Phytochemical reactions and biological activities of a traditional thai herbal formula for asthma treatment: free radical scavenging and inhibition of lung cancer cell proliferationAbstract
Lung cancer ranks as the third leading cause of cancer-related deaths in Thailand. Since there is currently no definitive cure, many researchers have turned to natural products in search of potential inhibitors of cancer cell proliferation. This study aimed at investigating the phytochemical reactions and biological activities of seven individual medicinal herbs (A–G) and a traditional Thai polyherbal formula (H). The herbs were extracted using ethanol (A1–H1) and water (A2–H2) as solvents. The resulting extracts were chemical reaction analyzed for the presence of 7 phytochemical groups. Free radical scavenging activity was evaluated using the DPPH assay. The inhibitory effect on lung cancer cell proliferation was assessed using the resazurin microplate assay. The H extracts contained all seven key phytochemical groups. However, both H1 and H2 demonstrated DPPH scavenging activity with SC50 values of 115.39 and 616.61 µg/ml, respectively. Only extracts D1, F1, and D2 had been able to inhibit the proliferation of small cell lung cancer with the Selectivity Index (SI) of 2.08, >1.29, and 7.11, respectively. Although the ethanol extract of the polyherbal formula had exhibited a relatively low level of bioactivity in this study, it also showed low cytotoxicity. Therefore, further investigations into its effects on other cancer cell lines or additional biological activities are recommended.
Keywords:
phytochemical reactions; biological activities; traditional Thai herbal
Resumo
O câncer de pulmão é a terceira principal causa de mortes relacionadas ao câncer na Tailândia. Como ainda não existe cura definitiva, muitos pesquisadores têm recorrido a produtos naturais na busca por possíveis inibidores da proliferação de células cancerígenas. Este estudo teve como objetivo investigar as reações fitoquímicas e as atividades biológicas de sete ervas medicinais individuais (A–G) e uma fórmula tradicional tailandesa de ervas (H). As ervas foram extraídas utilizando etanol (A1–H1) e água (A2–H2) como solventes. Os extratos resultantes foram analisados quanto à presença dos seguintes grupos fitoquímicos: alcaloides, terpenoides, esteroides, flavonoides, cumarinas, taninos/fenólicos e açúcares redutores. A atividade sequestradora de radicais livres foi avaliada usando o ensaio DPPH. O efeito inibitório sobre a proliferação de células de câncer de pulmão foi determinado pelo ensaio de microplacas com resazurina. Entre todas as amostras, os maiores rendimentos de extração foram obtidos a partir dos extratos etanólicos e aquosos da erva B. Ambos os extratos continham todos os sete grupos fitoquímicos principais. No entanto, os extratos da fórmula polifitoterápica H1 e H2 demonstraram atividade sequestradora de DPPH com valores de SC50 de 115.39 ± 2.16 µg/ml e 616.61 ± 3.53 µg/ml, respectivamente. Apenas os extratos D1, F1 e D2 foram capazes de inibir a proliferação de células de câncer de pulmão de pequenas células, com Índices de Seletividade (IS) de 2,08 >1.29 e 7.11, respectivamente. Embora o extrato etanólico da fórmula polifitoterápica tenha apresentado um nível relativamente baixo de bioatividade neste estudo, também demonstrou baixa citotoxicidade. Portanto, são recomendadas investigações adicionais sobre seus efeitos em outras linhagens de células cancerígenas ou em outras atividades biológicas.
Palavras-chave:
reações fitoquímicas; atividades biológicas; ervas tradicionais tailandesas
1. Introduction
Cancer can occur in people of all ages and genders worldwide, and its prevalence is continuously rising. Although cancer is a non-communicable disease, it is considered to be a serious illness due to its high mortality rate. In Thailand, cancer has been the leading cause of death. In 2022, there were approximately 140,000 new cancer cases reported—about 400 cases per day. The five most common types of cancer in Thailand have been colorectal cancer, liver and bile duct cancer, lung cancer, prostate cancer, and oral cancer. Hence, the search for effective herbal remedies for cancer treatment remains an ongoing mission among medical researchers (National Cancer Institute, 2024).
