Preliminary Study on Total Component Analysis and In Vitro Antitumor Activity of Eucalyptus Leaf Residues

Eucalyptus globulus is widely introduced and cultivated in Yunnan province. Its foliage is mainly used to extract eucalyptus oil, but the by-product eucalyptus residue has not been fully utilized. Based on the above reasons, in this study, we sought to explore the comprehensive utilization potential of eucalyptus resources. The total composition of eucalyptus residue was analyzed by ultra performance liquid chromatography-time-of-flight mass spectrometry (UPLC-Q/TOF MS), and the active components and nutrient components of eucalyptus leaf residue were determined by chemical methods and liquid phase techniques. Meanwhile, the antitumor activity of triterpenoids in eucalyptus leaves was evaluated by tetramethylazazole blue colorimetric assay (MTT). The results of qualitative analysis indicated that 55 compounds were identified from eucalyptus residue, including 28 phloroglucinols, 17 terpenoids, 3 flavonoids, 5 fatty acids, 1 amino acid and 2 polyphenols. Among them, the pentacyclic triterpenoids, in eucalyptus residue, were mainly oleanane type and urthane type. The results of quantitative determination indicated that the content of triterpenoid compounds was 2.84% in eucalyptus residue, which could be enhanced to 82% by silicone separation. The antitumor activity results showed that triterpenoid compounds have moderate inhibitory effects on human breast cancer cell MDA-MB-231, gastric adenocarcinoma cell SGC-7901 and cervical cancer cell Hela. The half maximal inhibitory concentration (IC50) was 50.67, 43.12 and 42.65 μg/mL, respectively. In this study, the triterpenoids from eucalyptus leaf residues were analyzed to reveal that the triterpenoids from eucalyptus leaf have antitumor effects and have potential to be developed as antitumor drugs.


Introduction
Eucalypt is the general name of the species of genus Angophora, Corymbia, and Eucalyptus of the Myrtaceae family, containing a total of 945 species and varieties that are important economic species naturally distributed in countries such as Australia [1].Known for their fast growth, high yield, strong adaptability, and wide-ranging applications, eucalyptus trees have been introduced to China for over a century.Currently, more than 600 counties in over 20 provinces across China cultivate eucalyptus plantations [2].Commonly planted eucalyptus species include Eucalyptus globulus, Eucalyptus citriodora, Eucalyptus grandis, and Eucalyptus urophylla, with major production regions in Guangdong, Guangxi, Yunnan, and Hainan [3].
One significant application of eucalyptus trees is for the production of eucalyptus essential oil.According to statistics, eucalyptus essential oil is a crucial export commodity for China, consistently ranking among the top four in export share from 2016 to 2020.Eucalyptus essential oil is well recognized for its anti-inflammatory, analgesic, anticancer, and antiviral properties, making it a valuable resource in the pharmaceutical industry [4].
Notably, Eucalyptus globulus cultivated in Yunnan has a high content of 1,8-cineole in its eucalyptus essential oil, exceeding 70%, making it a primary source for domestic eucalyptus essential oil production [5].However, the residue after the extraction of eucalyptus essential oil, namely eucalyptus leaf residue, is not fully utilized.At present, eucalyptus residue is usually burned, which causes serious waste of resources and environmental pollution [6].
In this study, we focus on exploring the potential comprehensive utilization of eucalyptus leaf residue.Firstly, compounds, extracted from eucalyptus leaf residue with organic solvents of different polarity, were analyzed by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS) and chemical methods.Subsequently, the extract was separated and purified by silica gel column chromatography, and high purity triterpenoids were obtained.Then, the efficacy of triterpenoids was evaluated through in vitro antitumor activity, aiming to provide insights into the pharmacological research of active components in eucalyptus leaf residue and offer a basis for the comprehensive utilization of eucalyptus leaf resources.

