5% phosphoric acid, 1% sulfanilamide) and Griess B (0.1% N-ethylenediamine) in equal volumes. The collected supernatant was mixed with an equal volume of Griess reagent and incubated at room temperature. The absorbance was measured at 540 nm using a Versamax microplate reader (Molecular Devices, USA). NO concentrations were quantified based on a standard calibration curve generated using sodium nitrite (NaNO₂) at various concentrations.

Western blot

Western blotting was performed to assess inflammation-related protein expression in RAW264.7 cells treated with Cannabis root fractions. Cells (5 × 10⁵ cells/well) were seeded in a 6-well plate, incubated for 24 h, and pretreated with Cannabis root fractions and cannabinoid receptor antagonists for 1 h before LPS stimulation (1 µg/mL, 0.1 µg/mL). After 24 h, cells were lysed in RIPA buffer (Thermo Fisher Scientific, Rockford, IL, USA) supplemented with 1% protease and phosphatase inhibitors (Gendepot, Barker, TX, USA). Protein concentration was determined using a BCA kit (Thermo Fisher Scientific, USA). Proteins (30 µg) were separated by 10% SDS-PAGE, transferred onto PVDF membranes, and blocked with 5% BSA. Membranes were incubated overnight at 4 °C with primary antibodies (1:500–1:1000 dilution), followed by washing and incubation with secondary antibodies (1:2000 dilution) for 2 h at 4 °C. Protein detection was performed using the SuperSignal ECL substrate (Thermo Fisher Scientific, USA) and imaged with the Fusion Solo system (Vilber Lourmat, Collegien, France). The primary antibodies used were anti-Cyclooxygenase-2 (COX-2), anti-Extracellular signal-regulated kinase (ERK), anti-P-ERK, anti-Phospho-Signal transducer and activator of transcription 3 (STAT3), anti-P-STAT3, and anti-Nuclear factor kappa B (NF-κB) (Cell Signaling Technology, Berkeley, MA, USA), anti-CB1 and CB2 (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and Lamin B, iNOS and β-actin (Santa Cruz, CA, USA). Secondary antibodies were sourced from Santa Cruz (Santa Cruz, USA).

Real-time quantitative PCR (RT-qPCR)

Total RNA was extracted from RAW264.7 cells exposed to inflammatory conditions using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from the isolated RNA using the AccuPower RT PreMix kit (Bioneer, Korea) following the provided protocol, with a final reaction volume of 20 µL. The primer sequences used for real-time PCR amplification are listed in Table 1. Quantitative PCR (qPCR) was performed using the SYBR Green Master Mix (Roche, Switzerland) under the following cycling conditions: initial denaturation at 95 °C for 10 s, annealing at 56–61 °C for 20 s, and extension at 72 °C for 20–60 s, repeated for 45 cycles. Fluorescence detection and quantification of gene expression levels were conducted using the Roche LightCycler 480 system (Roche Applied Science, USA).

Table 1.

Primer sequences used for quantitative Real-time PCR

Gene	Forword / Reverse (5’ → 3’)	Accession number
iNOS	5’-GAG ACA GGG AAG TCT GAA GCA C-3’	NM_010927.4
	5’-CCA GCA GTA GTT GCT CCT CTT C-3’
IL-6	5’-ATC CAG TTG CCT TCT TGG GAC TGA-3’	NM_001314054.1
	5’-TAA GCC TCC GAC TTG TGA AGT GGT − 3’
IL-1β	5’-CTG AAC TCA ACT GTG AAA TGC CA-3’	NM_008361.4
	5’-AAA GGT TTG GAA GCA GCC CT-3’
CB1	5’-TTC CAC CGC AAA GAT AGT − 3’	NM_007726.5
	5’-TGA AGG AGG CTG TAA CCC-3’
CB2	5’-AGT GTG ACC ATG ACC TTC AC-3’	NM_009924.5
	5’-TCC AGA GGA CAT ACC CAT AG-3’
β-actin	5’-GAC GGC CAG GTC ATC ACT ATT G-3’	NM_007393.5
	5’-CCA CAG GAT TCC ATA CCC AAG A-3’
GAPDH	5’-GGT GAA GGT CGG TGT GAA CG-3’	NM_001289726.2
	5’-CTC GCT CCT GGA AGA TGG TG-3’