Thailand is rich in medicinal plants that have been traditionally used to treat respiratory disorders, such as Phyllanthus emblica (Indian gooseberry), Citrus aurantiifolia (lime), and Solanum trilobatum. A polyherbal formula consisting of seven medicinal plants—Cyperus rotundus L. (nutgrass), Drypetes roxburghii Wall., Cassia garrettiana Craib, Caesalpinia sappan L. (sappan wood), Schefflera leucantha R.Vig., Millingtonia hortensis (cork tree), and Derris scandens (Roxb.) Benth.—has traditionally been used to nourish the lungs, relieve cough, asthma, bronchitis, and treat tonsillitis (Wirasathien and Tangkiatkumjai, 2019; Local Thai Herbal Knowledge Center – Dr. Phorn, 2024). Although several of these individual herbs have previously been studied for their anticancer properties, the complete formula has never been investigated for its phytochemical composition or biological activities.
Therefore, this study aimed at examining the anticancer activity of the polyherbal formula used in asthma treatments, which was composed of the aforementioned seven herbs, against small lung cancer cells. The study also evaluated its antioxidant activity through DPPH radical scavenging, along with preliminary phytochemical reactions.
2. Material and Methods
2.1. Part 1: sample preparation and extraction
2.1.2. Plant sample preparation
In November of 2024, seven dried medicinal plants and one herbal formula were purchased from a traditional herbal medicine shop in the Mueang District of Phetchaburi Province, Thailand. The seven plants and the herbal formula, which were used for treating asthma, were assigned the following sample codes: A – C. rotundus, B – D. roxburghii, C –C. garrettiana, D –C. sappan, E –S. leucantha, F –M. hortensis, G – D. scandens, and H – the herbal formula consisting of a mixture of the seven plants (A–G) (Local Thai Herbal Knowledge Center – Dr. Phorn, 2024). All plant samples were authenticated by a botanist and stored at the Research Unit of Natural Products and Chemistry at the Department of Chemistry in the Faculty of Science and Technology at Phetchaburi Rajabhat University.
2.1.3. Extraction of active compounds from herbs and the formula
Two types of extracts were prepared: Firstly, there were the ethanolic extracts (A1–H1). The samples A–H were soaked in ethanol for 7 days, and this process was repeated three times. The extracts were filtered and concentrated using a rotary evaporator until a viscous, solvent-free residue was obtained. The crude ethanolic extracts were stored at 6°C for further biological activity testing. Secondly, there were the aqueous extracts (A2–H2). The samples A–H were boiled in water for 15 minutes, and the extracts were then filtered and concentrated in a water bath until a thick, water-free residue was obtained. Both the ethanolic and the aqueous extracts were stored at 6°C for subsequent phytochemical screening and biological activity evaluation.
2.2. Part 2: Phytochemical screening reactions
Prior to the preliminary screening of phytochemical groups, 50 mg of each extract was dissolved in 50 ml of its respective extraction solvent. The extracts were then subjected to qualitative phytochemical tests to determine the presence of seven major phytochemical groups: alkaloids, terpenoids, steroids, flavonoids, coumarins, tannins/phenolics, and reducing sugars (Pinoargote-Chang et al., 2025; Singchai, 2024). Qualitative results are displayed as + (presence) and − (absence) of phytochemicals.
2.3. Part 3: Bioactivity testing
2.3.1. Antioxidant activity using the DPPH radical scavenging assay
The antioxidant activity was evaluated using the DPPH radical scavenging assay based on the UV-Visible spectrophotometry technique (Singchai, 2024). The percentage of DPPH radical scavenging activity was calculated using the following Equation 1:
In which: As is the absorbance of the sample or standard solution and Ab is the absorbance of the solvent control. Any samples that had exhibited more than 50% scavenging activity were further tested to determine their SC50 values by performing triplicate assays using serial dilutions. Ascorbic acid (vitamin C) was used as the positive control.