Qualitative Analysis of Components in Eucalyptus Leaf Residue
As described in Section 3.3, the chemical components in n-hexane and 70% ethanol extracts of eucalyptus leaf residue were separated and identified using UPLC-Q/TOF MS.Figures 1 and 2 display the total ion chromatograms of n-hexane and 70% ethanol extracts of eucalyptus leaf residue in negative ion mode.From the figures, it can be observed that both extracts are primarily composed of phenolic compounds and triterpenoids, with similar compositions but varying in component concentrations.A total of 55 compounds were identified in this study, including 28 phloroglucinols, 17 terpenoids, 3 flavonoids, 5 fatty acids, 2 polyphenols, and 1 amino acid.The specific components are listed in Tables 1 and 2.

Triterpenoids
Triterpenoids are a class of molecules with the molecular formula (C 5 H 8 ) n that are derivatives of isoprene.Depending on the number of isoprene units in the molecular structure, they can be classified as monoterpenes (n = 2), sesquiterpenes (n = 3), diterpenes (n = 4), triterpenes (n = 6), tetraterpenes (n = 8), and polyterpenes (n > 8), and are widely distributed in nature.Most terpenoids in nature are oxygen-containing derivatives, typically alcohols, aldehydes, ketones, carboxylic acids, esters, and glycosides.
The compounds were identified through the analysis of secondary mass spectrometry fragment ion information, literature review, and the understanding of mass spectrometry fragmentation patterns.According to Tables 1 and 2, a total of 17 terpenoids were identified in eucalyptus leaf residue, mainly comprising monoterpenes, sesquiterpenes, and triterpenes.Terpenoids in negative ion mode were primarily present in the form of [M − H] − .Among them, nine compounds belonging to monoterpenes and sesquiterpenes were identified, including eucalyptol, eucaglobulin, eucalmaidin D, and some formyl-substituted terpene components.Eight triterpenoids were identified, including five triterpenes mainly of the dammarane and ursane types, and three containing coumaryl substitutions [7].
Analysis of pentacyclic triterpenoids: Taking compound peak 10 (Figure 1) as an example, its retention time was 13.46 min, and the quasi-molecular ion peak in negative ion mode was [M − H] − at m/z 455.In the secondary mass spectrum, the sub-ion  ] − at m/z 363, with an additional ion peak at m/z 248 due to the Retro-Diels-Alder reaction.Based on mass spectrometric information and literature [8], it was inferred that compound 10 is dammarane/ursane/betulinic acid, and its fragmentation pattern is shown in Figure 3  peak in negative ion mode was [M − H] − at m/z 633.In the secondary mass spectrum, a triterpene product ion peak [M−H−C9H6O3] − at m/z 471 was observed, corresponding to the loss of O-p-coumaryl (162 Da), and an additional ion peak at m/z 248 due to the Retro-Diels-Alder reaction.Based on the above information and literature [9], it was inferred that peak 37 is 3-O-trans-p-Coumaroyltormentic acid, and its fragmentation pattern is shown in Figure 4.  Based on mass spectrometric information and literature [8], it was inferred that compound 10 is dammarane/ursane/betulinic acid, and its fragmentation pattern is shown in Figure 3.
Analysis of coumaryl-substituted pentacyclic triterpenoids: Taking compound peak 37 (Figure 1) as an example, its retention time was 23.62 min, and the quasi-molecular ion peak in negative ion mode was [M − H] − at m/z 633.In the secondary mass spectrum, a triterpene product ion peak [M−H−C9H6O3] − at m/z 471 was observed, corresponding to the loss of O-p-coumaryl (162 Da), and an additional ion peak at m/z 248 due to the Retro-Diels-Alder reaction.Based on the above information and literature [9], it was inferred that peak 37 is 3-O-trans-p-Coumaroyltormentic acid, and its fragmentation pattern is shown in Figure 4.