Immunofluorescence

Immunofluorescence staining was performed to evaluate the effect of Cannabis root fractions on LPS-induced nuclear translocation of NF-κB (p65) in RAW264.7 cells. Cells were seeded at a density of 2 × 10⁵ cells/mL in Lab-Tek II chamber slides (Nalge Nunc, IL, USA) and treated with LPS (1 µg/mL) and the fraction (40 µg/mL). After treatment, cells were fixed with 4% formaldehyde at room temperature for 10 min. Permeabilization was conducted using 0.1% Triton X-100 for 10 min at room temperature, followed by blocking with 1% BSA for 60 min. The primary antibody targeting the NF-κB p65 subunit was diluted to 2 µg/mL in 1% BSA and incubated at room temperature for 3 h. After incubation, cells were washed twice with 1× PBS (pH 7.4). For nuclear counterstaining, mounting medium containing 4’,6-diamidino-2-phenylindole (DAPI) (Vector Lab Inc., USA) was applied. Fluorescence images were captured using a fluorescence microscope (BX50, Olympus, Japan) to visualize NF-κB nuclear localization.

Lipid extraction for LC-MS/MS analysis

Lipid extraction for LC-MS/MS analysis was performed according to the described protocol [26]. RAW264.7 macrophages were seeded in T-75 flasks at a density of 2 × 10⁶ cells/flask and incubated for 24 h, followed by co-treatment with the respective fractions. After 1 h of incubation, lipopolysaccharide (LPS) was added at a 0.1 µg/mL, and the cells were further incubated for 24 h. At the end of incubation, the culture medium was aspirated, and the cells were washed once with PBS before being harvested. The collected cells were centrifuged at 1,000 g for 3 min to pellet the cells, which were then resuspended in 1 mL methanol and homogenized. Subsequently, 2 mL of ice-cold chloroform and 1 mL of water were added, followed by overnight incubation at 4 °C with continuous agitation using a shaker. The samples were then centrifuged at 2,000 g for 10 min to separate the lower organic phase, which was subsequently dried to obtain lipid extracts. The extracted lipids were resuspended in 1 mL isopropyl alcohol and subjected to LC-MS/MS analysis.

LC-MS/MS analysis conditions for 2-AG

The analysis was conducted using a UHPLC System NASCA2 (Osaka Soda, Nishi-ku, Osaka, Japan). Chromatographic separation was performed on a Waters Acquity UPLC BEH C18 reverse-phase column (1.7 μm, 2.1 × 100 mm). A gradient elution method was employed using mobile phase A of 5 mM ammonium formate with 0.1% formic acid in water and mobile phase B of 0.1% formic acid in acetonitrile. The flow rate was set at 0.3 mL/min, with an injection volume of 5 µL. The column temperature was set at 40 °C. The mobile phase gradient was as follows: 0–1.5 min, A: 40%; 1.5–3 min, A: 5% (held for 7 min); 7.1–10 min, A: 40%.

Mass spectrometric detection was conducted using a QTRAP 4500 system (AB Sciex, Framingham, MA, USA) equipped with an electrospray ionization (ESI) source operating in positive ion mode. The system parameters were set as follows: curtain gas at 30, GS1 and GS2 at 50, ion spray voltage at 5.5 kV, declustering potential at 86 V, and collision energy at 20 V. The ion source temperature was maintained at 450 °C. Quantification of 2-AG was performed using multiple reaction monitoring (MRM) mode, with an MRM transition of m/z 379.3 → 287.2. The calibration curve exhibited linearity, with a coefficient of determination (R²) of 0.99998.

Statistical analysis

All experiments were repeated at least three times. Results are presented as the mean ± standard deviation (SD) or standard error of the mean (SEM). Statistical significance was evaluated using one-way ANOVA followed by Tukey’s post-hoc test, performed with GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA).

Results

Morphological and phytochemical characterization of Cannabis roots by batches

A sensory evaluation was conducted to assess the batch differences in Cannabis roots based on cultivation regions (Fig. 1A). The taproot of Cannabis has multiple lateral roots and numerous young rootlets. It measures over 12 cm in length, with the taproot having an approximate diameter of 1 cm. The outer surface exhibits a pale yellowish-brown to light grayish-brown coloration with transverse striations. No distinct morphological differences were observed in Cannabis roots based on cultivation regions. The phytochemical composition of Cannabis root was analyzed based on origins to evaluate morphological characteristics and key marker compounds, including Friedelin, Epifriedelanol, and Stigmasterol. No noticeable differences in these compounds were observed among the four samples (Fig. 1B).

