Solvent extracts of an herbal formula containing Petasites hybridus and Tanacetum parthenium inhibited the lung development in zebrafish embryos and demonstrated cytotoxicity in human lung cancer cell lines

1. Introduction

Inflammation is the innate defense against tissue injuries, which is activated by a complicated biological response to damage caused either by infections or chemicals (Maione et al., 2016). Anti-inflammatory drugs can modify the underlying mechanisms of inflammation to alleviate tissue injury and enhance patients’ well-being. The medicines used to combat inflammation, such as non-steroidal anti-inflammatory drugs (NSAIDs), are known to have adverse effects and often act in a non-selective manner (Bell et al., 2018).

Natural anti-inflammatory products isolated from medicinal plants could be the best alternative for synthetic anti-inflammatory drugs to avoid NSAID-related toxicities (Meghji et al., 2021; Yakhchali et al., 2021). Butterbur is a commercial herbal formulation containing ingredients from two medicinal plants, Petasites hybridus and Tanacetum parthenium. Pyrrolizidine alkaloids, which are toxic compounds found in Petasites hybridus, have been removed during the preparation of the Butterbur (Giles et al., 2005). Butterbur is used as a food supplement to enhance the blood flow to the brain, thereby supporting neurological function (NOWFOODS 2024).

Scientists study lung toxicity using cell (in vitro) or animal models (in vivo). Although in vitro assays are valuable, in vivo studies frequently yield more clinically relevant findings that can be extrapolated to humans. The zebrafish (Danio rerio), a small freshwater fish, is widely utilized in biomedical research due to its physiological and genetic similarities with humans. The swim bladder is an organ in teleost fish that is structurally and functionally analogous to mammalian lungs, and their development occurs in a similar way. They both develop as an extension from the esophagus, with the glottis in the same location (Daniels et al., 2004).

Despite differences in respiratory organ structure, the Sonic Hedgehog (Shh) signaling is needed for embryonic lung organogenesis across humans, rodents, and zebrafish (Bellusci et al., 1997; Winata et al., 2009; Fernandes-Silva et al., 2017). The Shh pathway is indispensable to regulate critical morphogenic mechanisms, such as cellular proliferation, differentiation, migration, and persistence for embryonic lung formation. The abnormal activation of SHH gives rise to many solid tumors in humans (Giroux-Leprieur et al., 2018).

Lung cancer is among the deadliest types of cancer in the world and is regarded as a leading factor in cancer-related death in both men and women, taking millions and half-lives each year. The prevalence and mortality of lung cancer have risen dramatically in recent decades among the Saudi population, considering lung cancer is the most lethal type in Saudi Arabia (Bray et al., 2018; Almatroudi 2021).

This study was designed to investigate the pulmonary toxicity of Butterbur during embryonic development by using zebrafish as an in vivo pulmonary toxicity model and to identify the major bioactive molecules through mass spectroscopy analysis. This study also explores the binding efficiency of major constituents present in the extracts with zebrafish Shh protein using the in silico molecular docking approach. Additionally, the cytotoxicity of Butterbur extracts was explored by in vitro cell viability assays to determine the potential of Butterbur extracts as an anticancer remedy against lung cancer.

2. Materials and Methods

3. Results

3.2 In-vivo toxicity of butterbur extracts

The toxicity of each solvent extract on the zebrafish embryos varied depending on the extract’s nature and concentration; however, all the extracts exhibited moderate levels of toxicity, with LC50 values higher than 100 µg/mL (Fig. 1 and Table 2). The n-hexane extract demonstrated the highest level of toxicity, with LC₅₀ values of 123.77± 0.57 µg/mL. The LC₅₀ of the methanol extract was ≥ 343.46 ± 0.55 µg/mL.

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Fig. 1.

The dose-response of zebrafish embryos towards different extracts of Butterbur. The data presented is the average value of three replicates ± SD.

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Table 2. Toxicity of zebrafish embryos against different extracts of butterbur.

Sr. No.	Extract	(µg/mL)*
1	Ethyl acetate	129.49±0.35
2	n-hexane	123.77±0.57
3	Chloroform	175.25±1.03
4	Methanol	343.46± 0.55

* LC50 Means of three biological replicates ± standard deviation.