Phloroglucinols
Phloroglucinol and its derivatives are a unique class of compounds specific to Eucalyptus plants, characterized by distinctive structures.They often combine with monoterpenes, sesquiterpenes, and diterpenes to form novel compounds.Due to the diverse structures of the associated monoterpenes, sesquiterpenes, and diterpenes, a wide variety of phloroglucinol derivatives are formed.According to literature reports, phloroglucinol derivatives are mainly classified into three types of compounds: phloroglucinols, phloroglucinol dimers, and hybrids formed by phloroglucinols with monoterpenes, sesquiterpenes, or diterpenes.In eucalyptus leaf residue, a total of 28 phloroglucinol components were identified.Some are phloroglucinol dimers, primarily including Macrocarpal A and Sideroxylonal A/B/C [10][11][12].Others are hybrids formed by phloroglucinols with monoterpenes, sesquiterpenes, or diterpenes, mainly including eucalyptone, eucalyptal, Macrocarpal A/B/D/E, Macrocarpal C, and Macrocarpal I/J [7,12,13].
Analysis of phloroglucinol dimers: Taking compound peak 13 (Figure 1) as an example, its retention time was 15.14 min, and the quasi-molecular ion peak in negative ion mode was [M − H] − at m/z 499.In the secondary mass spectrum, the quasi-molecular ion peak [M−H−CO] − at m/z 471, formed by the removal of CO, was observed.This ion further dehydrated to form the [M−H−CO−H 2 O] − at m/z 453 sub-ion.Additionally, the base peak at m/z 249 of isopentyl dimethylphloroglucinol [C 13 H 13 O 5 ] − was observed.Based on the above information and literature [14], it was inferred that compound 13 is Sideroxylonal A/B/C, and its fragmentation pattern is shown in Figure 5a.Combining the fragmentation pattern of the compound with literature data [15,16], it was inferred that the compound was Eucalmaidin D/cypellogin A/B, and its fragmentation pattern is shown in Figure 6.
Molecules 2024, 29, x FOR PEER REVIEW 12 of 17 that the compound was Eucalmaidin D/cypellogin A/B, and its fragmentation pattern is shown in Figure 6.

Quantitative Analysis of Components in Eucalyptus Leaf Residue
Based on the quantitative method in Section 3.4.4,the active components (total sugars, total terpenes, phloroglucinol derivatives) and nutritional components (crude fiber, crude protein, crude fat, crude ash, moisture) of eucalyptus leaf residue were determined, with a total content of 76.99%.The content of each component in the eucalyptus leaf residue is shown in Table 3.

Quantitative Analysis of Components in Eucalyptus Leaf Residue
Based on the quantitative method in Section 3.4.4,the active components (total sugars, total terpenes, phloroglucinol derivatives) and nutritional components (crude fiber, crude protein, crude fat, crude ash, moisture) of eucalyptus leaf residue were determined, with a total content of 76.99%.The content of each component in the eucalyptus leaf residue is shown in Table 3.The content of terpenoids in eucalyptus leaf residue accounted for 2.84% of the raw material.Studies displayed that pentacyclic triterpenes, found in Eucalyptus, have inhibitory effects on cancer cells in the esophagus, lung [17], liver [18], breast [19], pancreas [20], colon, and stomach.The anti-tumor mechanism was mainly achieved by blocking the cell cycle of tumor cells, regulating the protein expression of related genes, inhibiting cell proliferation, inducing cell differentiation, and regulating the body's immune response.In the early stages of cancer treatment, patients might choose treatment drugs with minimal adverse effects or high efficiency at lower doses [21].Therefore, the pentacyclic triterpenes, from eucalyptus leaves, are expected to be developed as candidate anti-tumor drugs.
The content of phloroglucinol derivatives in eucalyptus leaf residue accounted for 1.93% of the raw material.Research has shown that phloroglucinol derivatives have good biological activities such as antibacterial, antiviral, and anti-tumor effects, with broad prospects for medical research, and are expected to become new anti-tumor and antibacterial drugs [22].
Eucalyptus leaf residue contains a small amount of flavonoids.Studies indicated that flavonoids in eucalyptus leaves have antibacterial, anti-cardiovascular, anti-inflammatory, analgesic and significant antioxidant properties, which could be developed for new, natural antioxidant-containing functional foods and applied widely in the pharmaceutical field [23].
The content of crude fiber in eucalyptus leaf residue was 27.6%, and the content of crude protein was 5.64%.Due to the high content of crude fiber, it could be used as fiber feed (the content of crude fiber ≥ 18%) in the feed industry.

