Potential natural hydrosol blend TGLON suppresses the proliferation of five cancer cell lines and also ameliorates idiopathic pulmonary fibrosis in mouse model

Potential natural hydrosol blend TGLON suppresses the proliferation of five cancer cell lines and also ameliorates idiopathic pulmonary fibrosis in mouse model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Potential natural hydrosol blend TGLON suppresses the proliferation of five cancer cell lines and also ameliorates idiopathic pulmonary fibrosis in mouse model Wei-Hsiang Huang, Mei-Lin Chang, Ching-Che Lin, Chih-Peng Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4440611/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Previous studies on natural substances for tumor cell inhibition have predominantly concentrated on efficacy, often overlooking safety concerns. This research utilizes "The Greatest Love of Nature (TGLON), a proprietary blend of plant hydrosols, to investigate its potential inhibitory effects on a variety of cancer cell lines while ensuring its relative safety to normal lung cells (MRC-5). Furthermore, initial animal studies have demonstrated that TGLON can mitigate the progression of idiopathic pulmonary fibrosis without acute oral toxicity, underscoring its potential utility as a nutritional supplement. Methods: TGLON, a hydrosol blend developed with specific proportions, underwent initial validation for its constituent composition and safety profile. The blend capacity to inhibit five different tumor cell types and alleviate pulmonary fibrosis was evaluated using cell viability assays and controlled animal experiments. Results: Dosages were determined to be safe at dilutions greater than 80-fold. At this concentration, TGLON exhibited inhibitory effects on 40.7% of lung cancer cells, 84% of liver cancer cells, 49.8% of breast cancer cells, 38% of stomach cancer cells, and inhibited 52% of leukemic lymphoblasts at a 200-fold dilution. Oral administration of TGLON was found to be safe and effective in reducing Bleomycin-induced pulmonary fibrosis in animal models. Conclusions: Under conditions deemed relatively safe, TGLON demonstrates bioactive properties, such as inhibiting five distinct types of human tumor cells and alleviating pulmonary fibrosis in animal models. These findings highlight its substantial potential as a nutritional supplement for improving overall patient health. hydrosol blend TGLON cancer cells reduce pulmonary fibrosis natural products Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction An analysis of cancer incidence rates from 185 countries has revealed significant patterns in prevalence: lung cancer rates stand at 14.5% in men and 8.4% in women; liver cancer is present in 6.3% of men; breast cancer incidence is at 24.2% in women; and stomach cancer occurs at rates of 7.2% in men and 4.1% in women. Notably, the occurrence of these cancers is often associated with hematological malignancies, such as lymphoblastic leukemia, which disrupt the normal blood milieu and hematopoietic functions, potentially reducing immune system efficacy and increasing susceptibility to pathogenic infections [ 1 ]. During the Coronavirus Disease 2019 (COVID-19) global pandemic, this virus, which primarily targets lung epithelial cells via the Angiotensin-converting enzyme (ACE) 2 receptor, was observed to replicate rapidly post-infection, affecting additional lung cells. Such a process can lead to acute complications, including Idiopathic Pulmonary Fibrosis (IPF), a condition characterized by the excessive synthesis and pathological accumulation of Extracellular Matrix (ECM) proteins in the alveoli, ultimately resulting in pulmonary fibrosis [ 2 – 4 ]. In our study, we meticulously selected aromatic plants rich in volatile secondary metabolites, leveraging their natural growth methods that eschew the need for pesticides, thereby reducing soil environmental impact and supporting sustainable development efforts in preserving terrestrial ecosystems [ 5 , 6 ]. Investigations into the extracts of these plants have indicated their efficacy in moderating the progression of IPF [ 7 , 8 ]. Additionally, these extracts have shown significant anti-proliferative properties against various cancer cell lines. For instance, Cymbopogon Nardus (CN) has exhibited marked cytotoxicity against prostate cancer cells [ 9 ]. Litsea Cubeba (LC) has demonstrated potent cytotoxic activities against human cancers of the lung, liver, breast, and oral cavity [ 10 , 11 ]. Chamaecyparis Formosensis (CF) has been found to be cytotoxic to human liver cancer cells [ 12 ]. Calocedrus Formosana (CAF) has shown notable effectiveness against human lung and bladder cancer cells [ 13 , 14 ]. Cinnamomum Camphora (CC) has targeted colon cancer cells with significant cytotoxic effects [ 15 ]. Eucalyptus Robusta smith (ER) has exerted cytotoxicity against human pancreatic cancer cells [ 16 ]. Similarly, Cinnamomum Zeylanicum (CZ) has been active against human liver and breast cancer cells [ 17 , 18 ]. Cunninghamia Lanceolata (CL) has displayed cytotoxic activity against both human lung and liver cancers [ 19 ]. Melaleuca Alternifolia (MA) has proven effective against lung, breast, and prostate cancer cells [ 20 ]. Cinnamomum Micranthum (CM) has shown cytotoxic effects on murine leukemia cells [ 21 ]. Cryptomeria Japonica (CJ) has revealed cytotoxic effects against human oral and lung cancer cells [ 22 , 23 ]. Acacia Confusa (AC) has been identified as cytotoxic to human breast cancer cells [ 24 ]. It is important to note that while these aromatic plants are recognized for their anti-cancer properties, their potential toxicity to normal cells necessitates careful consideration. For example, essential oils from Melaleuca alternifolia, at a 14,000-fold dilution, have been shown to inflict substantial damage, up to 50%, on normal human lung cells [ 25 ]. Consequently, identifying an optimal dosage that effectively inhibits cancer cell growth while preserving the integrity of normal cells has become a pivotal aspect of our research. Recently, there has been an increasing focus on the study of Essential Oil Blends, particularly exploring their antibacterial, antifungal, and antiviral properties, as well as their potential in alleviating diarrhea in animal models. This paradigm shift marks a significant advance in innovative research, suggesting that meticulous control in processing and formulation may enhance the safety profiles of these products [ 26 , 27 ]. However, it is important to note in our research that, while volatile secondary metabolites from aromatic plants are concentrated in essential oils through steam distillation, they may still pose considerable risks to normal cells. Therefore, our investigation has shifted toward exploring hydrosol products derived from steam distillation. We have developed a specific hydrosol blend, named The Greatest Love of Nature (TGLON), representing a pioneering effort in using hydrosol blends to study tumor cell inhibition, acute toxicity, and the attenuation of IPF. Methods Preparation of hydrosol blend and chemicals In this research, a diverse array of plant species was cultivated in the forested and agricultural regions of Taiwan, at altitudes spanning 500 to 1000 meters. Hydrosols were extracted from a variety of plants utilizing steam distillation. These plants included CN, LC, CF, CAF, CC, ER, CZ, CL, MA, CM, CJ, and AC, and the extracts were used to design a novel hydrosol mixture named TGLON. The formulation of TGLON is strategically based on the anti-cancer properties of its key constituents such as Terpinen-4-ol, Camphor, and δ-Cadinene. Terpinen-4-ol, a primary compound in the monoterpenol category, is particularly noted for its efficacy in inhibiting the proliferation of tumor cells. To optimize the therapeutic potential, TGLON was meticulously formulated, focusing predominantly on monoterpenols while ensuring a balanced integration of sesquiterpenes, other oxides, monoterpenes, sesquiterpenols, and ketones, and simultaneously minimizing the content of irritating aldehydes. The specific formulation ratios are CN 8%, LC 6%, CF 40%, CAF 10%, CC 8%, ER 6%, CZ 2%, CL 4%, MA 2%, CM 10%, CJ 2%, and AC 2%. This proprietary hydrosol blend is slated for further investigative research to assess its ability to inhibit tumor cell growth and mitigate the progression of IPF under more controlled and safer experimental conditions [ 28 – 31 ]. In this study, a variety of human cell lines were sourced, encompassing MRC-5 lung cells, A-549 lung cancer cells, HepG2 liver cancer cells, MCF-7 breast cancer cells, MKN-45 stomach cancer cells, and MOLT-4 Leukemic Lymphoblasts. These were obtained from the Bioresource Collection and Research Center (BCRC) in East Dist, HSZ, TWN, ensuring the authenticity and viability of the cells for research purposes. Additionally, all chemicals employed in our analysis were of analytical reagent grade, acquired from ECHO CHEMICAL CO., LTD., Toufen City, ZMI, TWN. This ensured the highest level of purity and consistency in our experimental procedures. Throughout the various stages of analysis, ultrapure water was utilized exclusively, guaranteeing minimal interference from contaminants and thus maintaining the integrity of our experimental results. Gas chromatography mass spectrometry (GC-MS) Revise according to Adams description of the literature [ 32 ]. Using a GC7890, followed by a 5977B MSD (Agilent Technologies, Inc., Santa Clara, CA, USA), which functions as a quadrupole mass spectrometer. The injection port operates in split mode at a constant temperature of 300°C. The chromatographic separation column is a DB-5 MS, with a length of 30 m, an inner diameter of 0.25 mm, and a film thickness of 0.25 µm (Agilent Technologies, Inc., Santa Clara, CA, USA). The carrier gas is helium with a purity of ≥ 99.999%, and the flow rate is set at 1 mL/min. The temperature ramp conditions are from 50°C to 280°C, with a ramp rate of 3°C per minute, followed by a 5-minute isothermal hold. For the calculation of the Kovats Index (KI), we meticulously recorded the Retention Times (RT) of both the test sample and the C8-C40 alkane standard mixture. The mass spectrometer operates under Electron-Impact Ionization (EI) at 70 eV. Mass spectral data acquired through the analysis utilizing the 2.64 Automated Mass Spectral Deconvolution and Identification System (AMDIS) developed by the National Institute of Standards and Technology (NIST). Identification is accomplished by comparing the GC-MS spectral data and KI of polar and nonpolar compounds with reference compounds from the NIST database. In vitro cytotoxicity assay Improving methodology according to Alley et al. [ 33 ]. We initially seeded 3000 cells into each well of a 96-well culture plate. These wells were each supplemented with 100µL of culture medium and incubated for a period of 24 hours. Following the incubation, medium was removed, and test samples were introduced into the wells at various dilution factors: 20X, 40X, 80X, 160X, 320X, 640X, 1280X for the MRC-5, A-549, HepG2, MCF-7, and MKN-45 cell lines, and 20X, 200X, 2000X, 20000X, 40000X, 80000X, 200000X for the MOLT-4 cell line. These were then