Spermacoce alata Aubl. Essential Oil: Chemical Composition, In Vitro Antioxidant Activity, and Inhibitory Effects of Acetylcholinesterase, α-Glucosidase and β-Lactamase

Spermacoce alata Aubl. is widely available in the market as traditional Chinese medicine and animal feed, due to its properties of clearing heat and treating malaria and its high-protein and crude fiber content. In this study, the essential oil of S. alata was obtained through hydrodistillation. GC–MS and GC–FID methods were used to identify the chemical components and their relative abundance. Furthermore, the antioxidant capacity was measured using DPPH, ABTS, and FRAP assays, and the inhibitory effects of acetylcholinesterase, α-glucosidase, and β-lactamase were also evaluated. A total of 67 compounds were identified, with the major constituents being palmitic acid (30.74%), linoleic acid (16.13%), and phenylheptatriyne (8.07%). The essential oil exhibited moderate antioxidant activity against DPPH (IC50 > 10 mg/mL), while the IC50 value for the ABTS assay was 3.84 ± 2.12 mg/mL and the FRAP assay value was 87.22 ± 12.22 µM/g. Additionally, the essential oil showed moderate anti-acetylcholinesterase activity (IC50 = 286.0 ± 79.04 μg/mL), significant anti-α-glucosidase activity (IC50 = 174.7 ± 13.12 μg/mL), and potent anti-β-lactamase activity (IC50 = 37.56 ± 3.48 μg/mL). The results suggest that S. alata has the potential for application in pharmacology, warranting further exploration and investigation.


Introduction
Antioxidants are compounds that can prevent, delay, or reverse oxidation reactions by donating electrons to free radicals, thereby preventing cell dysfunction caused by free radicals [1].They are commonly used to prevent the oxidation of lipids and proteins and have been shown to play an essential role in preventing and controlling many diseases in the body, such as diabetes mellitus, neurodegenerative diseases, inflammation, and cancer, by mitigating the adverse effects of oxidative stress [2,3].While widely used synthetic antioxidants are effective, concerns exist regarding their potential adverse effects on human health, prompting a long-standing search for natural antioxidant sources [4].Plant essential oils have shown promising results in this regard and are increasingly in demand as natural alternatives to synthetic antioxidants [5,6], highlighting the significance of extracting antioxidant constituents from plants.
Alzheimer's disease, a neurodegenerative disease associated with impaired memory and other cognitive functions, is considered one of the most severe threats to older people [7].One of the principal therapeutic approaches for Alzheimer's disease centers on inhibiting acetylcholinesterase activity [8].Acetylcholinesterase is primarily responsible for breaking down acetylcholine, rendering it inactive and halting nerve signaling.Therefore, acetylcholinesterase inhibitors are commonly used in the treatment of Alzheimer's disease and other related conditions [9].Hung et al. found that plant essential oils possess inhibitory activity against acetylcholinesterase, which could profoundly influence the treatment strategies for Alzheimer's disease [10].In addition, studies have also shown that oxidative stress and free radical damage may be the initial indicators of Alzheimer's disease, and antioxidants can mitigate oxidative stress by protecting cells from Aβ-induced neurotoxicity or inhibiting the formation and stabilization of amyloid-β fragments (fAβ).Therefore, antioxidants play an essential role in managing and controlling Alzheimer's disease [11].Alzheimer's disease can be treated by anti-acetylcholinesterase and antioxidant methods [12].
Diabetes mellitus (DM) is a common chronic metabolic disorder characterized by hyperglycemia caused by the disturbance of carbohydrate metabolism [13].α-Glucosidase is a key glucoside hydrolase that catalyzes the hydrolysis of disaccharides and oligosaccharides into absorbable monosaccharides in the final step of carbohydrate digestion [14,15].Therefore, glucosidase inhibitors can effectively treat diabetes mellitus by delaying the breakdown of carbohydrates, inhibiting glucose absorption, and reducing blood sugar levels.However, acarbose, the most widely used drug, can cause abdominal discomfort, such as bloating and diarrhea, in nearly 20% of patients, making it essential to find alternatives [16].Previous studies have found that terpenoid compounds such as β-pinene, γ-terpinene, α-terpineol, and linalool from plant extracts and essential oils exhibited α-glucosidase inhibitory effects, making them potential treatments for diabetes mellitus [17,18].
β-lactam antibiotics are widely used to treat bacterial infectious diseases [19,20].They work by forming acylase complexes with penicillin-binding proteins (PBPs), destroying the integrity of the cell wall and eventually leading to cell lysis, thereby inhibiting transpeptidase activity [21].However, β-lactam antibiotics exert a strong selective pressure on bacteria, driving their evolution to produce a suite of enzymes capable of effectively degrading or inactivating β-lactam antibiotics, consequently endowing bacteria with increasing tolerance [22,23].Therefore, β-lactamase inhibitors need to be urgently developed [19,24].Hayanni and Shora found that various natural products from plants have an inhibitory effect on β-lactamase activity [25].Therefore, the extraction of β-lactamase inhibitors from plants might offer a potential treatment strategy for bacterial infections resistant to β-lactam antibiotics.
Plant essential oils, derived from flowers, fruits, stems, roots, and other parts of plants, are natural substances with a wide range of biological activities, such as antiacetylcholinesterase activity, anti-α-glucosidase activity, antibacterial activity, and antioxidant properties, which have promoted their application in the pharmacology, cosmetics, and food industries [26,27].Essential oils have been used for traditional medicinal purposes since antiquity, and the recent rise in interest in green consumerism has led to a preference for natural products over synthetic ones [28].In addition to their medicinal value, plant essential oils can be applied in the breeding industry to maintain animal health, enhance animal production capacity, and improve the quality of livestock products [29].Given the practical value of essential oils, further exploration of their potential capacity extracted from natural plants is warranted.
The genus Spermacoce comprises approximately 250 to 300 species distributed in tropical and subtropical regions [30].It is found in Mexico, South America, Africa, Asia, and Australia [31].Spermacoce alata is an herbaceous plant with pubescent stems, elliptical leaves, smooth surfaces, and membranous stipules.Known for clearing heat, detoxication, and high-quality feeding value, S. alata has broad application prospects.As a traditional medicine, S. alata treats malaria in Nepal [32].In Nigeria, they are used to treat human schistosomiasis [33].The compounds of S. alata can reduce chronic low-grade inflammation, hepatic lipid toxicity, oxidative stress, and insulin resistance by regulating the activity of metabolic transcription factors and taking advantage of the prebiotic activity, free radicalscavenging ability, and immunomodulatory properties of secondary metabolites to prevent and treat metabolic syndromes such as diabetes mellitus, fatty liver disease, atherosclerosis, and cardiovascular disorders, which have broad therapeutic prospects [34,35].In previous studies, it has also exhibited potent anti-leukemia activity [36].In agricultural production, S. alata can also be used as feed for livestock, with studies showing that adding S. alata to feed can significantly enhance the antioxidant capacity of chicken, extend the shelf life of meat products, and positively influence overall broiler production [37].
The clinical effectiveness and practical applications of S. alata primarily result from the various biological activities of its chemical components.However, no comprehensive investigation was conducted on the essential oil of S. alata.Therefore, the present study aims to investigate the antioxidant activity, anti-acetylcholinesterase, anti-α-glucosidase, and anti-β-lactamase activities of essential oil from S. alata.