3.3 Zebrafish’ swim bladder formation and growth were affected by butterbur extracts

The sublethal dose optimization: A sublethal concentration for each extract (approximately 50–100 µg/mL, corresponding to ½ to ⅔ of the LC₅₀ for each respective extract) was selected for swim bladder analysis. Treated larvae were carefully examined using standard developmental staging criteria (Kimmel et al., 1995) and showed no other signs of teratogenicity or developmental delay except for the swim bladder defect. This indicates the effect was specific to swim bladder organogenesis and not a result of general developmental arrest. The size and shape of the inflated swim bladder (black arrow) of mock (0.5% v/v methanol) treated larvae (n=150 ± 1.64) have been shown in Figs. 2(a, b). The swim bladder formation and development were not affected in zebrafish embryos treated with the methanol extract of Butterbur (150 µg/mL), as the size and growth of the swim bladder were correlated with mock-treated embryos, Figs. 2(c, d) (n=150 ±1.34) at 4dpf. The zebrafish embryos treated with the ethyl acetate extract of Butterbur have been shown in Figs. 2(e, f). The size of the swim bladder in treated embryos (n=150±0.70) was smaller than control embryos at the same development stage. It is evident from Fig. 2(f) that a small swim bladder was present in treated embryos, which means that swim bladder formation was not affected in the ethyl acetate extract-treated larvae, but it did affect the growth of the swim bladder. However, on the other hand, the n-hexane extract of Butterbur affected the formation as well as growth of the swim bladder in treated zebrafish embryos. As shown in Figs. 2(g, h), the swim bladder was not formed in embryos which were treated with (50µg/mL n= 150) n-hexane extract of Butterbur at 5dpf (experiment end point). There could be a possibility that the delay in swim bladder development in treated larvae could be due to the development delay caused by Butterbur treatment. Hence, treated larvae were carefully examined for developmental staging criteria, following the zebrafish developmental staging criteria explained by (Kimmel et al., 1995) and (Parichy et al., 2009). The developmental staging hallmarks for the inflated swim bladder at 4d pf (The open mouth, appearance of gut beneath the swim bladder, completion of larval pigment pattern, Figs. 2g and h) were all present in treated embryos except the swim bladder, which means swim bladder organogenesis was severely affected in these larvae. The chloroform extract of Butterbur did not affect the organogenesis but the growth of swim bladder but albeit the affect was mild as the size of swim bladder was not severely affected in chloroform extract treated embryos (Figs. 2i and j).

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Fig. 2.

Zebrafish swim bladder formation and growth were affected by the extracts of butterbur. The representative live images of zebrafish embryos mock treated (a &b) with methanol (c & d), ethyl acetate (e & f), n-hexane (g & h), and chloroform (i & j) extracts of Butterbur The ethyl acetate and chloroform extract perturbed the growth, while the n-hexane extract blocked the formation and growth of the swim bladder (SB pointed by the black arrow). In each lane, the lower panel is the high-magnification image of the same embryos has been shown in the upper panel.

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3.5 Chemical characterization of butterbur solvent extracts

GC-MS spectra of the chemical compounds identified in the n-hexane extract have been shown in Fig. 3, and details of the compounds (name, molecular formula and weight, retention time, etc.) have been provided supplementary Table S1. A total of three compounds were identified in n-hexane extract, the most abundant compounds were (8.beta.-h)-3,4a,5-trimethyl-4,4a,5,6,7,8a9-octahydronaphthol[3-.bifuran-2(4h)-one (30.54%), which had a retention time of 19.9 min. The second compound, which was abundant in n-hexane extract, is 9,12-Octadecadienoic acid (11.59 %), with a retention time of 21.25 min. The third major compound identified by GC-MS analysis is hexadecanoic acid (8.94 %).

Table 1

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Fig. 3.

Chromatogram of N-hexane extract of Butterbur.

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The chemical constituents identified in the chloroform extract of Butterbur have been presented in Table S2. GC-MS spectra can be seen in Fig. 4. Eight (8) compounds where I identified from the chloroform extract of Butterbur. The major compounds were 1,2,3-Propanetriol, diacetate (44.56 %, retention time 7.95 min) and 4-Hydroxytetradec-2-ynal (27.10%, retention time 20.35 min).