incubated for an additional 72 hours. Post-incubation, the culture medium was carefully aspirated, and each well was washed using 150µL of Phosphate Buffered Saline (PBS). To each well, 100µL of MTT reagent (0.5 mg/mL) was added. The wells were then covered with aluminum foil to prevent light exposure and placed in a CO2 incubator for a duration of 30 minutes. Following this incubation step, the MTT reagent was aspirated, and the wells were once again washed with PBS. This was succeeded by the addition of 100µL of Dimethyl Sulfoxide (DMSO) per well, facilitating the dissolution of formazan crystals. After ensuring thorough mixing of the contents, the absorbance at 540 nm wavelength was quantitatively assessed using an ELISA reader. In vivo Acute Oral Toxicity Study In this meticulously conducted study, Crl: CD Sprague Dawley (SD) rats provided by BioLASCO Taiwan Co., Ltd., Nangang District, TPE, TWN, were selected as the model organisms. The rats were chosen based on specific criteria: age (8 to 9 weeks), with male rats in the weight range of 260 to 300 grams and female rats between 180 to 230 grams, to establish a uniform baseline for our research. The preference for SD rats was due to their demonstrated responsiveness to a wide range of toxicological agents, rendering them suitable for our toxicological studies. Ethical considerations took precedence in our study, ensuring all animal experiments adhered to the highest standards. Our protocol received thorough evaluation and approval from the Institutional Animal Care and Use Committee (IACUC), designated with the IACUC number 2020-R501-051 and approval date of December 28, 2020. We adhered rigorously to the internationally recognized guidelines for the care and use of laboratory animals, ensuring ethical and responsible conduct in our scientific research. before commencing the study, all rats were subjected to a mandatory quarantine and acclimatization period of no less than three days within our animal facility. Regarding their housing, we placed three rats in each polycarbonate cage. These cages were meticulously labeled with essential details such as group number, study number, dosage level, sex, animal ID, and scheduled euthanasia date. We maintained controlled environmental conditions, with room temperature set at 22 ± 3°C and relative humidity at 50 ± 20%, including a 12-hour automated light/dark cycle (lights on at 7:00 am, off at 19:00 pm). The rats diet consisted of PicoLab® Rodent Diet 20 (sourced from LabSupply Inc., St. Louis, MO, USA), and they had continuous access to sterilized tap water. Detailed housing conditions are depicted in Fig. 1 . Throughout the study, the rats received a daily oral administration of a 50% (v/v) TGLON solution or Water for injection (WFI) oral administration, each at a volume of 10 mL. We conducted observations on Days 1, 8, and 15, focusing on changes in average weight, potential mortality, or any clinical signs. The study culminated on Day 15 with a comprehensive dissection to examine for any additional anatomical damages [ 34 ]. In vivo Pulmonary Fibrosis Study In this rigorously designed study, we employed 18 male C57BL/6 mice, supplied by BioLASCO Taiwan Co., Ltd., Nangang District, TPE, TWN, as our biological models. These mice were specifically selected based on their age range of 8 to 9 weeks and a weight bracket of 20 to 25 grams, to maintain uniformity in our research baseline. The animal experimentation protocol underwent a stringent evaluation and received approval from the IACUC, assigned the approval number 2021-R501-049, with an approval date of January 14, 2021. This procedure ensured adherence to the highest ethical standards. Each mouse was uniquely identified using ear tags and corresponding cage numbers. for housing, the mice were accommodated in polycarbonate cages, with a grouping of three mice per cage. These cages were outfitted with identification cards that meticulously recorded details such as cage number, trial number, dosage group, sex, and animal identification number. We meticulously maintained environmental conditions at a stable room temperature of 22 ± 3°C and a relative humidity of 50 ± 20%. The light exposure was controlled through a 12 hour automatic light/dark cycle, with illumination periods from 7:00 am to 19:00 pm. The dietary needs of the mice were met with PicoLab® Rodent Diet 20 (provided by LabSupply Inc., St. Louis, MO, USA) and were freely accessible throughout the duration of the study. Additionally, sterilized tap water was supplied ad libitum in their cages. for detailed housing conditions, refer to Fig. 2 and Tables 1 – 2 . Table 1 Treatment information of IPF model. No. Sample Pre-treatment (Day − 14 ~ -1) Bleomycin a Induction (Day 0) Treatment (Day 0–20) Necropsy (Day 21) 1 Saline Oral, daily None Oral, daily 6 Males 2 Saline Oral, daily Single dose, IT Oral, daily 6 Males 3 Test article Oral, daily Single dose, IT Oral, daily 6 Males a Bleomycin (3U/kg) administration intratracheally (IT) on day 0. Test article: TGLON. IPF, Idiopathic Pulmonary Fibrosis. Table 2 Test article and vehicle control administration. Items Controls Route Oral Gavage (PO) Frequency Once daily Dose 5 mL/kg of 100% Test article Concentration of dosing solution 50% (v/v) diluted in saline Dosing Volume 10 mL/kg Test article: TGLON. PO, per os. In the IPF experiment, each mouse initially received an oral dose of either 50% (v/v) TGLON solution or 10mL Saline daily for a period of 2 weeks. This was followed by an intratracheal administration of 50µL Bleomycin (3U/kg) to induce IPF. Post-induction, the oral administration of TGLON 50% (v/v) or Saline 10mL was continued for an additional 3 weeks, culminating in dissection to evaluate the resultant effects [ 35 , 36 ]. Statistical Analysis The mean values, ± standard deviations were calculated using MS Excel 2007. Data was analysed by using Analysis of Variance (ANOVA) and differences among the means were deter- mined for significance at p < 0.05 and p < 0.01 using Duncan multiple range test by SPSS (version 16.0). Results GC-MS analysis of TGLON Please refer to Tables 3 – 4 . For details on the 23 chemical compounds identified by TGLON. These compounds are listed in descending order by area percentage as follows: Terpinen-4-ol (10.72%), Camphor (8.73%), δ-Cadinene (7.58%), α-Terpineol (6.93%), Safrole (6.87%), 1,8-Cineole (6.08%), cis-Myrtanol (4.22%), (-)-Myrtenol (3.94%), γ-Terpinene (3.17%), α-Muurolene (2.9%), tau-Cadinol (2.82%), α-Cedrene (2.47%), α-Pinene (2.46%), tau-Muurolol (2.46%), α-Cedrol (2.16%), γ-Muurolene (2.15%), D-Limonene (1.93%), α-Terpinene (1.54%), α-Elemol (1.45%), para-Cymene (1.42%), Borneol (1.12%), β-Citronellal (1.11%), and β-Elemene (0.94%). Additionally, these compounds have been further categorized and sorted by area percentage from highest to lowest: monoterpenols (26.93%), sesquiterpenes (16.04%), other oxides (12.95%), monoterpenes (10.52%), sesquiterpenols (8.89%), ketones (8.73%), and aldehydes (1.11%). Table 3 GC-MS analysis of TGLON. PK # Constituent Type KI RT % Area Method of Identification 1 α-Pinene Monoterpene 939 9.606 2.46 MS;KI;RC 2 α-Terpinene Monoterpene 1017 12.221 1.54 MS;KI 3 para-Cymene Monoterpene 1024 12.455 1.42 MS;KI;RC 4 D-Limonene Monoterpene 1029 12.603 1.93 MS;KI;RC 5 1,8-Cineole Other oxide 1031 12.669 6.08 MS;KI;RC 6 γ-Terpinene Monoterpene 1059 13.552 3.17 MS;KI 7 Camphor Ketone 1146 16.174 8.73 MS;KI;RC 8 β-Citronellal Aldehyde 1153 16.412 1.11 MS;KI 9 Borneol Monoterpenol 1169 16.83 1.12 MS;KI;RC 10 Terpinen-4-ol Monoterpenol 1177 17.177 10.72 MS;KI;RC 11 α-Terpineol Monoterpenol 1188 17.555 6.93 MS;KI;RC 12 (-)-Myrtenol Monoterpenol 1195 17.734 3.94 MS;KI 13 cis-Myrtanol Monoterpenol 1253 19.559 4.22 MS;KI 14 Safrole Other oxide 1287 20.361 6.87 MS;KI 15 β-Elemene Sesquiterpene 1390 23.258 0.94 MS;KI 16 α-Cedrene Sesquiterpene 1411 23.86 2.47 MS;KI 17 γ-Muurolene Sesquiterpene 1479 26.013 2.15 MS;KI 18 α-Muurolene Sesquiterpene 1500 26.373 2.9 MS;KI 19 δ-Cadinene Sesquiterpene 1523 26.569 7.58 MS;KI 20 α-Elemol Sesquiterpenol 1549 27.173 1.45 MS;KI 21 α-Cedrol Sesquiterpenol 1601 28.519 2.16 MS;KI;RC 22 tau-Cadinol Sesquiterpenol 1640 29.365 2.82 MS;KI 23 tau-Muurolol Sesquiterpenol 1642 29.663 2.46 MS;KI PK #, Peak Number; RT, Retention Times; KI, Kovats Index; MS, mass spectrum; RC, reference compound. Table 4 Analogs of TGLON Type % Area Sesquiterpenes 16.04 Monoterpenes 10.52 Monoterpenols 26.93 Sesquiterpenols 8.89 Ketones 8.73 Aldehydes 1.11 Other oxides 12.95 Viability of TGLON in MRC-5, A-549, HepG2, MCF-7, MKN-45, and MOLT-4 cells Please refer to Fig. 3 A for the survival rates of MRC-5 normal lung cells across a range of dilutions. The survival rate at a 20-fold dilution was 2.7%; at 40-fold, it increased to 40.9%; at 80-fold, it rose to 95.4%; at 160-fold, it reached 99.2%; at 320-fold, it was 98%; at 640-fold, it approached 99.1%; and at 1280-fold, it exceeded 100%, registering at 101%. Establishing a benchmark survival rate of 90% or higher, dilutions of TGLON above 80-fold are deemed safe for MRC-5 normal cells. Consequently, for tumor cells, a similar standard will apply, setting the survival rate benchmark at dilutions greater than 80-fold. Please refer to Fig. 3 B for the viability of A-549 lung cancer cells at varying dilutions. At an 80-fold dilution, the cell survival rate was 59.3%, indicating an inhibition of 40.7%. At 160-fold, the survival rate increased to 77%, with a 23% inhibition. At 320-fold, it reached 84%, with a 16% inhibition. At 640-fold, the survival rate was 94.1%, with a 5.9% inhibition, and at 1280-fold, it was 93.2%, with a 6.8% inhibition. For HepG2 liver cancer cells, please consult Fig. 3 C. At an 80-fold dilution, the survival rate was 16%, with an inhibition of 84%. At 160-fold, it increased to 28.3%, with an inhibition of 71.7%. At 320-fold, the survival rate was 63.5%, with a 36.5% inhibition. At 640-fold, it rose to 96.4%, with a 3.6% inhibition, and at 1280-fold, it was 93.8%, with a 6.2% inhibition. Regarding the viability of MCF-7 breast cancer cells, refer to Fig. 3 D. At an 80-fold dilution, the survival rate was 50.2%, demonstrating a 49.8% inhibition. For the viability of MKN-45 stomach cancer cells, consult Fig. 3 E. At an 80-fold dilution, the survival rate was 62%, with a 38% inhibition. At 160-fold, it was 72%, with a 28% inhibition. At 320-fold, the survival rate improved to 79%, with a 21% inhibition. At 640-fold, it reached 83%, with a 17% inhibition. Please consult Fig. 3 F for the viability analysis of MOLT-4 leukemic lymphoblasts. Unlike tumor cells originating from solid organs, MOLT-4 cells undergo malignant proliferation within the human body, leading to malignant disorders of the hematologic and bone marrow systems, which are closely associated with leukemia. At a 200-fold dilution, the survival rate of these cells was 48%, reflecting a 52% inhibition. At a 2,000-fold dilution, the survival rate improved to 64%, indicating a 36% inhibition. 