Fig. 1.

Comparison of Cannabis root by batches. A Sensory evaluation of Cannabis root by produced locations. B Thin layer chromatography (TLC) by batches. EP: Epifriedelin, F-1: Friedelin, F-2: Friedelin, S: Stigmasterol, IF: Cannabis root from Imha open fields, IG: Cannabis root from Imha greenhouses, PF: Cannabis root from Pungcheon open fields, OF: Cannabis root from Okcheon open fields

Identification of phytochemical constituents in Cannabis root fractions

In subsequent experiments, Cannabis root cultivated in Imha-myeon was extracted using 70% ethanol and fractionated based on solvent polarity. To verify the presence of key marker compounds, TLC analysis was conducted (Fig. 2A). The fractions were analyzed at a concentration of 0.1 g/mL, with a total migration distance of 14 cm. The Rf values of the identified compounds were 0.943 for Friedelin, 0.893 for Epifriedelanol, and 0.743 for Stigmasterol. TLC analysis revealed that Friedelin, Epifriedelanol, and Stigmasterol were detected in CSRH and CSREA fractions, while only Stigmasterol was detected in CSRB, and compounds were not detected in CSRW. Due to incomplete separation of Friedelin in TLC, HPLC analysis was performed, confirming its presence at 11.535 min in all fractions, including CSRH, CSREA, CSRB, and CSRW (Fig. 2B).

Fig. 2.

Comparison of bioactive components in Cannabis roots across fractions. A TLC by Cannabis root fractions. B High performance liquid chromatography (HPLC) fingerprinting analysis of Cannabis root fractions. From the top, the compounds and fractions are arranged as follows: Friedelin, Cannabis Sativa Root Hexane fraction (CSRH), Cannabis Sativa Root Ethyl Acetate fraction (CSREA), Cannabis Sativa Root Butanol fraction (CSRB), and Cannabis Sativa Root Water fraction (CSRW)

Antioxidant compound content and antioxidant activity of Cannabis root fractions

The total phenolic, tannin, flavonoid content, and free radical scavenging activity of the four fractions of Cannabis root were evaluated (Table 2). CSRH contained 28.344 µg/mg of total phenolics, 36.050 µg/mg of total tannins, and 47.773 µg/mg of total flavonoids. CSREA exhibited the highest antioxidant content, with 58.151 µg/mg of total phenolics, 156.216 µg/mg of total tannins, and 105.510 µg/mg of total flavonoids. CSRB contained 27.477 µg/mg of total phenolics and 49.216 µg/mg of total tannins, while CSRW contained 14.687 µg/mg of total phenolics and 14.800 µg/mg of total tannins. Flavonoids were not detected in CSRB and CSRW.

Table 2.

Analysis of total phenolic, tannin, and flavonoid contents in Cannabis sativa root extract fractions, including DPPH assay evaluation

Phenolic content (µg/mg)				Antioxidant activity
	TPC	TTC	TFC	DPPH (IC50) (µg/mL)
CSRH	28.344 ± 0.009	36.050 ± 1.548	47.773 ± 4.476	183.614
CSREA	58.151 ± 0.018	156.216 ± 7.008	105.510 ± 3.954	30.219
CSRB	27.477 ± 0.001	49.216 ± 1.057	-	148.748
CSRW	14.687 ± 0.007	14.800 ± 7.211	-	-

Comparative analysis revealed that CSREA contained 2.05 to 3.96 times higher levels of phenolics, 4.33 to 10.56 times more tannins, and 2.21 times more flavonoids than the other fractions, confirming its superior antioxidant compound content. The second highest fraction for total phenolics was CSRH and for total tannins was CSRB. The DPPH radical scavenging activity was assessed based on IC50 values, which were 183.614 µg/mL for CSRH, 30.219 µg/mL for CSREA, and 148.748 µg/mL for CSRB. CSREA demonstrated the most potent radical scavenging activity, followed by CSRB, CSRH, and CSRW.