Table 2

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Table 3

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Fig. 4.

Chromatogram of n-hexane extract of Butterbur.

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The chemical constituents identified in the ethyl acetate extract of Butterbur have been presented in S3. GC-MS spectra have been shown in Fig. 5. The top three major compounds (depending on the percentage in total) were 1) 1-Hexyl-2-nitrocyclon-hexane (38.57 %, retention time of 19.30 min), 2) 2-Hydroxy-6-methyl benzalcehyde (13.78 % and retention time of 12.15 min), and 3) 1,2,3-Propanetriol, monoacetate (10.59 %, retention time 6.97 min).

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Fig. 5.

Chromatogram of ethyl acetate extract of Butterbur.

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The chemical constituents identified in the methanol extract of Butterbur have been presented in Table S4, and the corresponding GC-MS spectra have been shown in Fig. 6. The major compounds identified in the methanol extract were: 1) E-2-Octadecadecen-1-ol (17.90%, retention time 20.25 min), 2) Hypoxanthine (17.13%, retention time 13.04 min), and 3) 5-(hydroxymethyl)-2-furancarboxalcehyde (16.46%, retention time 9.97 min).

Table 4

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Fig. 6.

Chromatogram of the methanol extract of Butterbur.

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3.7 In silico molecular docking of candidate molecules to zebrafish sonic hedgehog (shh) protein

The major compounds (petasalbin, petasin, Octadecadienoic Acid, 1-Hexyl-2- Nitrocyclon-hexane and, 1,2,3-Propanetriol, Diacetate) in each solvent extract were selected as ligands for binding to the zebrafish Sonic Hedgehog (Shh) protein. The 3D ligand structures were retrieved from PubChem and converted to MOL2. CBDock2 was used to perform molecular docking of ligands with the target protein. The objective was to assess the binding significance of various compounds with the target protein. CBdock2 is a molecular docking tool designed for predicting the binding modes and affinities of small molecules with target proteins. It utilizes a combination of machine learning techniques, physics-based scoring functions, and conformational sampling algorithms to efficiently explore the ligand-binding space (Liu et al., 2022). As presented in Table 4 and Fig. 7, among the tested compounds, petasin exhibited a notably high binding affinity (−6.4 kcal/mol) with zebrafish Shh.

Table 4. Protein Ligand Interactions residues along with their binding affinity.

Ligand name	Binding affinity	Protein-ligand interaction residues
1H2N	-5.5	GLU63, ARG72, GLU188, GLN100, VAL191, ALA192, GLU75, THR99, LEU76
123PDA	-4.8	GLU142, LEU76, THR99, ILLE66, GLU75, VAL191, GLU188, ALA187, GLU63, ARG72
OctaDA	-5.5	PHE135, GLU176, HIS182, LEU139, SER138, GLU53, TYR174
Petasin	-6.4	PHE135, HIS134, SER138, GLU136, GLU137, GLU53, TRP172, HIS182, TYR174, GLU176, HIS180

1H2N: 1-Hexyl-2- Nitrocyclon-hexane, 123PDA: 1,2,3-Propanetriol, Diacetate: OctaDA Octadecadienoic Acid.

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Fig. 7.

(a) shows a summary of interactions of amino acid residues of the target protein with compound 1H2N. It forms a hydrogen bond only with THR99 with a distance of 3.11 angstrom. (b) represents the interactions with the compound 123PDA. It shows hydrophilic interaction with GLU63, ARG72, with the distance of 2.23,3.16 Angstroms, respectively. (c) depicts the interaction with the compound octaDA along with the hydrophilic interaction with LEU139 (3.09 Angstrom), GLU53 (1.93 Angstrom). (d) shows interaction of compound petasin with hydrophilic interaction with HIS180 (3.07 Angstrom).

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We also performed the molecular docking using the online free molecular docking tool SwissDock using its latest 2024 user-friendly version (Bugnon et al., 2024). Docking was performed by choosing “attractive cavities.” The results obtained from the SwissDock have also demonstrated that petasin and petasalbin are the molecules that are the best binding candidates to the zebrafish Shh protein. Screenshots of the docking results are shown in Figs. S1-5.