2.3.2. Anti-proliferative activity against small lung cancer cells (NCI-H187)
The anti-proliferative activity against NCI-H187 small lung cancer cells was evaluated by experts at the Bioactive Compound Screening Laboratory of the National Center for Genetic Engineering and Biotechnology, using the Resazurin Microplate Assay (REMA) (Brien et al, 2000). The cancer cells were cultured in 96-well plates containing complete RPMI-1640 medium, which had been supplemented with 15% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 2.5 g/L glucose, and 2.2 g/L sodium bicarbonate. The cells were incubated at 37°C in a 5% CO2 atmosphere. Subsequently, 6.7 × 104 cells were transferred to 384-well culture plates, and 5 μl of the extract at a concentration of 50 μg/ml was added, followed by 45 μl of cell suspension. The plates were then incubated under optimal conditions (37°C, 5% CO2) for 5 days. After incubation, 0.0625 μl of resazurin solution was added, and the plates were incubated again at 37°C in 5% CO2 for 4 hours.
The fluorescence was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm, using the bottom-reading mode of a SOFT Max fluorometer (Molecular Devices, USA). The experiment was performed in duplicate, and the cell viability was calculated.
Doxorubicin and ellipticine were used as positive controls, while dimethyl sulfoxide (DMSO) served as the negative control. The percentage of cell growth inhibition was calculated using Equation 2:
In which: FUT and FUC are the average fluorescence intensities per well for the test extract or the positive control and the negative control, respectively.
The selectivity index (SI) indicates the cytotoxic selectivity of the extracts against small lung cancer cells (NCI-H187) versus normal cells (vero cell). SI was IC50 of plant extracts in a vero cell line divided by IC50 of the same plant extract in small lung cancer cell line (Krzywik et al., 2020).
2.4. Statistical analysis
The basic data was analyzed using descriptive statistics, including percentages, means, and standard deviations. All experiments were performed in triplicate. The differences between the groups were compared using the t-test, with a level of statistical significance of 0.05.
3. Results
Seven dried single herbal components (A–H), which are used in the formulation for asthma treatment, were investigated. In this formula, Herb E was the main component, accounting for 33.33%, followed by Herb C at 25.00%. The remaining five herbs contributed equally to the formulation, each comprising 8.33%.
The herbs A to G and the traditional formula (H) were extracted using ethanol and water. Among the ethanol extracts, B1 yielded the highest extractive value at 20.66±0.02% wt/wt. For the aqueous extracts, B2 showed the highest extractive value at 22.44±0.05% wt/wt. The extractive yields of both the ethanol and aqueous extracts showed statistically significant differences at the level of 0.05, as shown in Table 1.
The analysis of the 7 chemical groups found in the ethanol extracts (A1 to H1) revealed the presence of important chemical constituents in all 7 groups, including alkaloids, terpenoids, steroids, flavonoids, coumarins, tannins/phenolics, and reducing sugars, except for B1, which did not contain flavonoids. With regard to the aqueous extracts, B2, C2, D2, F2, and G2 exhibited all the chemical groups, while A2 contained all groups except for alkaloids and terpenoids, as shown in Table 2.
In this study, several ethanol extracts showed the ability to scavenge DPPH free radicals. Extract C1 exhibited the highest potential to scavenge DPPH free radicals, with the lowest SC50 value of 25.57±0.28 μg/ml, which is lower than that of vitamin C (as shown in Table 3). The scavenging activity of all the extracts was calculated using the standard calibration curve with an R2 value of the linear line approaching zero, ranging between 0.9804 and 0.9976. Furthermore, the scavenging activity of the water and ethanol extracts showed significant statistical differences at the level of 0.05, as shown in Table 3.
The test for anti-small lung cancer cell activity in this study found that only extracts D1, F1, and D2 had exhibited more than 50% inhibition of cancer cell growth at a concentration of 50 μg/ml. The extracts, D1 and D2, were able to inhibit the growth of lung cancer cells with IC50 values of 2.87 and 2.66 μg/ml, respectively, and exhibited cytotoxicity against normal cells with IC50 values of 5.96 and 18.90 μg/ml, respectively. The extract F1 inhibited the growth of lung cancer cells with an IC50 value of 38.72 μg/ml and exhibited cytotoxicity against vero cells with an IC50 value greater than 50 μg/ml. The positive control substances were doxorubicin and ellipticine, with IC50 values for inhibition of lung cancer cell growth of 0.0497 and 0.842 μg/ml, respectively (as shown in Table 4).