In Vitro Antitumor Activity
In order to evaluate the antitumor activity of triterpenoids (content of 82.55%) from eucalyptus leaf residue, fluorouracil (5-FU) was chosen as the positive control drug.MDA-MB-231, SGC-7901, and Hela tumor cells were selected as experimental subjects, and the triterpenoids were tested for activity using the MTT (4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) colorimetric method.
The results, as shown in Table 4, demonstrated a certain antitumor activity of triterpenoids from eucalyptus leaf residue.Compared to positive drug (5-FU), they demonstrated moderate inhibitory activity against MDA-MB-231, SGC-7901, and Hela cells, with half-maximal inhibitory concentration (IC 50 ) values of 50.67, 43.12, and 42.65 µg/mL, respectively.Additionally, Wu et al. [24] studied the growth inhibition rate of mango saibao total triterpenes (TTC) on H 22 liver cancer cells.Results showed tumor inhibition rates of 42.65% and 43.63% for the high-dose groups (200 mg/kg and 400 mg/kg), respectively.Liang et al. [25] investigated the effects of total triterpenes from Celastrus orbiculatus on the proliferation, apoptosis, and invasion of human esophageal cancer Eca-109 cells.Studies revealed an inhibitory effect, with a maximum inhibition rate of 44.69% at a drug concentration of 160 µg/mL, indicating a dose-response relationship.Ma et al. [26] explored the antitumor activity of different doses of triterpenoid compounds from Laurencia, and results showed an inhibition rate of 39.1% in the high-dose group (63.29%, 100 mg/kg).Compared to positive drug (5-FU) and triterpenoid compounds from other plant sources, the antitumor activity of the triterpenoids from eucalyptus leaf residue was stronger than that of other plant triterpenes, and weaker than that of positive drugs (5-FU).To summarize, triterpenoids found in eucalyptus leaf residue exhibit moderate antitumor activity and hold potential for further development.The data from this study provided insights and theoretical support for pharmaceutical research on the effective components of eucalyptus leaves.

Materials and Reagents
Eucalyptus leaves were collected from Yunnan.The eucalyptus leaf residue was obtained after the extraction of essential oil from eucalyptus leaves by the method of steam distillation.The residue was dried, ground, sifted through a 40-60 mesh sieve, and stored for extraction.
Ethanol and n-hexane from Yongda Reagent (Tianjin, China); Acetonitrile from Fisher (Waltham, MA, USA); Deionized water (18.2MΩ•cm) from Millipore Milli-Q plus (Haverhill, MA, USA) system.Solvents were of analytical grade for extraction purposes and LC/MS grade for UPLC-Q/TOF MS.

Sample Preparation and Enrichment
Preparation of Analysis Samples: Firstly, 10 g eucalyptus leaf residue was weighed and transferred to a conical flask, followed by the addition of 60 mL of n-hexane for extraction at 50 • C for 3 h.The extract was then filtered and concentrated to obtain an n-hexane concentrate with a yield of 4.38%.Subsequently, the remaining residue was further extracted with 60 mL of 70% ethanol at 80.0 • C for 3 h, after which the extract was filtered and concentrated to obtain a 70% ethanol concentrate with a yield of 10.56%.Then, the n-hexane and 70% ethanol extracts were redissolved, diluted to 3.0 mg/mL, and filtered through a 0.22 µm membrane into the liquid phase injection vial for UPLC-Q/TOF MS analysis.
Isolation of Triterpenoid Components: Firstly, 10 g eucalyptus leaf residue was weighed and transferred to a conical flask, followed by the addition of 60 mL of 70% ethanol for extraction at 85 • C for 3 h.The extract was then filtered and concentrated to obtain ethanol extract.Subsequently, the triterpenoids, with high purity (content > 80%), were obtained from ethanol extract by silica gel column chromatography using a mixture of n-hexane and ethyl acetate (v:v, 2:1) as eluent, and were used for in vitro antitumor experiments.Due to the different polarities of n-hexane and 70% ethanol extracts, different elution solvents were used to ensure the complete elution of components.n-hexane extract was eluted and separated using 0.1% formic acid in water and isopropanol: acetonitrile (v:v, 1:1) as the mobile phase, and 70% ethanol extract was eluted and separated using 0.1% formic acid in water and acetonitrile as the mobile phase.