72% at 20,000-fold with a 28% inhibition, 85% at 40,000-fold with a 15% inhibition, and 92.9% at 80,000-fold with a 7.1% inhibition. Acute toxicity assay of TGLON in rats Please refer to Table 5 . Over a two-week period, oral administration of a 50% (v/v) TGLON solution at a dosage of 10 mL/kg resulted in no mortality, clinical signs, or discomfort among the rats. Moreover, no pathological alterations were noted in the gross necropsy lesions upon dissection. Table 5 Mortality, clinical observations and gross findings in rats. Gender Male Female Group 1 2 1 2 Dose Level (mL/kg) 0 a 5 0 5 Mortality 0/5 0/5 0/5 0/5 Treatment-ralated Clinical Signs 0/5 0/5 0/5 0/5 Treatment-ralated Gross Necropsy Lesion 0/5 0/5 0/5 0/5 N/N: Total number of abnormal or dead animals observed/Total number of animals examined. a Water for injection (WFI). For details on the body weights of male rats, see Table 6 . On Day 1, the body weight for Group 1 (WFI) was 287.5 ± 13.8g, and for Group 2 (TGLON) it was 286.9 ± 14.1g, demonstrating consistency in experimental conditions. By Day 15, the weights had increased to 341.3 ± 24.3g for Group 1 and 336.5 ± 26.6g for Group 2. The final weight gain was 53.8 ± 16.0g for Group 1 and 49.6 ± 18.6g for Group 2. Similarly, refer to Table 6 for the female rats. On Day 1, the weights were 208.6 ± 10.0g for Group 1 and 205.6 ± 10.0g for Group 2, maintaining consistency in the experimental setup. By Day 15, the weights rose to 239.6 ± 8.1g for Group 1 and 238.6 ± 17.8g for Group 2. The final weight gains were 31.0 ± 8.6g for Group 1 and 33.0 ± 11.1g for Group 2. These results confirm that continuous oral administration of the 50% (v/v) TGLON solution at 10 mL/kg does not impact the body weight of either male or female rats. Table 6 Body weight variations of rats. Body Weights Gender Male Female Group 1 2 1 2 Dose Level (mL/kg) 0 a 5 0 5 Day 1 287.5 ± 13.8 286.9 ± 14.1 208.6 ± 10.0 205.6 ± 10.0 Day 8 317.6 ± 19.5 320.2 ± 21.7 225.6 ± 10.0 227.9 ± 16.3 Day 15 341.3 ± 24.3 336.5 ± 26.6 239.6 ± 8.1 238.6 ± 17.8 Final Gain 53.8 ± 16.0 49.6 ± 18.6 31.0 ± 8.6 33.0 ± 11.1 Final Gain: Body weight on Day 15 minus body weight on Day 1 (Mean ± SD, N = 5). a Water for injection (WFI). Investigation of TGLON in treating IPF in mice Please refer to Fig. 4 . Following Bleomycin-induced IPF, the body weight of mice in the saline group decreased to 80.11 ± 2.56% by Day 9, representing a reduction of approximately 20%. In contrast, mice that received an oral dose of a 50% (v/v) TGLON solution at 10 mL/kg exhibited a smaller decrease to 89.49 ± 2.38%, about an 11% reduction. This outcome demonstrates that a two-week pre-treatment with TGLON can effectively reduce weight loss. Refer to Figs. 5 A & 5 B. In the context of Bleomycin-induced IPF, the Inflammation Index and Fibrosis Index for the saline-treated group were recorded at 3.52 ± 0.45 and 5.21 ± 0.56, respectively. These indices confirm fibrotic damage to the lung tissue, leading to altered lung structure with the formation of fibrotic bands or small clusters (Fig. 6 ). Conversely, the group treated with a 50% (v/v) TGLON solution at 10 mL/kg showed lower indices of 1.52 ± 0.33 for Inflammation and 2.36 ± 0.2 for Fibrosis (Figs. 5 A & 5 B). This indicates that although there was moderate thickening of the lung tissue, it did not significantly impair the lung structure, thus moderating the severity of Bleomycin-induced IPF (Fig. 6 ). Discussion The monoterpenol group exhibits anti-inflammatory properties, while the ester group enhances the synthesis of elastin and possesses anti-wrinkle features. Given that similar structures tend to display comparable bioactivities, we have classified the GC-MS results into analogs. This method facilitates the organization of the complex components [ 37 – 40 ]. TGLON is primarily composed of monoterpenols, supported by sesquiterpenes, other oxides, monoterpenes, sesquiterpenols, and ketones, with a proportion of low-irritancy aldehydes enhancing the blend. This formulation meets our anticipated specifications. In TGLON, Terpinen-4-ol, Camphor, and δ-Cadinene are crucial components recognized for their ability to inhibit cancer cells [ 29 – 31 ]. However, when Terpinen-4-ol dominates the composition of essential oils, it results in 50% cytotoxicity to MRC-5 normal cells at a dilution of 14,000 times [ 25 ]. Similarly, Camphor as the primary component inflicts 50% damage to MRC-5 normal cells at a dilution of 238,000 times [ 41 ]. Extracts where δ-Cadinene is the principal component also cause 50% damage to MRC-5 normal cells at a dilution of 3,900 times [ 42 ]. Despite this, TGLON demonstrates a high survival rate of 95.4% in MRC-5 cells at an 80-fold dilution, underscoring its safety and marking a significant advancement for hydrosol blends. The enhanced safety profile of TGLON for MRC-5 cells may stem from Terpinen-4-ol ability to inhibit the production of lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α), interleukins IL-1β, IL-6, IL-10, and prostaglandin E2 (PG-E2) [ 38 , 43 ]. Additionally, Camphor effectively reduces the expression of IL-1β, IL-6, and TNF-α [ 44 ], while δ-Cadinene curbs the production of LPS-induced nitric oxide (NO) [ 45 ]. TGLON demonstrates bioactivity in inhibiting A-549 lung cancer cells, likely due to components such as terpinen-4-ol, camphor, δ-Cadinene, 1,8-Cineole, γ-Terpinene, and particularly cedrol. Cedrol is noted for its ability to induce apoptosis in A-549 cells by reducing mitochondrial transmembrane potential (MTP) and downregulating phosphatidylinositol 3'-kinase (PI3K)/Akt expression [ 46 – 48 ]. Additionally, TGLON may inhibit HepG2 liver cancer cells, likely due to the presence of terpinen-4-ol, γ-Terpinene, and α-pinene [ 49 , 50 ]. It is also capable of suppressing MCF-7 breast cancer cells through the actions of Terpinen-4-ol, α-Terpineol, Camphor, and Safrole [ 30 , 51 – 53 ]. Furthermore, camphor, 1,8-cineole, and α-pinene contribute to its potential to inhibit MKN-45 stomach cancer cells [ 54 ]. TGLON may also inhibit MOLT-4 leukemic lymphoblasts, potentially through Terpinen-4-ol, which impacts cell survival by causing loss of mitochondrial membrane potential and releasing cytochrome c into the cytosol. This triggers caspase-8 activation, leading to the cleavage of cytosolic Bid, which activates mitochondria in a process linked to the downregulation of Bcl-2 protein and upregulation of caspase-3 [ 55 ]. In an acute toxicity assay, the oral lethal dose (LD 50 ) of Terpinen-4-ol administered to rats was determined to be 1.3 g/mL [ 56 ]. The LD 50 for camphor, similarly administered orally, was recorded at 1.31 g/mL [ 57 ]. Nonetheless, no mortality or symptoms of toxicity were observed in rats following the administration of a 50% (v/v) TGLON solution at a dosage of 10 mL/kg (effective dose 5 mL/kg), and their body weights continued to grow normally. This indicates that TGLON is safer than monomeric compounds [ 58 , 59 ]. The effectiveness of TGLON in alleviating IPF is consistent with that of U.S. FDA-approved drugs Nintedanib and Pirfenidone used for IPF treatment. The mechanism by which TGLON ameliorates IPF may involve active components from the monoterpenol group, such as α-Terpineol, (-)-Myrtenol, Borneol, cis-Myrtanol, and Terpinen-4-ol, as well as β-Citronellal from the aldehyde group. These constituents are believed to reduce the expression of Malondialdehyde (MDA), Alpha-Smooth Muscle Actin (α-SMA), Transforming Growth Factor-Beta (TGF-β), Cyclooxygenase-2 (COX-2), Prostaglandin E2 (PGE2), and Tumor Necrosis Factor-Alpha (TNF-α), while enhancing the expression of superoxide dismutase (SOD) [ 8 ]. Conclusion TGLON has demonstrated the capability to inhibit various human tumor cell lines, including A-549, HepG2, MCF-7, MKN-45, and MOLT-4, without adversely affecting normal human cells. Animal research has shown that the sustained oral administration of a 50% (v/v) TGLON solution at 10 mL/kg for three weeks can safely mitigate IPF. Additionally, the pesticide-free cultivation of TGLON plants not only supports environmental sustainability but also highlights its potential for development into a food or dietary supplement. Declarations Funding This work was financially supported by Holy Tree Biomedical Co., Ltd. and the Guidance Association of Taiwan Aromaplants in Taiwan. Availability of Data and Material Due to the ongoing application for a market permit for the nutritional supplement, the datasets utilized and analyzed in this study are not available to the public. Nonetheless, they can be accessed from the corresponding author upon a reasonable request. Ethics approval Experimental protocols adopted were based on World Health Organization Guidelines for care and use of laboratory animals. The experimental usage of the animals was approved by the Ethics Committee of the National Ethics Committee for Control and Supervision of Experiments on Animals. Consent for publication All authors have agreed to publish this manuscript. Conflict of interest This research has successfully secured a patent for its innovative invention, highlighting its significant contributions to the field. The acquired patents are officially registered under the numbers CN106668571B in China, TWI605818B in Taiwan, and US10314872B2 in the United States, demonstrating the international recognition of this scientific breakthrough. Furthermore, all authors consent to Holy Tree Biomedical Co., Ltd. (Mei-Lin Chang) to commercializing products related to this research. References Ferlay J, Colombet M, Soerjomataram I, Mathers C, Parkin DM, Piñeros M, Znaor A, Bray F. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int J Cancer. 2019;144(8):1941–53. Mylvaganam RJ, Bailey JI, Sznajder JI, Sala MA. Recovering from a pandemic: pulmonary fibrosis after SARS-CoV-2 infection. Eur Respir Rev. 2021;30(162):210194. 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Derelanko MJ, Hollinger MA. Handbook of toxicology. 2 ed. Boca Raton: Chemical Rubber Company; 2001. Gilhodes J-C, Julé Y, Kreuz S, Stierstorfer B, Stiller D, Wollin L. Quantification of pulmonary fibrosis in a bleomycin mouse model using automated histological image analysis. PLoS ONE. 2017;12(1):e0170561. Limjunyawong N, Mitzner W, Horton MR. A mouse model of chronic idiopathic pulmonary fibrosis. Physiol Rep. 2014;2(2):e00249. Ben Ammar R, Mohamed ME, Alfwuaires M, Abdulaziz Alamer S, Bani Ismail M, Veeraraghavan VP, Sekar AK, Ksouri R, Rajendran P. Anti-Inflammatory Activity of Geraniol Isolated from Lemon Grass on Ox-LDL-Stimulated Endothelial Cells by Upregulation of Heme Oxygenase-1 via PI3K/Akt and Nrf-2 Signaling Pathways. Nutrients 2022;14(22). Nogueira MN, Aquino SG, Rossa Junior C, Spolidorio DM. Terpinen-4-ol and alpha-terpineol (tea tree oil components) inhibit the production of IL-1β, IL-6 and IL-10 on human macrophages. Inflamm Res. 2014;63(9):769–78. 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Camphor and menthol as anticancer agents: Synthesis, structure-activity relationship and interaction with cancer cell lines. Anticancer Agents Med Chem. 2023;23(6):614–23. Assmann CE, Cadoná FC, Bonadiman BSR, Dornelles EB, Trevisan G, Cruz, IBMd. Tea tree oil presents in vitro antitumor activity on breast cancer cells without cytotoxic effects on fibroblasts and on peripheral blood mononuclear cells. Biomed Pharmacother. 