Anti-inflammatory effects of Cannabis root fractions in RAW264.7

To assess the cytotoxicity of the fractions, a cell viability assay was performed in RAW264.7 cells (Fig. 3A). Treatment with the fractions alone or together with LPS did not lead to significant reduction in cell viability. Based on these results, the concentration of the fractions for subsequent experiments was set at 20 and 40 µg/mL.

Fig. 3.

Comparison of anti-inflammatory effects among CSR fractions. A Assessment of cell viability using fractionated Cannabis Sativa Root (CSR) samples. Results are presented as means ± SEM. Statistical analysis was conducted using one-way ANOVA with Tukey’s test. B Effects of the CSR fractions on Nitric oxide (NO) production in Lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Results are presented as means ± SEM. #### p < 0.0001 vs. non-treated control and ** p < 0.01 vs. LPS-treated control. C Effects of the CSR fractions on inflammatory mediators in LPS-stimulated RAW264.7 cells. D Effects of the CSR fractions on mRNA levels of pro-inflammatory cytokines were determined by Real-time quantitative PCR (RT-qPCR). Results are presented as means ± SEM. ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. non-treated control and * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. LPS-treated control. NC, non-treated control; IND, LPS-treated group; PC, 500 µM Dexamethasone-treated group

The effect of Cannabis root fractions on LPS-induced nitric oxide (NO) production was examined using the Griess reagent assay (Fig. 3B). NO levels were 68.44 µM in the LPS-treated group, 45.61 µM in the CSRH high-dose group, 38.54 µM in the low-dose CSREA group, and 23.54 µM in the CSREA high-dose group, corresponding to NO reductions of 33.36%, 43.69%, and 65.60%, respectively. CSRB and CSRW did not exhibit significant NO inhibition.

To further investigate anti-inflammatory activity, the expression levels of iNOS and COX-2 were analyzed using Western blotting (Fig. 3C). CSREA was the only fraction that showed a statistically significant reduction in iNOS expression, decreasing by 81.56% at high concentrations. COX-2 expression increased in CSRH in a dose-dependent manner, while CSREA exhibited a non-significant decrease at high concentrations. CSRB and CSRW did not show notable changes in COX-2 expression.

To evaluate the regulatory effects of Cannabis root fractions on LPS-induced inflammatory cytokines, the mRNA expression levels of iNOS, IL-1β, and IL-6 were quantified using qRT-PCR (Fig. 3D). iNOS expression showed a decreasing trend with CSRH and CSREA, although statistical significance was not observed, whereas CSRB and CSRW exhibited an increase. For IL-1β and IL-6, CSREA was the only fraction that significantly reduced mRNA expression.

Effects of Cannabis root fractions on NF-κB activity

To investigate the inhibitory effects of Cannabis root fractions on NF-κB activation, the effects were examined in LPS-treated macrophages. However, immunoblot analysis showed no significant reduction of NF-κB expression by CSR fractions (Fig. 4A). Furthermore, immunofluorescence imaging confirmed that NF-κB activity was not effectively inhibited by EA40 treatment as demonstrated by immunofluorescence imaging (Fig. 4B).

Fig. 4.

Comparison of NF-κB activity affected by different CSR fractions. A Protein levels of Nuclear factor kappa B (NF-κB) in the nucleus and cytosol. B Visualization of NF-κB translocation via immunofluorescence staining. Results are presented as means ± SEM. ## p < 0.001 vs. non-treated control

Effects of Cannabis root fractions on endocannabinoid-related inflammatory markers

The impact of Cannabis root fractions on LPS-induced MAPK and JAK-STAT3 signaling pathways was assessed. For ERK, significant inhibition was observed in CSRH and CSREA, with CSRH high-dose treatment reducing ERK by 39.20% and CSREA inhibiting ERK by 38.90% at low doses and 49.67% at high doses (Fig. 5A).

Fig. 5.

Comparison of effects on the endocannabinoid system-specific pathways among CSR fractions. A and B The expression levels of Phospho-Extracellular signal regulated kinase (p-ERK) and Phospho-Signal transducer and activator of transcription 3 (p-STAT3) were assessed by western blot. Results are presented as means ± SEM. #### p<0.0001 vs non-treated control and * p< 0.05, ** p< 0.01, *** p< 0.001 vs LPS-treated control

For STAT3, CSRH exhibited an increasing trend at high concentrations. CSREA significantly reduced STAT3 expression, with reductions of 59.89% at low doses and 76.50% at high doses. CSRB reduced STAT3 expression by 50.21% at low doses and 49.58% at high doses, while CSRW reduced STAT3 by 51.40% at low doses and 62.64% at high doses, showing significant suppression (Fig. 5B).