Mass Spectrometry Conditions
Electrospray ionization source (ESI) in negative ion mode was used to collect MS E data.Calibration solutions were 200 pg/µL leucine enkephalin solution and 0.5 mmol/L sodium formate solution.The scan range was m/z 50-1200, with a scan time of 0.5 s.In negative ion mode, the capillary voltage was 2.5 kV, cone voltage was 40 V, ion source temperature was 120 • C, and high-purity N 2 was used as the auxiliary spray ionization and desolvation gas.The desolvation gas temperature was 400 • C, and the flow rate was 800 L/h [27,28].

Data Analysis
Mass spectrometry data were collected and processed using Masslynx V4.1 software.The UNIFI scientific information system was used for data browsing, storage, and comprehensive analysis.Component identification was performed by extracting MS spectra and related MS/MS information, based on built-in mass spectrometry analysis platforms, including ChemSpider online databases (LIPID, Hmbd.ca, etc.) and traditional Chinese medicine databases (TCM Chinese [UNIFI 1.7]), combined with literature information [29][30][31].

Quantitative Methods
Total sugar, total phenol, total triterpenoid, and moisture content in the extracts were determined using the phenol-sulfuric acid method, Folin-phenol method, vanillinaluminum chloride method, and 105 • C oven method, respectively.Crude fiber, crude protein, crude fat, and crude ash content in eucalyptus leaf residue were determined according to [GB/T 5009.

Efficacy Experiments In Vitro Antitumor Activity Experiments
Cancer cells (human breast cancer cells MDA-MB-231, human gastric adenocarcinoma cells SGC-7901, and human cervical cancer cells Hela) were cultured in DMEM medium at 37 • C and 5% CO 2 until logarithmic growth phase.Cells in logarithmic growth phase were digested with trypsin, and 10 µL of cell suspension was transferred to a 96-well plate.After 24 h of adherent culture, 100 µL of different concentrations of triterpenoid solution (mass concentrations of 0, 6.25, 12.5, 25, 50, and 100 µg/mL) was added.After 48 h of incubation, 100 µL of CCK-8 reaction reagent (10:1) was added, mixed, and incubated for an additional 1.5 h at 37 • C and 5% CO 2 .The liquid in the wells was then removed, and 200 µL of 0.1% dimethyl sulfoxide (DMSO) was added to each well, followed by oscillation for 10 min to dissolve the crystals [36,37].Absorbance values (OD values) were detected at 450 nm using a microplate reader.The negative control group was the DMSO, and each experimental group was set up in 5 replicate wells.The inhibition rate of triterpenoids on cancer cells was calculated using Formula (1):

Statistical Analysis
GraphPad Prism 8 statistical software was used for statistical analysis, and Microsoft Excel 2010 was used to assist in calculating the half-maximal inhibitory concentration (IC 50 ) values.

Conclusions
This study employed UPLC-Q/TOF MS to isolate and identify the chemical components in eucalyptus leaf residue.A total of 55 chemical components were preliminarily identified, including phloroglucinol derivatives, terpenoids, flavonoids, polyphenols, organic acids, amino acids, and 77% of the chemical components of eucalyptus leaf residue were revealed.Furthermore, the residue was abundant in phloroglucinols and triterpenoid compounds, with contents of 1.93% and 2.84%, respectively, making it a valuable natural source for these compounds.Additionally, the triterpenoid compounds in eucalyptus leaf residue exhibited moderate inhibitory effects on MDA-MB-231, SGC-7901, and HeLa cells, indicating their potential as promising candidates for anticancer drug development.This research offers a framework for pharmaceutical studies on the active components of eucalyptus leaves, thereby establishing a theoretical foundation for the rational development and utilization of eucalyptus leaf residue resources.
[M−H−HCHO−H 2 O] − at m/z 407 was observed, indicating the loss of formaldehyde and water.Further removal of C 2 H 6 formed the sub-ion peak [M−H−HCHO−H 2 O−C 2 H 6 ] − at m/z 377, and subsequent removal of CH 2 resulted in the sub-ion peak [M−H−HCHO−H 2 O-C 2 H 6 −CH