2018;103:1253–61. Ghavam M. In vitro biological potential of the essential oil of some aromatic species used in Iranian traditional medicine. Inflammopharmacology. 2022;30(3):855–74. Khaw-on P, Banjerdpongchai R. Induction of Intrinsic and Extrinsic Apoptosis Pathways in the Human Leukemic MOLT-4 Cell Line by Terpinen-4-ol. Asian Pac J Cancer Prev. 2012;13(7):3073–6. Unknown. Fragrance raw materials monographs. Food Chem Toxicol. 1982;20(6):637–852. Unknown. Camphor USP. Food Cosmetics Toxicol. 1978;16:665–71. Liu GY, Budinger GRS, Dematte JE. Advances in the management of idiopathic pulmonary fibrosis and progressive pulmonary fibrosis. BMJ. 2022;377:e066354. Bonella F, Spagnolo P, Ryerson C. Current and Future Treatment Landscape for Idiopathic Pulmonary Fibrosis. Drugs. 2023;83(17):1581–93. Additional Declarations Competing interest reported. This research has successfully secured a patent for its innovative invention, highlighting its significant contributions to the field. The acquired patents are officially registered under the numbers CN106668571B in China, TWI605818B in Taiwan, and US10314872B2 in the United States, demonstrating the international recognition of this scientific breakthrough. Furthermore, all authors consent to Holy Tree Biomedical Co., Ltd. (Mei-Lin Chang) to commercializing products related to this research. Supplementary Files PatentnumberCN106668571B.pdf PatentnumberTWI605818B.pdf PatentnumberUS10314872B2.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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SD, sprague dawley; PO, \u003cem\u003eper os.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4440611/v1/a540953c750c05c07764841a.jpg"},{"id":57801781,"identity":"0f9159a1-5abb-43d0-ac01-29b119f34dcb","added_by":"auto","created_at":"2024-06-05 22:23:36","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":80055,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental procedures of IPF mice model. Test article: TGLON.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4440611/v1/2ba7502468f0c1b449d7192e.jpg"},{"id":57801783,"identity":"da31ef32-fbba-4214-94a4-35087732a009","added_by":"auto","created_at":"2024-06-05 22:23:36","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":372579,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of TGLON on (A) MRC-5 cells; (B) A-549 cells; (C) HepG2 cells; (D) MCF-7 cells; (E) MKN-45 cells; (F) MOLT-4 cells. Blank: untreated group as blank; Data are presented as means ± SE.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4440611/v1/07c451db2af9456fb299f8f6.jpg"},{"id":57802238,"identity":"3c215a4d-2f72-4174-b989-49e414ccbe5b","added_by":"auto","created_at":"2024-06-05 22:31:36","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":140172,"visible":true,"origin":"","legend":"\u003cp\u003eBody weight changes in Bleomycin treated mice. Data are presented as means ± SE. #: indicates \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 compared to Saline-treated group, *: indicates \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 Compered to Belo+Saline treated group. Bleo: Bleomycin.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4440611/v1/9cef8459c88860dbf6b575b9.jpg"},{"id":57801786,"identity":"313229b1-f68a-4824-80fe-e48bc93cdae6","added_by":"auto","created_at":"2024-06-05 22:23:36","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":170379,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Inflammation index and (B) Fibrosis Index of lung in Bleomycin treated mice. Data are presented as means ± SE.\u003c/p\u003e\n\u003cp\u003e**: indicates \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 Compared to Belo+Saline treated group. Bleo: Bleomycin.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4440611/v1/6652cbcdffda7651b754d9cb.jpg"},{"id":57802239,"identity":"875b1eff-535e-4905-8588-2cf117ad7197","added_by":"auto","created_at":"2024-06-05 22:31:36","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":687957,"visible":true,"origin":"","legend":"\u003cp\u003eHematoxylin and eosin stain of lung tissue in Bleomycin treated mice.\u003c/p\u003e\n\u003cp\u003e'X' refers to the specific magnification used when observing lung tissue.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4440611/v1/32541f2123073cf5c0ca55de.jpg"},{"id":65552048,"identity":"53407c12-d250-44d1-bb77-3e4a8487db0a","added_by":"auto","created_at":"2024-09-30 01:16:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2373431,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4440611/v1/34e232bb-f9f4-476b-9f7e-336440b6f9b1.pdf"},{"id":57801787,"identity":"dcd45484-da81-41a8-9cb7-ed6b143a609d","added_by":"auto","created_at":"2024-06-05 22:23:36","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":5566752,"visible":true,"origin":"","legend":"","description":"","filename":"PatentnumberCN106668571B.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4440611/v1/e227bb3e1e8b84623b1a4a92.pdf"},{"id":57802241,"identity":"de10ef1b-dcc8-47a0-a124-83106b8690d4","added_by":"auto","created_at":"2024-06-05 22:31:36","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":7940423,"visible":true,"origin":"","legend":"","description":"","filename":"PatentnumberTWI605818B.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4440611/v1/249e3e1e45e059d1430ba873.pdf"},{"id":57802240,"identity":"f989fff6-c311-423b-8b98-50a1c75d0f3b","added_by":"auto","created_at":"2024-06-05 22:31:36","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":1260448,"visible":true,"origin":"","legend":"","description":"","filename":"PatentnumberUS10314872B2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4440611/v1/665b433314b3a6eb38fb73a9.pdf"}],"financialInterests":"Competing interest reported. This research has successfully secured a patent for its innovative invention, highlighting its significant contributions to the field. The acquired patents are officially registered under the numbers CN106668571B in China, TWI605818B in Taiwan, and US10314872B2 in the United States, demonstrating the international recognition of this scientific breakthrough. Furthermore, all authors consent to Holy Tree Biomedical Co., Ltd. (Mei-Lin Chang) to commercializing products related to this research.","formattedTitle":"Potential natural hydrosol blend TGLON suppresses the proliferation of five cancer cell lines and also ameliorates idiopathic pulmonary fibrosis in mouse model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAn analysis of cancer incidence rates from 185 countries has revealed significant patterns in prevalence: lung cancer rates stand at 14.5% in men and 8.4% in women; liver cancer is present in 6.3% of men; breast cancer incidence is at 24.2% in women; and stomach cancer occurs at rates of 7.2% in men and 4.1% in women. Notably, the occurrence of these cancers is often associated with hematological malignancies, such as lymphoblastic leukemia, which disrupt the normal blood milieu and hematopoietic functions, potentially reducing immune system efficacy and increasing susceptibility to pathogenic infections [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. During the Coronavirus Disease 2019 (COVID-19) global pandemic, this virus, which primarily targets lung epithelial cells via the Angiotensin-converting enzyme (ACE) 2 receptor, was observed to replicate rapidly post-infection, affecting additional lung cells. Such a process can lead to acute complications, including Idiopathic Pulmonary Fibrosis (IPF), a condition characterized by the excessive synthesis and pathological accumulation of Extracellular Matrix (ECM) proteins in the alveoli, ultimately resulting in pulmonary fibrosis [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our study, we meticulously selected aromatic plants rich in volatile secondary metabolites, leveraging their natural growth methods that eschew the need for pesticides, thereby reducing soil environmental impact and supporting sustainable development efforts in preserving terrestrial ecosystems [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Investigations into the extracts of these plants have indicated their efficacy in moderating the progression of IPF [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Additionally, these extracts have shown significant anti-proliferative properties against various cancer cell lines. For instance, \u003cem\u003eCymbopogon Nardus\u003c/em\u003e (CN) has exhibited marked cytotoxicity against prostate cancer cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. \u003cem\u003eLitsea Cubeba\u003c/em\u003e (LC) has demonstrated potent cytotoxic activities against human cancers of the lung, liver, breast, and oral cavity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. \u003cem\u003eChamaecyparis Formosensis\u003c/em\u003e (CF) has been found to be cytotoxic to human liver cancer cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. \u003cem\u003eCalocedrus Formosana\u003c/em\u003e (CAF) has shown notable effectiveness against human lung and bladder cancer cells [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. \u003cem\u003eCinnamomum Camphora\u003c/em\u003e (CC) has targeted colon cancer cells with significant cytotoxic effects [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. \u003cem\u003eEucalyptus Robusta\u003c/em\u003e smith (ER) has exerted cytotoxicity against human pancreatic cancer cells [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Similarly, \u003cem\u003eCinnamomum Zeylanicum\u003c/em\u003e (CZ) has been active against human liver and breast cancer cells [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. \u003cem\u003eCunninghamia Lanceolata\u003c/em\u003e (CL) has displayed cytotoxic activity against both human lung and liver cancers [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. \u003cem\u003eMelaleuca Alternifolia\u003c/em\u003e (MA) has proven effective against lung, breast, and prostate cancer cells [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. \u003cem\u003eCinnamomum Micranthum\u003c/em\u003e (CM) has shown cytotoxic effects on murine leukemia cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. \u003cem\u003eCryptomeria Japonica\u003c/em\u003e (CJ) has revealed cytotoxic effects against human oral and lung cancer cells [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. \u003cem\u003eAcacia Confusa\u003c/em\u003e (AC) has been identified as cytotoxic to human breast cancer cells [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. It is important to note that while these aromatic plants are recognized for their anti-cancer properties, their potential toxicity to normal cells necessitates careful consideration. For example, essential oils from Melaleuca alternifolia, at a 14,000-fold dilution, have been shown to inflict substantial damage, up to 50%, on normal human lung cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConsequently, identifying an optimal dosage that effectively inhibits cancer cell growth while preserving the integrity of normal cells has become a pivotal aspect of our research. Recently, there has been an increasing focus on the study of Essential Oil Blends, particularly exploring their antibacterial, antifungal, and antiviral properties, as well as their potential in alleviating diarrhea in animal models. This paradigm shift marks a significant advance in innovative research, suggesting that meticulous control in processing and formulation may enhance the safety profiles of these products [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, it is important to note in our research that, while volatile secondary metabolites from aromatic plants are concentrated in essential oils through steam distillation, they may still pose considerable risks to normal cells. Therefore, our investigation has shifted toward exploring hydrosol products derived from steam distillation. We have developed a specific hydrosol blend, named The Greatest Love of Nature (TGLON), representing a pioneering effort in using hydrosol blends to study tumor cell inhibition, acute toxicity, and the attenuation of IPF.