Effects of Cannabis root fractions on endocannabinoid receptor expression

The interaction between Cannabis root fractions and the endocannabinoid system was assessed by evaluating expression of cannabinoid receptors (CB1 and CB2) using qRT-PCR and Western blotting. As shown in Fig. 6A, LPS stimulation significantly upregulated CB1 and CB2 mRNA expression, whereas treatment with CSREA and CSRB resulted in a significant reduction in their expression levels. At the protein level, the expression of CB1 and CB2 was also reduced following CSRH and CSREA treatment, as confirmed by Western blot analysis (Fig. 6B).

Fig. 6.

Comparison of the effects on the expression of endocannabinoid receptors by CSR fractions. A mRNA levels of endocannabinoid receptor were determined by RT-qPCR. B Endocannabinoid receptor protein levels were determined by western blot. Results are presented as means ± SEM. # p<0.05 vs non-treated control and ** p< 0.01, *** p< 0.001 vs LPS-treated control

Quantification of intracellular 2-AG in macrophages treated with Cannabis root fractions

Lipid components were extracted from macrophages treated with LPS and Cannabis root fractions, followed by LC-MS/MS analysis to quantify 2-AG levels. The regression equation of the 2-AG calibration curve was determined as y = 3.30683e5x − 23.10459, with a correlation coefficient of r² = 0.99998, indicating excellent linearity. The retention time of 2-AG was confirmed to be 4.99 min. Table 3 presents the 2-AG concentrations in cells treated with different fractions (with or without 0.1 µg/ml LPS) based on the calibration curve.

Table 3.

Intracellular levels of 2-AG in LPS-stimulated macrophages following treatment with ethanol-extracted fractions from Cannabis root

Sample	Concentration (ng/mL)	Precursor ion (m/z)	Product ion (m/z)	Retention Time (min)
NC	2.825	379.3	287.2	4.98
IND	5.171	379.3	287.2	4.98
CSRH+	5.683	379.3	287.2	4.98
CSREA+	3.187	379.3	287.2	4.98
CSRB+	3.67	379.3	287.2	4.98
CSRW+	5.541	379.3	287.2	4.98
CSREA	9.633	379.3	287.2	4.99
PC+	4.431	379.3	287.2	4.99

LPS treatment (0.1 µg/mL) resulted in a 1.830-fold increase in 2-AG levels compared to the untreated group. The CSRH + and CSRW+ exhibited slight increases of 1.099-fold and 1.072-fold, respectively, compared to the LPS-induced group. In contrast, CSREA + and CSRB+ showed 43.93% and 29.0% reductions, respectively. Dexamethasone treatment led to a 14.4% reduction in 2-AG levels compared to the LPS-induced group. Notably, CSREA treatment alone (without LPS) resulted in a 3.401-fold increase compared to the normal control, a 1.863-fold increase compared to the negative control and a 3.023-fold increase compared to the inflamed state treated with CSREA (CSREA+).

Inflammatory response following cannabinoid receptor antagonist treatment

To evaluate the inflammatory response upon cannabinoid receptor inhibition, SR141716A (1 µM, CB1 antagonist) or AM630 (300 nM, CB2 antagonist) was administered, and the levels of NO production and iNOS protein expression were assessed (Fig. 7A, B). The results indicated that NO levels and iNOS expression exhibited a similar decreasing trend to CSREA treatment alone, with no statistically significant differences observed between CSREA and antagonist-treated groups.

Fig. 7.