Fiuure 1-5

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4. Discussion

Lung inflammation is a hallmark of acute lung injury, and the majority of acute and chronic lung diseases are associated with lung inflammation. (Kunihiko and Stephan 2014). Several herbal formulations are used to cure lung inflammations (Kim et al., 2017; Zhou et al., 2021). These formulations possess anti-oxidant, anti-inflammatory, and anti-apoptotic properties (Zhang et al., 2023). However, these herbal formulations were merely investigated to determine if they can cause pulmonary toxicity during embryonic development. Use of suitable experimental animal models for lung toxicity could be helpful to determine the safety of anti-inflammatory herbal formulations.

Petasites hybridus and Tanacetum parthenium have been traditionally used to cure respiratory diseases and lung inflammation (Lee et al., 2004; Pareek et al., 2011; Urda et al., 2022). These two medicinal plants are used to prepare an herbal formula, “Butterbur,” which has been used in this study to evaluate the safety profile on embryonic development. However, in vivo developmental toxicity, especially the effect of these two plants on embryonic lung development, is largely unknown before this study. The acute embryonic toxicity assay has indicated that Butterbur extract induced mild to moderate levels of toxicity in zebrafish embryos at high doses (100 µg/mL). The safety profile of Butterbur as an herbal formula has never been reported before; however, the Petasites hybridus has been reported to be safe in animal models of toxicity and also in human clinical trials (Diener et al., 2018; Borlak et al., 2022). Similarly, feverfew (T. parthenium) has been reported to be either harmless or having mild and transient side effects in experimental animal models and clinical trials (Ernst and Pittler 2001), as pyrrolizidine alkaloids, which are toxic compounds found in Petasites hybridus, have been removed during the preparation of the Butterbur (Giles et al., 2005).

Zebrafish has been successfully used to model some of the human respiratory diseases, such as Bronchopulmonary Dysplasia (BPD)/Alveolar Hypoplasia (Lee et al., 2019). However, Zebrafish also have limitations as a lung disease model, top of these is that zebrafish do not have true alveoli, and Gill breathing dominates in zebrafish, but lung/swim bladder studies are still valid, hence it is recommended that the results of zebrafish studies must be validated in mammals. Researchers can accelerate discoveries in pulmonary medicine by combining zebrafish discovery-phase work with mammalian validation studies.

This study used the swim bladder development in zebrafish embryos as a model of in vivo lung development. The swim bladder development in zebrafish starts from 32 hpf and occurs in three developmental phases: i) epithelial budding, ii) mesodermal layer growth, and iii) inflation of chambers at specific times until 4.5 days post fertilization (Winata et al., 2009). Swim bladder development in zebrafish has been proposed as a model of lung injury (Lee et al., 2019). Researchers have used zebrafish swim bladders to study human mucosal and fungus infections (Gratacap et al., 2013; Voelz et al., 2015). The zebrafish swim bladder was also used as an in vivo injury-repair model for lung disease (Perrin et al., 1999).

The results have indicated that Butterbur extracts at sublethal doses have influenced the swim bladder development in zebrafish larvae either by interrupting the organogenesis (organ formation) or the development/ growth of the swim bladder. Zebrafish embryos treated with n-hexane extract of Butterbur did not have a swim bladder until 5 to 6 dpf, without inducing any other teratogenicity or development delay. Notably, the chloroform extract demonstrated greater potency in non-cancerous HUVEC cells than in the lung cancer lines, indicating a potential lack of selectivity that would limit its therapeutic utility and warrants further investigation. We did not find any published reports about the effect of butterbur as an herbal formula or its individual ingredients on lung development so far.