4. Discussion
The traditional Thai herbal remedy used to nourish the lungs, relieve cough, and treat asthma consisted of seven individual herbs Local Thai Herbal Knowledge Center – Dr. Phorn, 2024), which were A through G. In some formulas, which were specifically used to treat asthma, herb F was excluded from the composition (Pitiphorn, 2002). However, in this study, the researcher focused on a formula in which all seven herbs had been included, with herb E as the main component, accounting for 8.33% of the formulas (as shown in Table 1). This proportion was determined to be consistent with a boiled asthma remedy used by traditional herbalists in Nakhon Si Thammarat Province, Southern Thailand. Additionally, it was found that herb E made up 50% of the boiled herbal remedy used for asthma treatment by local healers in Surat Thani Province, Southern Thailand (Jitjum et al., 2019). Similarly, in China, herb E is also used to relieve coughing and treat asthma (Deepa et al., 2024).
The seven groups of compounds tested in this study comprised alkaloids, terpenoids, steroids, flavonoids, coumarins, tannins/phenolics, and reducing sugars. This research presents examples of specific reactions for each compound group. Alkaloids were found in all plant samples extracted with both ethanol and water, except for A2, which was the only extract that did not form an orange precipitate or a dark brown color when tested with Dragendorff’s reagent. However, the compound, Rotundine A, was detected in the A1 extract, which showed a positive reaction with Dragendorff’s reagent (Kandikattu et al., 2021).
In this study, it was found that all ethanol and aqueous extracts (Deachathai, 2016, Deepa, et al., 2024; Babiaka et al., 2021), except for A2, had not contained terpenoids as one of their components. Testing with concentrated sulfuric acid in acetic acid resulted in observable color changes—ranging from colorless to blue or green in the presence of steroids, and red or reddish-brown when terpenoids were present. An example of a compound that gave a positive result with this reagent was β-sitosterol, which was found in extract E1. This reaction is reported (Xiong et al., 2007; Sarath and Sudha, 2019).
All extracts contained flavonoids as one of their components. However, in the case of extract B1, when tested using concentrated hydrochloric acid with magnesium metal as a catalyst, the expected red or reddish-pink coloration did not appear. A positive example of this test was observed in extract A1, which contained the flavonoid, Quercetin. The reaction is reported (Babiaka et al., 2021). In addition, when reacted with the base sodium hydroxide (NaOH) and heated to stimulate the reaction, only the D extract produced a red-colored compound. This color change is a characteristic chemical feature of the main active compound known as brazilein, a red pigment. This is a chemical hallmark of the conjugated tetraring phenolic group (Rajput et al., 2022). When it reacts with NaOH, it forms a red compound known as mono sodium conjugated tetraring phenolic (Madhiri and Panda, 2018).
All extracts contained coumarins as one of their components. When tested with sodium hydroxide solution (NaOH) and subjected to heat, fluorescence was observed. For example, the G1 extract contained scandenin, which exhibited a blue fluorescence under this test. This reaction is a chemical characteristic that is specific to the coumarin group (Poorna et al., 2020). The reaction resulted in the formation of a trans-conjugated aromatic compound (López-Castillo et al., 2013). When ferric chloride (FeCl3) was added to all extracts, the resulting solutions turned dark blue, dark brown, or deep brown. These color changes indicated the presence of tannins or phenolic compounds. For instance, the A1 extract contained Afzelechin, a phenolic compound (Babiaka et al., 2021). When the tests were conducted with ferric chloride, a dark brown solution was produced, which corresponded to the formation of an iron-phenolic complex (Abdelfatah et al., 2021).