Evaluation of pro-inflammatory mediators in macrophages treated with CSREA of Cannabis root along with CB1 or CB2 antagonists. A Evaluation of the ability of CSREA to suppress NO production. Results are presented as means ± SD. #### p<0.0001 vs non-treated control and ****p< 0.0001 vs LPS-treated control. B Measurement of inflammatory mediators in RAW264.7 cells treated with CSREA was performed using Western blot analysis. C Pro-inflammatory cytokines mRNA levels were determined by RT-qPCR. Results are presented as means ± SEM. #### p<0.0001 vs non-treated control and ** p< 0.01, *** p< 0.001, **** p< 0.0001 vs LPS-treated control, $ p< 0.001 vs EA40, p< 0.0001 vs EA40. NC, non-treated control; IND, LPS-treated group; EA40+1, co-treated with CSREA and Cannabinoid receptor 1 (CB1) antagonist; EA40+2, co-treated with CSREA and Cannabinoid receptor 2 (CB2) antagonist

The effects of cannabinoid receptor antagonists on pro-inflammatory cytokine expression were further examined (Fig. 7C). iNOS expression was significantly increased upon CB1 antagonist treatment compared to CSREA alone. Similarly, IL-1β and IL-6 expression were significantly upregulated following CB1 and CB2 antagonist treatment, compared to the levels observed in the CSREA-treated group.

Effects of cannabinoid receptor antagonist treatment on inflammatory pathways

To investigate the impact of cannabinoid receptor antagonism on inflammatory signaling pathways, macrophages were co-treated with Cannabis root fractions and cannabinoid receptor antagonists, followed by Western blot analysis. The ERK and STAT3 inflammatory pathways, which were activated by LPS stimulation, were significantly inhibited by CSREA treatment. However, when CB1 antagonist was co-treated, ERK activation was significantly restored compared to CSREA treatment alone (Fig. 8A). In contrast, STAT3 activation remained unchanged following cannabinoid receptor antagonist treatment (Fig. 8B).

Fig. 8.

Effects of CB1 or CB2 antagonists on Endocannabinoid system-specific pathways. A P-ERK, B P-STAT3 protein levels were determined by western blot. Results are presented as means ± SEM. #### p<0.0001 vs non-treated control and **** p< 0.0001 vs LPS-treated control, $$ p< 0.01 vs EA40

Discussion

In this study, fractionated extracts of Cannabis root were screened for their pharmaceutical potential. Compared to previously studied 70% ethanol extracts, fractionation led to a higher concentration of antioxidant compounds and improved radical scavenging activity [27]. The analysis of phenolic content indicated that CSRH and CSREA contained the highest levels of active compounds, with CSREA exhibiting the most potent radical scavenging activity (Table 2).

Cannabis root contains triterpenoid compounds such as Friedelin, Epifriedelanol, and Stigmasterol, which have direct antioxidant and anti-inflammatory effects [10]. Notably, Stigmasterol is known to modulate inflammation and mitigate immune responses [28], while Friedelin has been reported to inhibit inflammation and promote tissue regeneration [29]. In this study, TLC and HPLC were utilized to analyze Friedelin content in Cannabis root extracts (Fig. 2). TLC results showed that Friedelin was exclusively detected in CSRH and CSREA, whereas HPLC analysis confirmed its presence in all fractions even in minor quantities.

CSREA significantly reduced LPS-induced NO production (Fig. 3B) and the expression of pro-inflammatory cytokines (Fig. 3D); however, it did not affect NF-κB activation under the tested conditions (Fig. 4). These findings suggest that the anti-inflammatory activity of CSREA may be mediated through alternative signaling mechanisms beyond the canonical NF-κB pathway. Subsequent analyses revealed that CSREA notably attenuated ERK and STAT3 phosphorylation, indicating possible involvement of the MAPK and JAK-STAT signaling cascades. To explore the role of the ECS in mediating these responses, co-treatment experiments with CB1 and CB2 receptor antagonists were performed.

ERK plays a crucial role in inflammatory responses, cell survival, and tissue recovery [30]. In this study, CSREA treatment significantly inhibited LPS-induced ERK activation, thereby attenuating inflammatory responses and cytokine expression (Fig. 5A). These findings align with previous studies showing that ERK signaling interacts with ECS [31].

Co-treatment with CB1 and CB2 antagonists attenuated the anti-inflammatory effects of CSREA, as indicated by a statistically significant increase in pro-inflammatory markers (Fig. 7C). This finding supports the involvement of cannabinoid receptor signaling particularly via CB1 and CB2, in mediating CSREA’s regulatory effects. Notably, ERK activation was affected by CB1 antagonism, while STAT3 signaling remained unchanged, implying differential regulatory mechanisms (Fig. 8). These results suggest that the ECS may selectively modulate ERK signaling, whereas STAT3 might be influenced through receptor-independent or secondary pathways in LPS-stimulated macrophages.