Figure 4 .Figure 3 .
Figure 4. Fragmentation pathway of 3-O-trans-p-Coumaroyltormentic acid.2.1.2.PhloroglucinolsPhloroglucinol and its derivatives are a unique class of compounds specific to Eucalyptus plants, characterized by distinctive structures.They often combine with monoterpenes, sesquiterpenes, and diterpenes to form novel compounds.Due to the diverse structures of the associated monoterpenes, sesquiterpenes, and diterpenes, a wide variety of

Figure 4 .
Figure 4. Fragmentation pathway of 3-O-trans-p-Coumaroyltormentic acid.2.1.2.PhloroglucinolsPhloroglucinol and its derivatives are a unique class of compounds specific to Eucalyptus plants, characterized by distinctive structures.They often combine with monoterpenes, sesquiterpenes, and diterpenes to form novel compounds.Due to the diverse structures of the associated monoterpenes, sesquiterpenes, and diterpenes, a wide variety of
In the secondary mass spectrum, base peaks [M−H−H2O] − at m/z 453 and [M−H−CHO] − at m/z 443, formed by dehydration and removal of CHO, respectively, were observed.Additionally, the base peak at m/z 249 of isopentyl dimethylphloroglucinol [C13H13O5] − was detected.The fragmentation pathways observed were consistent with the mass spectrometric fragmentation reported for Macrocarpal A/B/D/E, suggesting that compound 11 is composed of Macrocarpal A/B/D/E, and its fragmentation pattern is shown in Figure5b.

Figure 5 .Figure 5 .
Figure 5. Fragmentation pathway of Sideroxylonal A (a) and Macrocarpal A (b).2.1.3.FlavonoidsFlavonoids are widely present in plants in nature and belong to the secondary metabolites of plants.Their basic skeleton is C6-C3-C6, and depending on the different substituents, they can be classified as flavones, anthocyanins, chalcones, flavonols, and so on.Different types of flavonoid glycosides and aglycones exhibit characteristic fragmentation patterns.Flavonoid aglycones are prone to undergoing neutral losses (such as the loss of sugar, -CH3, -CO, -H2O, -CH2O, and other free radicals) or undergoing Retro-Diels-Alder (RDA) reactions at the glycoside position, producing characteristic fragment ions.In eucalyptus leaf residue, three flavonoid compounds were identified, including Sideroxylin, Eucalmaidin D/cypellogin A/B, and leptospermone.The fragmentation patterns of these flavonoids are consistent with the literature-reported data for O-flavonoid glycosides.Analysis of flavonoids: Taking compound 4 (Figure2) as an example, its retention time was 1.43 min, and the quasi-molecular ion peak in negative ion mode was [M − H] − at m/z 629.In the secondary mass spectrum, the base peak [M−H−C10H14O2] − at m/z 463, formed by the loss of olivanic acid, was observed.This fragment ion further lost deoxyhexose to form the base peak [M−H−C10H14O2−C6H10O4] − at m/z 301 (quercetin).Combining the fragmentation pattern of the compound with literature data[15,16], it was inferred

Figure 2 .
. Total ion chromatography (TIC) of n-hexane extracts of Eucalyptus leaf residue in negative modes.Total ion chromatography (TIC) of 70% ethanol extracts of Eucalyptus leaf residue in negative modes. Figure 2. Total ion chromatography (TIC) of 70% ethanol extracts of Eucalyptus leaf residue in negative modes.
Figure 1.Total ion chromatography (TIC) of n-hexane extracts of Eucalyptus leaf residue in negative modes.Figure 1.

Table 1 .
Compounds identified from Eucalyptus leaf n-hexane extracts by UPLC-Q/TOF MS.

Table 3 .
Summary of the proportions of components in eucalyptus leaves.

Table 3 .
Summary of the proportions of components in eucalyptus leaves.

Table 4 .
Inhibitory effect of the triterpenoids and positive control drug against human cancer cell MDA-MB-231, SGC-7901 and Hela.