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of hydrosol blend and chemicals\u003c/h2\u003e \u003cp\u003eIn this research, a diverse array of plant species was cultivated in the forested and agricultural regions of Taiwan, at altitudes spanning 500 to 1000 meters. Hydrosols were extracted from a variety of plants utilizing steam distillation. These plants included CN, LC, CF, CAF, CC, ER, CZ, CL, MA, CM, CJ, and AC, and the extracts were used to design a novel hydrosol mixture named TGLON. The formulation of TGLON is strategically based on the anti-cancer properties of its key constituents such as Terpinen-4-ol, Camphor, and δ-Cadinene. Terpinen-4-ol, a primary compound in the monoterpenol category, is particularly noted for its efficacy in inhibiting the proliferation of tumor cells. To optimize the therapeutic potential, TGLON was meticulously formulated, focusing predominantly on monoterpenols while ensuring a balanced integration of sesquiterpenes, other oxides, monoterpenes, sesquiterpenols, and ketones, and simultaneously minimizing the content of irritating aldehydes. The specific formulation ratios are CN 8%, LC 6%, CF 40%, CAF 10%, CC 8%, ER 6%, CZ 2%, CL 4%, MA 2%, CM 10%, CJ 2%, and AC 2%. This proprietary hydrosol blend is slated for further investigative research to assess its ability to inhibit tumor cell growth and mitigate the progression of IPF under more controlled and safer experimental conditions [\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, a variety of human cell lines were sourced, encompassing MRC-5 lung cells, A-549 lung cancer cells, HepG2 liver cancer cells, MCF-7 breast cancer cells, MKN-45 stomach cancer cells, and MOLT-4 Leukemic Lymphoblasts. These were obtained from the Bioresource Collection and Research Center (BCRC) in East Dist, HSZ, TWN, ensuring the authenticity and viability of the cells for research purposes. Additionally, all chemicals employed in our analysis were of analytical reagent grade, acquired from ECHO CHEMICAL CO., LTD., Toufen City, ZMI, TWN. This ensured the highest level of purity and consistency in our experimental procedures. Throughout the various stages of analysis, ultrapure water was utilized exclusively, guaranteeing minimal interference from contaminants and thus maintaining the integrity of our experimental results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eGas chromatography mass spectrometry (GC-MS)\u003c/h2\u003e \u003cp\u003eRevise according to Adams description of the literature [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Using a GC7890, followed by a 5977B MSD (Agilent Technologies, Inc., Santa Clara, CA, USA), which functions as a quadrupole mass spectrometer. The injection port operates in split mode at a constant temperature of 300\u0026deg;C. The chromatographic separation column is a DB-5 MS, with a length of 30 m, an inner diameter of 0.25 mm, and a film thickness of 0.25 \u0026micro;m (Agilent Technologies, Inc., Santa Clara, CA, USA). The carrier gas is helium with a purity of \u0026ge;\u0026thinsp;99.999%, and the flow rate is set at 1 mL/min. The temperature ramp conditions are from 50\u0026deg;C to 280\u0026deg;C, with a ramp rate of 3\u0026deg;C per minute, followed by a 5-minute isothermal hold. For the calculation of the Kovats Index (KI), we meticulously recorded the Retention Times (RT) of both the test sample and the C8-C40 alkane standard mixture. The mass spectrometer operates under Electron-Impact Ionization (EI) at 70 eV. Mass spectral data acquired through the analysis utilizing the 2.64 Automated Mass Spectral Deconvolution and Identification System (AMDIS) developed by the National Institute of Standards and Technology (NIST). Identification is accomplished by comparing the GC-MS spectral data and KI of polar and nonpolar compounds with reference compounds from the NIST database.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eIn vitro cytotoxicity assay\u003c/h2\u003e \u003cp\u003eImproving methodology according to Alley et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. We initially seeded 3000 cells into each well of a 96-well culture plate. These wells were each supplemented with 100\u0026micro;L of culture medium and incubated for a period of 24 hours. Following the incubation, medium was removed, and test samples were introduced into the wells at various dilution factors: 20X, 40X, 80X, 160X, 320X, 640X, 1280X for the MRC-5, A-549, HepG2, MCF-7, and MKN-45 cell lines, and 20X, 200X, 2000X, 20000X, 40000X, 80000X, 200000X for the MOLT-4 cell line. These were then incubated for an additional 72 hours. Post-incubation, the culture medium was carefully aspirated, and each well was washed using 150\u0026micro;L of Phosphate Buffered Saline (PBS). To each well, 100\u0026micro;L of MTT reagent (0.5 mg/mL) was added. The wells were then covered with aluminum foil to prevent light exposure and placed in a CO2 incubator for a duration of 30 minutes. Following this incubation step, the MTT reagent was aspirated, and the wells were once again washed with PBS. This was succeeded by the addition of 100\u0026micro;L of Dimethyl Sulfoxide (DMSO) per well, facilitating the dissolution of formazan crystals. After ensuring thorough mixing of the contents, the absorbance at 540 nm wavelength was quantitatively assessed using an ELISA reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eIn vivo Acute Oral Toxicity Study\u003c/h2\u003e \u003cp\u003eIn this meticulously conducted study, Crl: CD Sprague Dawley (SD) rats provided by BioLASCO Taiwan Co., Ltd., Nangang District, TPE, TWN, were selected as the model organisms. The rats were chosen based on specific criteria: age (8 to 9 weeks), with male rats in the weight range of 260 to 300 grams and female rats between 180 to 230 grams, to establish a uniform baseline for our research. The preference for SD rats was due to their demonstrated responsiveness to a wide range of toxicological agents, rendering them suitable for our toxicological studies. Ethical considerations took precedence in our study, ensuring all animal experiments adhered to the highest standards. Our protocol received thorough evaluation and approval from the Institutional Animal Care and Use Committee (IACUC), designated with the IACUC number 2020-R501-051 and approval date of December 28, 2020. We adhered rigorously to the internationally recognized guidelines for the care and use of laboratory animals, ensuring ethical and responsible conduct in our scientific research. before commencing the study, all rats were subjected to a mandatory quarantine and acclimatization period of no less than three days within our animal facility. Regarding their housing, we placed three rats in each polycarbonate cage. These cages were meticulously labeled with essential details such as group number, study number, dosage level, sex, animal ID, and scheduled euthanasia date. We maintained controlled environmental conditions, with room temperature set at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C and relative humidity at 50\u0026thinsp;\u0026plusmn;\u0026thinsp;20%, including a 12-hour automated light/dark cycle (lights on at 7:00 am, off at 19:00 pm). The rats diet consisted of PicoLab\u0026reg; Rodent Diet 20 (sourced from LabSupply Inc., St. Louis, MO, USA), and they had continuous access to sterilized tap water. Detailed housing conditions are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThroughout the study, the rats received a daily oral administration of a 50% (v/v) TGLON solution or Water for injection (WFI) oral administration, each at a volume of 10 mL. We conducted observations on Days 1, 8, and 15, focusing on changes in average weight, potential mortality, or any clinical signs. The study culminated on Day 15 with a comprehensive dissection to examine for any additional anatomical damages [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003eIn vivo Pulmonary Fibrosis Study\u003c/h2\u003e \u003cp\u003eIn this rigorously designed study, we employed 18 male C57BL/6 mice, supplied by BioLASCO Taiwan Co., Ltd., Nangang District, TPE, TWN, as our biological models. These mice were specifically selected based on their age range of 8 to 9 weeks and a weight bracket of 20 to 25 grams, to maintain uniformity in our research baseline. The animal experimentation protocol underwent a stringent evaluation and received approval from the IACUC, assigned the approval number 2021-R501-049, with an approval date of January 14, 2021. This procedure ensured adherence to the highest ethical standards. Each mouse was uniquely identified using ear tags and corresponding cage numbers. for housing, the mice were accommodated in polycarbonate cages, with a grouping of three mice per cage. These cages were outfitted with identification cards that meticulously recorded details such as cage number, trial number, dosage group, sex, and animal identification number. We meticulously maintained environmental conditions at a stable room temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C and a relative humidity of 50\u0026thinsp;\u0026plusmn;\u0026thinsp;20%. The light exposure was controlled through a 12 hour automatic light/dark cycle, with illumination periods from 7:00 am to 19:00 pm. The dietary needs of the mice were met with PicoLab\u0026reg; Rodent Diet 20 (provided by LabSupply Inc., St. Louis, MO, USA) and were freely accessible throughout the duration of the study. Additionally, sterilized tap water was supplied ad libitum in their cages. for detailed housing conditions, refer to Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTreatment information of IPF model.