The phenotype of Butterbur extract (especially the n-hexane) treated embryo has indicated that these extracts might contain phytocompounds, which have negatively regulated the expression of signaling molecules that are needed for the swim bladder development in zebrafish. Hence, the extracts of Butterbur were processed for phytochemical analysis. Although variety of compounds have been reported from P. hybridus and T. parthenium plants when studied individually, however, no prior information was available regarding the nature of biomolecules present in Butterbur as an herbal formulation before this study The major compound identified in the n-hexane extract present at more than 30 %, was (8.BETA.-H)-3,4A,5-trimethyl-4,4a,5,6,7,8,8a,9-octahydronaphtho[2,3-b]furan-2(4h)-one. This is a novel compound, which was tentatively identified as a structural analogue of petasalbin from the n-hexane extract of Butterbur and has never been reported before. The compound search from the PubChem or ChemSpider databases using this chemical formula had retrieved zero results. This is a novel compound being reported for the first time from a Butterbur herbal formulation. The search for the nearest compound having a similar structure retrieved a compound known as “petasalbin,” which is a sesquiterpenoid found in butterbur species (Kulinowski et al., 2022). Petasin, a sesquiterpene known as a compound found in plants of the genus Petasites (Horwitz 2007), is a Furan derivative (Borlak et al., 2022). However, we could not detect Petasin in the Butterbur formulation. Petasin, another major compound reported in butterbur species, along with petasalbin, is a sesquiterpene found in P. hybridus (Kulinowski et al., 2022). We thus hypothesized that the major compounds which is present in n-hexane extract might be responsible for inhibiting swim bladder formation and development in zebrafish embryos. The phytochemistry of P. hybridus and T. parthenium has been extensively explored. Both plants contain sesquiterpenes as major constituents, and their biological properties are attributed to these sesquiterpenes (Shih et al., 2011). More than 200 distinct sesquiterpene structures have been documented in the Petasites genus. These can be categorized into three primary classes: eremophilane-type, furanoeremophilane-type, and bakkenolide-type. (Siegenthaler and Neuenschwander 1996). Several Sesquiterpene lactones have been reported from the plant T. parthenium (Kashkooe et al., 2024).

Hedgehog signaling is crucial for lung development in both mammals (Kugler et al., 2015; Fernandes-Silva et al., 2017; Zeng et al., 2022) and zebrafish (Winata et al., 2009; Zhang et al., 2022). Hence, we investigated whether the phytochemicals present in Butterbur extracts inhibited the Shh signaling. This was achieved through in silico studies by evaluating the binding affinity of Butterbur-derived molecules to zebrafish Shh. The results of molecular docking studies have indicated that petasalbin, petasin, and 9,12-Octadecadienoic acid exhibited the highest binding affinity to zebrafish Shh, as compared to other compounds. These in-silico studies suggest that the absence of swim bladder formation in zebrafish embryos may be due to inhibition of Shh signaling. Previous research has highlighted a strong transcriptomic similarity between the development of the swim bladder and the lungs (Zheng et al., 2011). However, further molecular studies are needed to check the expression of zebrafish Shh in zebrafish embryos treated with either petasin (as a pure compound) or crude Butterbur extracts.

The cytotoxicity of Butterbur extracts was tested against human lung cancer and primary cell lines. The methanol and ethyl acetate fraction of P. hybridus did not induce toxicity in the tested cell lines, whereas chloroform and hexane extracts have shown cytotoxic activity in lung cancer and normal cell lines. Based on the IC ₅₀ values, Notably, the cytotoxicity was notably more prominent in non-cancerous primary cells (HUVECs), suggesting a potential lack of selectivity that warrants caution for therapeutic development. The cytotoxicity of P. hybridus’s extracts has been reported against various cancer cell lines (Kim et al., 2015; Lyu et al., 2019; Tzoneva et al., 2021; Liu and Wang, 2022; Apostolova et al., 2023) but not against lung cancer. On the other hand, the anticancer potential of Tanacetum parthenium, either its crude extracts or its active ingredient “Parthenolide,” has been reported in many cancer cell lines, including lung cancer (Kashkooe et al., 2024). However, we have not come across any in vitro or in vivo study reporting the anticancer effect herbal formula Butterbur against human lung cancer. Hence, the cytotoxicity of the Butterbur in lung cancer is a novel finding of this study as well.

5. Conclusions

In conclusion, our study reveals that solvent extracts of Butterbur can induce specific developmental toxicity in the zebrafish swim bladder, potentially via inhibition of Shh signaling, and also exhibit cytotoxicity in human lung cell lines. These findings highlight a dual nature: a potential risk for pulmonary development (particularly in pregnant patients) and a potential source of anti-cancer compounds. This underscores the critical need to rigorously evaluate the safety and biological activity of complex herbal formulations. Future research should focus on isolating and characterizing the specific compounds responsible for these effects while assessing their therapeutic potential and toxicity in higher vertebrate models of lung cancer.