This study also employed different LPS concentrations to assess the impact of CSREA on inflammatory responses via the ECS. High-dose LPS (1 µg/mL) robustly activated pro-inflammatory signaling pathways, effectively inducing iNOS, COX-2, IL-1β, and IL-6 expression, establishing a suitable model for evaluating the anti-inflammatory effects of the fractions. In contrast, previous studies have reported that low-dose LPS treatment modulates transcriptional activity in macrophages and maintains the expression of both pro-inflammatory and anti-inflammatory cytokines [32]. Since low-dose LPS prevents excessive TLR4 activation, it may serve as a useful model for investigating ECS activation and its role in resolving inflammation. In this study, low-dose LPS (0.1 µg/mL) provided a moderate inflammatory stimulus, allowing for a more detailed analysis of CB1 and CB2 receptor-mediated signaling pathways (Figs. 7 and 8).

2-AG is a key endocannabinoid involved in inflammation, serving as a precursor to arachidonic acid and modulating immune responses [33]. Several studies have reported the anti-inflammatory effects of 2-AG [34, 35] demonstrating that endocannabinoid levels increase under inflammatory conditions to counteract excessive immune activation. Notably, inhibiting monoacylglycerol lipase (MAGL), the primary enzyme responsible for 2-AG degradation, has been shown to enhance anti-inflammatory and analgesic effects in acute inflammation models [36, 37]. Additionally, LPS-treated macrophages exhibited a two-fold increase in 2-AG levels, indicating its role in regulating inflammation [38]. Increased 2-AG levels in non-inflammatory conditions may promote inflammation resolution and tissue repair, thus supporting cellular homeostasis [39].

To investigate whether Cannabis root fractions regulate endocannabinoid levels, intracellular 2-AG levels were measured in LPS-treated macrophages. In the LPS (0.1 µg/mL) treated macrophages, 2-AG levels were increased, likely as a compensatory anti-inflammatory response [40]. However, co-treatment of CSREA and CSRB with LPS reduced 2-AG levels; notably, CSREA also significantly suppressed pro-inflammatory cytokine expression (Table 3 and Fig. 8). These results might suggest that CSREA mitigates inflammation, contributing to a reduction in elevated 2-AG levels. In contrast, in non-inflammatory conditions, CSREA increased 2-AG levels, implying that it modulates endocannabinoid metabolism, potentially enhancing tissue recovery.

COX-2 utilizes arachidonic acid as a substrate to synthesize prostaglandin E2 (PGE 2), a key inflammatory mediator [41]. In addition, COX-2 is involved in the metabolism of endocannabinoids, such as 2-AG and anandamide (AEA), resulting in attenuating their activity [42]. The results of this study demonstrated that COX-2 expression was not significantly reduced, distinguishing the mechanism of Cannabis root from other ordinary natural products that exert anti-inflammatory effects through the initial blocking of TLR4/MD2 signaling activation induced by LPS [43]. Notably, although endocannabinoid receptors do not directly interact with LPS, the endocannabinoid pathway indirectly suppresses excessive inflammatory responses by modulating the phenotype of immune cells, including macrophages and T cells, from a pro-inflammatory to an anti-inflammatory state [44, 45]. Inflammation triggered by LPS has been reported to upregulate CB1-biased expression and enhance downstream signaling; however, a compensatory mechanism is also activated, leading to an increased synthesis of anti-inflammatory endocannabinoids [46, 47].

In the present study, LPS stimulation activated COX-2, thereby inducing an inflammatory response, while the increased levels of 2-AG were interpreted as a compensatory mechanism against this inflammatory stimulus. These findings suggest that CSREA modulated inflammation via activation of the 2-AG pathway without directly inhibiting COX-2 expression. Further research on ECS-associated signaling molecules, such as adenylyl cyclase and cAMP, is required to elucidate the mechanisms on CSREA.

Activation of the endocannabinoid pathway has been reported to be more effective in resolving inflammation and promoting recovery than merely blocking acute inflammatory responses [48]. The activation of CB2-biased signaling can facilitate macrophage M2 polarization, leading to the upregulation of anti-inflammatory cytokines such as IL-4 and IL-10, contributing to its immunomodulatory efficacy [49]. Given the reported interplay between innate immune signaling via TLR pathways and the endocannabinoid system [48], a combined approach targeting both pathways is expected to yield more effective therapeutic outcomes for inflammatory diseases.