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePre-treatment\u003c/p\u003e \u003cp\u003e(Day \u0026minus;\u0026thinsp;14 ~ -1)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBleomycin \u003csup\u003ea\u003c/sup\u003e Induction\u003c/p\u003e \u003cp\u003e(Day 0)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003cp\u003e(Day 0\u0026ndash;20)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNecropsy\u003c/p\u003e \u003cp\u003e(Day 21)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSaline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOral, daily\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOral, daily\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6 Males\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSaline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOral, daily\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSingle dose, IT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOral, daily\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6 Males\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTest article\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOral, daily\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSingle dose, IT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOral, daily\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6 Males\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003e Bleomycin (3U/kg) administration intratracheally (IT) on day 0. Test article: TGLON. IPF, Idiopathic Pulmonary Fibrosis.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTest article and vehicle control administration.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eItems\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControls\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRoute\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOral Gavage (PO)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFrequency\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOnce daily\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 mL/kg of 100% Test article\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConcentration of dosing solution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50% (v/v) diluted in saline\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDosing Volume\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 mL/kg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTest article: TGLON. PO, \u003cem\u003eper os.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eIn the IPF experiment, each mouse initially received an oral dose of either 50% (v/v) TGLON solution or 10mL Saline daily for a period of 2 weeks. This was followed by an intratracheal administration of 50\u0026micro;L Bleomycin (3U/kg) to induce IPF. Post-induction, the oral administration of TGLON 50% (v/v) or Saline 10mL was continued for an additional 3 weeks, culminating in dissection to evaluate the resultant effects [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe mean values, \u0026plusmn; standard deviations were calculated using MS Excel 2007. Data was analysed by using Analysis of Variance (ANOVA) and differences among the means were deter- mined for significance at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 using Duncan multiple range test by SPSS (version 16.0).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003eGC-MS analysis of TGLON\u003c/h2\u003e\n\u003cp\u003ePlease refer to Tables\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. For details on the 23 chemical compounds identified by TGLON. These compounds are listed in descending order by area percentage as follows: Terpinen-4-ol (10.72%), Camphor (8.73%), \u0026delta;-Cadinene (7.58%), \u0026alpha;-Terpineol (6.93%), Safrole (6.87%), 1,8-Cineole (6.08%), cis-Myrtanol (4.22%), (-)-Myrtenol (3.94%), \u0026gamma;-Terpinene (3.17%), \u0026alpha;-Muurolene (2.9%), tau-Cadinol (2.82%), \u0026alpha;-Cedrene (2.47%), \u0026alpha;-Pinene (2.46%), tau-Muurolol (2.46%), \u0026alpha;-Cedrol (2.16%), \u0026gamma;-Muurolene (2.15%), D-Limonene (1.93%), \u0026alpha;-Terpinene (1.54%), \u0026alpha;-Elemol (1.45%), para-Cymene (1.42%), Borneol (1.12%), \u0026beta;-Citronellal (1.11%), and \u0026beta;-Elemene (0.94%). Additionally, these compounds have been further categorized and sorted by area percentage from highest to lowest: monoterpenols (26.93%), sesquiterpenes (16.04%), other oxides (12.95%), monoterpenes (10.52%), sesquiterpenols (8.89%), ketones (8.73%), and aldehydes (1.11%).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eGC-MS analysis of TGLON.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePK #\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eConstituent\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eType\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eKI\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRT\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e% Area\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMethod of Identification\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026alpha;-Pinene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMonoterpene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e939\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e9.606\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.46\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI;RC\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026alpha;-Terpinene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMonoterpene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1017\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e12.221\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.54\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epara-Cymene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMonoterpene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1024\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e12.455\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.42\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI;RC\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eD-Limonene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMonoterpene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1029\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e12.603\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.93\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI;RC\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1,8-Cineole\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOther oxide\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1031\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e12.669\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI;RC\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gamma;-Terpinene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMonoterpene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1059\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e13.552\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCamphor\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKetone\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1146\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e16.174\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8.73\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI;RC\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026beta;-Citronellal\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAldehyde\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1153\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e16.412\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBorneol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMonoterpenol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1169\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e16.83\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI;RC\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTerpinen-4-ol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMonoterpenol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1177\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e17.177\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e10.72\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI;RC\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026alpha;-Terpineol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMonoterpenol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1188\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e17.555\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.93\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI;RC\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e(-)-Myrtenol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMonoterpenol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1195\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e17.734\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.94\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ecis-Myrtanol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMonoterpenol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1253\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e19.559\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4.22\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSafrole\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOther oxide\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1287\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e20.361\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.87\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026beta;-Elemene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSesquiterpene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1390\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e23.258\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.94\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026alpha;-Cedrene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSesquiterpene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1411\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e23.86\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.47\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gamma;-Muurolene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSesquiterpene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1479\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e26.013\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026alpha;-Muurolene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSesquiterpene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1500\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e26.373\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026delta;-Cadinene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSesquiterpene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1523\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e26.569\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e7.58\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026alpha;-Elemol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSesquiterpenol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1549\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e27.173\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.45\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026alpha;-Cedrol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSesquiterpenol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1601\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e28.519\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI;RC\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e22\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003etau-Cadinol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSesquiterpenol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1640\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e29.365\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.82\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003etau-Muurolol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSesquiterpenol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1642\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e29.663\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.46\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMS;KI\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003ePK #, Peak Number; RT, Retention Times; KI, Kovats Index; MS, mass spectrum; RC, reference compound.