The extracts H1 and H2, obtained using different solvents, showed a statistically significant difference (p < 0.05) in the percentage yield of extract per weight of the dried plants. The ethanol extracts yielded a significantly higher amounts of extract compared to the water extracts. This trend was consistent with most individual herbal components, except for extract B, in which the aqueous extract yielded significantly more than the ethanol extract at the significance level of 0.05. The main herbal component in this formulation was herb E, followed by herb C, with the percentages of extract-to-dry weight at 33.33% and 25.00%, respectively. Both herbs yielded significantly more extract with ethanol compared to water (p < 0.05). Moreover, both the H1 extract and the H2 extract were found to contain all seven phytochemical groups, as summarized in Table 2.
The findings of this study indicated that the ethanol extract C1 had exhibited the highest DPPH radical scavenging activity, with an SC50 value of 25.57 ± 0.28 μg/ml (as shown in Table 3), which was statistically significantly different (p < 0.05) than the aqueous extract C2. This suggested that the bioactive compounds responsible for this activity possess a moderate polarity and are likely to be poorly soluble or insoluble in water. Additionally, this group of ethanol-extracted compounds demonstrated HIV-1 PR inhibitory activity, with an IC50 value of 15.6 μg/ml (Bunluepuech et al., 2016). However, in terms of anti-cancer potential, both ethanol extract C and water extract C exhibited relatively low inhibition rates against lung cancer cells at a concentration of 50 µg/ml, with inhibition rates of 19.70% and 17.74%, respectively.
The D2 extract, which was water-based, exhibited the highest DPPH radical scavenging activity among the water extracts of the other herbs, with an SC50 value of 50.24 ± 2.23 μg/ml (as shown in Table 3), which was significantly different (p < 0.05) from D1 (with an SC50 of 46.19 ± 1.11 μg/ml). Although the activity was nearly two times lower than Ascorbic acid, it is noteworthy that the methanol extract of D, as previously reported, showed 80% DPPH radical scavenging at 100 μg/ml (Artati et al., 2025). The results of this study aligned with those earlier findings for the methanol extract of D (Kankamol, 2021).
Moreover, both the D1 and D2 extracts showed growth inhibition of lung cancer cells, with Selectivity Index (SI) values of 2.08 and 7.11, respectively (as shown in Table 4). A higher SI value indicated greater safety to normal cells, while also exhibiting anti-cancer activity. The SI was calculated by dividing the IC50 of the extract against vero cell line by the IC50 of the same extract against the lung cancer cells (Krzywik et al., 2020). The results were consistent with the National Cancer Institute's classification of D1 and D2 as moderately toxic to cancer cells, since their log IC50 values fall within the range of 0 < log IC50 < 1.10 (Lumlerdkij et al., 2020). Additionally, the water and methanol extracts of plant D that had been previously reported at 50 µg/ml demonstrated 81.06 ± 0.29% and 100 ± 0.00% growth inhibition against lung cancer cells, respectively (Bukke et al., 2018; Kankamol, 2021). Currently, plant D is still undergoing medical research (Pashmina et al., 2024; Vij et al., 2023).
Although the F1 extract has not been extensively studied in scientific research (Janai et al., 2017, Khobjai, et al., 2018; Ashokkumar et al., 2025), this study found that the SC50 values of both the ethanol and aqueous extracts were greater than 1,000 µg/ml. The inhibition of lung cancer cell growth was also noteworthy. The F1 extract showed a log IC50 of 1.59 and a Selectivity Index (SI) > 1.29, indicating moderate biological activity in inhibiting lung cancer cells, in accordance with the National Cancer Institute's criteria for cancer cell toxicity (Lumlerdkij et al., 2020).
The A1 extract demonstrated very low DPPH radical scavenging activity with an SC50 of 585.35±11.91 µg/ml. This was consistent with previously reported values for 80% methanol and 80% ethanol extracts at the concentration of 500 µg/ml, which showed radical scavenging percentages of 61.30% and 43.98%, respectively (Singh, 2024). Additionally, extract A has been studied for various biological activities, including its ability to inhibit different cancer cell types (Singh, 2024). In this study, the A1 and A2 extracts only demonstrated about 20% inhibition of lung cancer at an initial concentration of 50 µg/ml, making it less promising for further investigation in inhibiting this type of cancer.