This study provides preliminary evidence that fractions derived from Cannabis root exert anti-inflammatory effects through non-canonical signaling pathways (Fig. 9). Unlike conventional herbal medicines that typically act by inhibiting TLR4 or COX-2 pathways, CSREA appeared to modulate inflammation via the endocannabinoid system (ECS) without directly suppressing COX-2 expression. The observed changes in 2-AG levels under different conditions suggest a selective involvement of the ECS. However, further investigation is needed to elucidate the underlying mechanisms. In particular, analysis of key endocannabinoids such as AEA, the use of a broader range of antagonists, and in vivo validation using knock-out model are required to gain a more comprehensive understanding of the regulatory pathways involved.

Fig. 9.

Schematic representation of the anti-inflammatory mechanism of CSREA. CSREA increased 2-AG production and modulated CB1/CB2 receptors, leading to the suppression of ERK phosphorylation and partial inhibition of STAT3 phosphorylation. These effects contributed to the downregulation of inflammatory mediators (iNOS, IL-1β, and IL-6). Notably, CSREA did not affect NF-κB signaling, suggesting that its anti-inflammatory action occurs via NF-κB-independent pathways

In addition, with the increasing production of Cannabis, its root, which is typically discarded as agricultural waste, may represent a new source of bioactive compounds. The findings of this study suggest that Cannabis root can be repurposed as a valuable material, and that such a by-product recycling strategy could contribute to environmental sustainability and the advancement of bio-based industries.

Conclusion

This study emphasises the therapeutic potential of Cannabis sativa root, a traditionally used but underexplored herbal resource, by demonstrating its anti-inflammatory effects via the endocannabinoid system. Among the fractions, CSREA showed the most potent antioxidant and anti-inflammatory activities, notably through the modulation of CB1 and CB2 receptors and increased 2-AG levels. CSREA significantly inhibited ERK phosphorylation via ECS activation. A reduction in STAT3 phosphorylation was also observed, which may occur through a distinct pathway. Notably, NF-κB signaling was not affected, implying that it is not a major contributor to the anti-inflammatory activity of CSREA. These findings suggest that Cannabis root, particularly the CSREA fraction derived from the Korean Cheungsam variety, may be considered a potential candidate for further preclinical investigation as a natural anti-inflammatory agent, given its modulatory effects on inflammation-related pathways. However, as the present study was limited to an acute in vitro inflammation model, further investigation in chronic and in vivo settings is necessary to comprehensively validate its therapeutic relevance.

Abbreviations

2-AG

2-arachidonoylglycerol

CB1

Cannabinoid receptor 1

CB2

Cannabinoid receptor 2

CSRH

Cannabis sativa root hexane fraction

CSREA

Cannabis sativa root ethyl acetate fraction

CSRB

Cannabis sativa root butanol fraction

CSRW

Cannabis sativa root water fraction

COX-2

Cyclooxygenase-2

DAPI 4′,6

Diamidino-2-phenylindole

DMEM

Dulbecco's Modified Eagle Medium

DPPH 2,2

Diphenyl-1-picrylhydrazyl

FBS

Fetal Bovine Serum

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

HPLC

High performance liquid chromatography

IL-1β

Interleukin 1 beta

IL-6

Interleukin 6

iNOS

Inducible nitric oxide synthase

LPS

Lipopolysaccharide

NF-κB

Nuclear factor kappa B

NO

Nitric oxide

P-ERK

Phospho-Extracellular signal regulated kinase

P-STAT3

Phospho-Signal transducer and activator of transcription 3

TLC

Thin layer chromatography

TLR4

Toll-like receptor 4

Authors' contributions

Conceptualization: D.-W.L. Validation: G.-R.Y. Formal analysis: H.-L.J. Investigation: H.-L.J., G.-R.Y. Data curation: H.J., M.-S.L. Writing – Original Draft: H.-L.J., D.-W.L. Writing – Review &amp; Editing: H.K., W.-H.P. Visualization: H.K. Supervision: W.-H.P. Funding acquisition: D.-W.L.

Funding

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number : RS-2023-KH139513).

Data availability

Data will be made available on request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.