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"char\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab4\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eAnalogs of TGLON\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eType\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e% Area\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSesquiterpenes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e16.04\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMonoterpenes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e10.52\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMonoterpenols\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e26.93\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSesquiterpenols\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8.89\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKetones\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8.73\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAldehydes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.11\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOther oxides\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e12.95\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eViability of TGLON in MRC-5, A-549, HepG2, MCF-7, MKN-45, and MOLT-4 cells\u003c/h2\u003e\n\u003cp\u003ePlease refer to Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA for the survival rates of MRC-5 normal lung cells across a range of dilutions. The survival rate at a 20-fold dilution was 2.7%; at 40-fold, it increased to 40.9%; at 80-fold, it rose to 95.4%; at 160-fold, it reached 99.2%; at 320-fold, it was 98%; at 640-fold, it approached 99.1%; and at 1280-fold, it exceeded 100%, registering at 101%. Establishing a benchmark survival rate of 90% or higher, dilutions of TGLON above 80-fold are deemed safe for MRC-5 normal cells. Consequently, for tumor cells, a similar standard will apply, setting the survival rate benchmark at dilutions greater than 80-fold.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlease refer to Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB for the viability of A-549 lung cancer cells at varying dilutions. At an 80-fold dilution, the cell survival rate was 59.3%, indicating an inhibition of 40.7%. At 160-fold, the survival rate increased to 77%, with a 23% inhibition. At 320-fold, it reached 84%, with a 16% inhibition. At 640-fold, the survival rate was 94.1%, with a 5.9% inhibition, and at 1280-fold, it was 93.2%, with a 6.8% inhibition. For HepG2 liver cancer cells, please consult Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC. At an 80-fold dilution, the survival rate was 16%, with an inhibition of 84%. At 160-fold, it increased to 28.3%, with an inhibition of 71.7%. At 320-fold, the survival rate was 63.5%, with a 36.5% inhibition. At 640-fold, it rose to 96.4%, with a 3.6% inhibition, and at 1280-fold, it was 93.8%, with a 6.2% inhibition. Regarding the viability of MCF-7 breast cancer cells, refer to Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD. At an 80-fold dilution, the survival rate was 50.2%, demonstrating a 49.8% inhibition. For the viability of MKN-45 stomach cancer cells, consult Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE. At an 80-fold dilution, the survival rate was 62%, with a 38% inhibition. At 160-fold, it was 72%, with a 28% inhibition. At 320-fold, the survival rate improved to 79%, with a 21% inhibition. At 640-fold, it reached 83%, with a 17% inhibition.\u003c/p\u003e\n\u003cp\u003ePlease consult Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF for the viability analysis of MOLT-4 leukemic lymphoblasts. Unlike tumor cells originating from solid organs, MOLT-4 cells undergo malignant proliferation within the human body, leading to malignant disorders of the hematologic and bone marrow systems, which are closely associated with leukemia. At a 200-fold dilution, the survival rate of these cells was 48%, reflecting a 52% inhibition. At a 2,000-fold dilution, the survival rate improved to 64%, indicating a 36% inhibition. 72% at 20,000-fold with a 28% inhibition, 85% at 40,000-fold with a 15% inhibition, and 92.9% at 80,000-fold with a 7.1% inhibition.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003eAcute toxicity assay of TGLON in rats\u003c/h2\u003e\n\u003cp\u003ePlease refer to Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. Over a two-week period, oral administration of a 50% (v/v) TGLON solution at a dosage of 10 mL/kg resulted in no mortality, clinical signs, or discomfort among the rats. Moreover, no pathological alterations were noted in the gross necropsy lesions upon dissection.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab5\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eMortality, clinical observations and gross findings in rats.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGender\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eMale\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eFemale\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGroup\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDose Level (mL/kg)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMortality\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTreatment-ralated Clinical Signs\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTreatment-ralated Gross Necropsy Lesion\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eN/N: Total number of abnormal or dead animals observed/Total number of animals examined. \u003csup\u003ea\u003c/sup\u003e Water for injection (WFI).\u003c/p\u003e\n\u003cp\u003eFor details on the body weights of male rats, see Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e. On Day 1, the body weight for Group 1 (WFI) was 287.5\u0026thinsp;\u0026plusmn;\u0026thinsp;13.8g, and for Group 2 (TGLON) it was 286.9\u0026thinsp;\u0026plusmn;\u0026thinsp;14.1g, demonstrating consistency in experimental conditions. By Day 15, the weights had increased to 341.3\u0026thinsp;\u0026plusmn;\u0026thinsp;24.3g for Group 1 and 336.5\u0026thinsp;\u0026plusmn;\u0026thinsp;26.6g for Group 2. The final weight gain was 53.8\u0026thinsp;\u0026plusmn;\u0026thinsp;16.0g for Group 1 and 49.6\u0026thinsp;\u0026plusmn;\u0026thinsp;18.6g for Group 2. Similarly, refer to Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e for the female rats. On Day 1, the weights were 208.6\u0026thinsp;\u0026plusmn;\u0026thinsp;10.0g for Group 1 and 205.6\u0026thinsp;\u0026plusmn;\u0026thinsp;10.0g for Group 2, maintaining consistency in the experimental setup. By Day 15, the weights rose to 239.6\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1g for Group 1 and 238.6\u0026thinsp;\u0026plusmn;\u0026thinsp;17.8g for Group 2. The final weight gains were 31.0\u0026thinsp;\u0026plusmn;\u0026thinsp;8.6g for Group 1 and 33.0\u0026thinsp;\u0026plusmn;\u0026thinsp;11.1g for Group 2. These results confirm that continuous oral administration of the 50% (v/v) TGLON solution at 10 mL/kg does not impact the body weight of either male or female rats.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab6\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eBody weight variations of rats.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth colspan=\"4\" align=\"left\"\u003e\n\u003cp\u003eBody Weights\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGender\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eMale\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eFemale\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGroup\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDose Level (mL/kg)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDay 1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e287.5\u0026thinsp;\u0026plusmn;\u0026thinsp;13.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e286.9\u0026thinsp;\u0026plusmn;\u0026thinsp;14.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e208.6\u0026thinsp;\u0026plusmn;\u0026thinsp;10.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e205.6\u0026thinsp;\u0026plusmn;\u0026thinsp;10.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDay 8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e317.6\u0026thinsp;\u0026plusmn;\u0026thinsp;19.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e320.2\u0026thinsp;\u0026plusmn;\u0026thinsp;21.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e225.6\u0026thinsp;\u0026plusmn;\u0026thinsp;10.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e227.9\u0026thinsp;\u0026plusmn;\u0026thinsp;16.3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDay 15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e341.3\u0026thinsp;\u0026plusmn;\u0026thinsp;24.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e336.5\u0026thinsp;\u0026plusmn;\u0026thinsp;26.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e239.6\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e238.6\u0026thinsp;\u0026plusmn;\u0026thinsp;17.8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFinal Gain\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e53.8\u0026thinsp;\u0026plusmn;\u0026thinsp;16.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e49.6\u0026thinsp;\u0026plusmn;\u0026thinsp;18.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e31.0\u0026thinsp;\u0026plusmn;\u0026thinsp;8.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e33.0\u0026thinsp;\u0026plusmn;\u0026thinsp;11.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eFinal Gain: Body weight on Day 15 minus body weight on Day 1 (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, N\u0026thinsp;=\u0026thinsp;5). \u003csup\u003ea\u003c/sup\u003e Water for injection (WFI).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003eInvestigation of TGLON in treating IPF in mice\u003c/h2\u003e\n\u003cp\u003ePlease refer to Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. Following Bleomycin-induced IPF, the body weight of mice in the saline group decreased to 80.11\u0026thinsp;\u0026plusmn;\u0026thinsp;2.56% by Day 9, representing a reduction of approximately 20%. In contrast, mice that received an oral dose of a 50% (v/v) TGLON solution at 10 mL/kg exhibited a smaller decrease to 89.49\u0026thinsp;\u0026plusmn;\u0026thinsp;2.38%, about an 11% reduction. This outcome demonstrates that a two-week pre-treatment with TGLON can effectively reduce weight loss.\u003c/p\u003e\n\u003cp\u003eRefer to Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA \u0026amp; \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB. In the context of Bleomycin-induced IPF, the Inflammation Index and Fibrosis Index for the saline-treated group were recorded at 3.