Previous reports have shown that a hydrocarbon ester extracted from the methanol extract of the leaves of E was able to inhibit the growth of lung cancer cells with an IC50 of 59.82±1.60 µg/ml (Ramakrishnan et al., 2022). This was determined to be consistent with the current study, in which the E1 extract had an IC50 greater than 50 µg/ml. Meanwhile, the cancer cell inhibition properties of the five other herbs, namely A, B, C, F, and G, as well as the H formulation, have not been extensively reported. In this study, the ethanol extracts (A1, B1, C1, E1, G1, H1) and the water extracts (A2, B2, C2, E2, F2, G2, H2) of the herbs were tested at a concentration of 50 µg/ml, but showed inhibition values of less than 50%, so the IC50 values were not determined. This study investigated the inhibition of lung cancer cell growth at various concentrations: 0.62, 1.85, 5.56, 16.67, and 50 µg/ml (see Figure 1). Regarding the H1 and H2 herbal formulations used for treating the lungs and asthma, the inhibition percentages at 50 µg/ml were 42.31% and 9.38%, respectively. These results correlated with the inhibition of lung cancer cell growth of the individual herbs studied, particularly because the primary components in the formulation were E and C. The ethanol extract showed a higher inhibition rate compared to the water extract, but both plants still had inhibition rates of less than 50%. Furthermore, since B contributed the highest weight percentage in both the ethanol and aqueous extracts, it is possible that the H1 and H2 formulations contained the highest chemical components from B, which demonstrated less than 50% inhibition at 50 µg/ml. This suggests that the cancer cell inhibition, which was observed in H1, may largely be attributed to the important components from the D1 and F1 extracts.
The results of the inhibition of small lung cancer cell (NCI-H187) growth and vero cells of extracts at varies concentration.
Although extracts A, B, F, and G have been reported to inhibit the growth of various types of cancer cells, such as leukemia (K562 and L1210 cells), lymphoma (L5178 cells), cervical cancer (HeLa and SiHa cells), breast cancer (Ehrlich’s ascites cells, MDA-MB 231), oral cancer (KB cells), pancreatic cancer (PANC-1), colon cancer (HCT-116, HCT15, DLD-1, SW48, SW480), and liver cancer (HCC S102) (Simorangkir et al., 2019; Balkrishna et al., 2020; Mohotti et al., 2020; Yuenyongsawad et al., 2014; Mannarreddy et al., 2017), there have been fewer reports on their inhibitory effects on lung cancer cell growth. Moreover, this herbal formulation has not been previously studied for its phytochemical properties and biological activities. This highlights the importance of the current study, since it explores the phytochemical composition and biological efficacy of this herbal remedy, especially its potential to inhibit lung cancer.
The Thai herbal formulation for treating lung conditions and asthma studied in this research primarily consisted of E (S. leucantha), with C as the secondary plant component, along with five other single herbs. The ethanolic extract (H1) of this formulation contained seven bioactive chemical groups that had been previously reported to exhibit biological activities. Notably, extracts C1, D1, and F1 showed significant biological activities, including DPPH scavenging and the inhibition of small lung cancer cell growth. Consequently, H1 also demonstrated these bioactivities, albeit at lower levels. Even though the water extract (H2) did not show significant biological activity in these areas, both H1 and H2 might possess bioactivity in other aspects (Simorangkir et al., 2019; Balkrishna et al., 2020; Ashokkumar et al., 2025; Artati et al., 2025; Yuenyongsawad et al., 2014; Mannarreddy et al., 2017). Therefore, further investigation is warranted in order to explore other potential effects.
Acknowledgements
This research, which was funded under the Science, Research, and Innovation budget from the Science, Research, and Innovation Promotion Fund for the fiscal year of 2025 (Contract number: W.W.N-038-2568), was conducted at the Division of Chemistry, Faculty of Science and Technology at Phetchaburi Rajabhat University.
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Data Availability Statement
The entire data set that supports the results of this study was published in the article itself.
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Edited by
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Editor:
Marcelo A. M. Esquisatto
The entire data set that supports the results of this study was published in the article itself.