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45 and 5.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56, respectively.\u003c/p\u003e\n\u003cp\u003eThese indices confirm fibrotic damage to the lung tissue, leading to altered lung structure with the formation of fibrotic bands or small clusters (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eConversely, the group treated with a 50% (v/v) TGLON solution at 10 mL/kg showed lower indices of 1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 for Inflammation and 2.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 for Fibrosis (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA \u0026amp; \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). This indicates that although there was moderate thickening of the lung tissue, it did not significantly impair the lung structure, thus moderating the severity of Bleomycin-induced IPF (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe monoterpenol group exhibits anti-inflammatory properties, while the ester group enhances the synthesis of elastin and possesses anti-wrinkle features. Given that similar structures tend to display comparable bioactivities, we have classified the GC-MS results into analogs. This method facilitates the organization of the complex components [\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. TGLON is primarily composed of monoterpenols, supported by sesquiterpenes, other oxides, monoterpenes, sesquiterpenols, and ketones, with a proportion of low-irritancy aldehydes enhancing the blend. This formulation meets our anticipated specifications.\u003c/p\u003e \u003cp\u003eIn TGLON, Terpinen-4-ol, Camphor, and δ-Cadinene are crucial components recognized for their ability to inhibit cancer cells [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, when Terpinen-4-ol dominates the composition of essential oils, it results in 50% cytotoxicity to MRC-5 normal cells at a dilution of 14,000 times [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Similarly, Camphor as the primary component inflicts 50% damage to MRC-5 normal cells at a dilution of 238,000 times [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Extracts where δ-Cadinene is the principal component also cause 50% damage to MRC-5 normal cells at a dilution of 3,900 times [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Despite this, TGLON demonstrates a high survival rate of 95.4% in MRC-5 cells at an 80-fold dilution, underscoring its safety and marking a significant advancement for hydrosol blends. The enhanced safety profile of TGLON for MRC-5 cells may stem from Terpinen-4-ol ability to inhibit the production of lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α), interleukins IL-1β, IL-6, IL-10, and prostaglandin E2 (PG-E2) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Additionally, Camphor effectively reduces the expression of IL-1β, IL-6, and TNF-α [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], while δ-Cadinene curbs the production of LPS-induced nitric oxide (NO) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTGLON demonstrates bioactivity in inhibiting A-549 lung cancer cells, likely due to components such as terpinen-4-ol, camphor, δ-Cadinene, 1,8-Cineole, γ-Terpinene, and particularly cedrol. Cedrol is noted for its ability to induce apoptosis in A-549 cells by reducing mitochondrial transmembrane potential (MTP) and downregulating phosphatidylinositol 3'-kinase (PI3K)/Akt expression [\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Additionally, TGLON may inhibit HepG2 liver cancer cells, likely due to the presence of terpinen-4-ol, γ-Terpinene, and α-pinene [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. It is also capable of suppressing MCF-7 breast cancer cells through the actions of Terpinen-4-ol, α-Terpineol, Camphor, and Safrole [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Furthermore, camphor, 1,8-cineole, and α-pinene contribute to its potential to inhibit MKN-45 stomach cancer cells [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTGLON may also inhibit MOLT-4 leukemic lymphoblasts, potentially through Terpinen-4-ol, which impacts cell survival by causing loss of mitochondrial membrane potential and releasing cytochrome c into the cytosol. This triggers caspase-8 activation, leading to the cleavage of cytosolic Bid, which activates mitochondria in a process linked to the downregulation of Bcl-2 protein and upregulation of caspase-3 [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn an acute toxicity assay, the oral lethal dose (LD\u003csub\u003e50\u003c/sub\u003e) of Terpinen-4-ol administered to rats was determined to be 1.3 g/mL [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The LD\u003csub\u003e50\u003c/sub\u003e for camphor, similarly administered orally, was recorded at 1.31 g/mL [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Nonetheless, no mortality or symptoms of toxicity were observed in rats following the administration of a 50% (v/v) TGLON solution at a dosage of 10 mL/kg (effective dose 5 mL/kg), and their body weights continued to grow normally. This indicates that TGLON is safer than monomeric compounds [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The effectiveness of TGLON in alleviating IPF is consistent with that of U.S. FDA-approved drugs Nintedanib and Pirfenidone used for IPF treatment. The mechanism by which TGLON ameliorates IPF may involve active components from the monoterpenol group, such as α-Terpineol, (-)-Myrtenol, Borneol, cis-Myrtanol, and Terpinen-4-ol, as well as β-Citronellal from the aldehyde group. These constituents are believed to reduce the expression of Malondialdehyde (MDA), Alpha-Smooth Muscle Actin (α-SMA), Transforming Growth Factor-Beta (TGF-β), Cyclooxygenase-2 (COX-2), Prostaglandin E2 (PGE2), and Tumor Necrosis Factor-Alpha (TNF-α), while enhancing the expression of superoxide dismutase (SOD) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTGLON has demonstrated the capability to inhibit various human tumor cell lines, including A-549, HepG2, MCF-7, MKN-45, and MOLT-4, without adversely affecting normal human cells. Animal research has shown that the sustained oral administration of a 50% (v/v) TGLON solution at 10 mL/kg for three weeks can safely mitigate IPF. Additionally, the pesticide-free cultivation of TGLON plants not only supports environmental sustainability but also highlights its potential for development into a food or dietary supplement.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by Holy Tree Biomedical Co., Ltd. and the Guidance Association of Taiwan Aromaplants in Taiwan.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDue to the ongoing application for a market permit for the nutritional supplement, the datasets utilized and analyzed in this study are not available to the public. Nonetheless, they can be accessed from the corresponding author upon a reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental protocols adopted were based on World Health Organization Guidelines for care and use of laboratory animals. The experimental usage of the animals was approved by the Ethics Committee of the National Ethics Committee for Control and Supervision of Experiments on Animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have agreed to publish this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research has successfully secured a patent for its innovative invention, highlighting its significant contributions to the field. The acquired patents are officially registered under the numbers CN106668571B in China, TWI605818B in Taiwan, and US10314872B2 in the United States, demonstrating the international recognition of this scientific breakthrough. Furthermore, all authors consent to Holy Tree Biomedical Co., Ltd. (Mei-Lin Chang) to commercializing products related to this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFerlay J, Colombet M, Soerjomataram I, Mathers C, Parkin DM, Pi\u0026ntilde;eros M, Znaor A, Bray F. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int J Cancer. 2019;144(8):1941\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMylvaganam RJ, Bailey JI, Sznajder JI, Sala MA. Recovering from a pandemic: pulmonary fibrosis after SARS-CoV-2 infection. 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Drugs. 2023;83(17):1581\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"hydrosol blend, TGLON, cancer cells, reduce pulmonary fibrosis, natural products","lastPublishedDoi":"10.21203/rs.3.rs-4440611/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4440611/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003ePrevious studies on natural substances for tumor cell inhibition have predominantly concentrated on efficacy, often overlooking safety concerns. This research utilizes \"The Greatest Love of Nature (TGLON), a proprietary blend of plant hydrosols, to investigate its potential inhibitory effects on a variety of cancer cell lines while ensuring its relative safety to normal lung cells (MRC-5). Furthermore, initial animal studies have demonstrated that TGLON can mitigate the progression of idiopathic pulmonary fibrosis without acute oral toxicity, underscoring its potential utility as a nutritional supplement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e TGLON, a hydrosol blend developed with specific proportions, underwent initial validation for its constituent composition and safety profile. The blend capacity to inhibit five different tumor cell types and alleviate pulmonary fibrosis was evaluated using cell viability assays and controlled animal experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eDosages were determined to be safe at dilutions greater than 80-fold. At this concentration, TGLON exhibited inhibitory effects on 40.7% of lung cancer cells, 84% of liver cancer cells, 49.8% of breast cancer cells, 38% of stomach cancer cells, and inhibited 52% of leukemic lymphoblasts at a 200-fold dilution. Oral administration of TGLON was found to be safe and effective in reducing Bleomycin-induced pulmonary fibrosis in animal models.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003eUnder conditions deemed relatively safe, TGLON demonstrates bioactive properties, such as inhibiting five distinct types of human tumor cells and alleviating pulmonary fibrosis in animal models. These findings highlight its substantial potential as a nutritional supplement for improving overall patient health.\u003c/p\u003e","manuscriptTitle":"Potential natural hydrosol blend TGLON suppresses the proliferation of five cancer cell lines and also ameliorates idiopathic pulmonary fibrosis in mouse model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-05 22:23:31","doi":"10.21203/rs.3.rs-4440611/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"23a183e1-f350-4260-a06b-e75dce3cfdae","owner":[],"postedDate":"June 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-18T12:38:07+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-05 22:23:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4440611","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4440611","identity":"rs-4440611","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}