Essential Oil Yield and Component Analysis
The essential oil of S. alata obtained by hydrodistillation was a green hydrophobic oily liquid.The average yield of essential oil was 0.10 mL/kg.In previous studies of the Rubiaceae family plants, the yields of the essential oils of R. tinctorum, C. glabra, and P. leiocarpa were 0.10 mL/kg, 0.08 mL/kg, 0.10 mL/kg, respectively [38][39][40].Moreover, the latest study found that five species of Rubiaceae plants yielded 0.24 mL/kg, 0.32 mL/kg, 0.08 mL/kg, 0.05 mL/kg, and 0.30 mL/kg, respectively [41].Collectively, the yield of our essential oil is similar to those mentioned above, which is consistent with the Rubiaceaecharacteristic yield.The total ion chromatogram (TIC) of S. alata is shown in Figure 1.
Molecules 2024, 29, x FOR PEER REVIEW 3 of 17 radical-scavenging ability, and immunomodulatory properties of secondary metabolites to prevent and treat metabolic syndromes such as diabetes mellitus, fatty liver disease, atherosclerosis, and cardiovascular disorders, which have broad therapeutic prospects [34,35].In previous studies, it has also exhibited potent anti-leukemia activity [36].In agricultural production, S. alata can also be used as feed for livestock, with studies showing that adding S. alata to feed can significantly enhance the antioxidant capacity of chicken, extend the shelf life of meat products, and positively influence overall broiler production [37].
The clinical effectiveness and practical applications of S. alata primarily result from the various biological activities of its chemical components.However, no comprehensive investigation was conducted on the essential oil of S. alata.Therefore, the present study aims to investigate the antioxidant activity, anti-acetylcholinesterase, anti-α-glucosidase, and anti-β-lactamase activities of essential oil from S. alata.

Essential Oil Yield and Component Analysis
The essential oil of S. alata obtained by hydrodistillation was a green hydrophobic oily liquid.The average yield of essential oil was 0.10 mL/kg.In previous studies of the Rubiaceae family plants, the yields of the essential oils of R. tinctorum, C. glabra, and P. leiocarpa were 0.10 mL/kg, 0.08 mL/kg, 0.10 mL/kg, respectively [38][39][40].Moreover, the latest study found that five species of Rubiaceae plants yielded 0.24 mL/kg, 0.32 mL/kg, 0.08 mL/kg, 0.05 mL/kg, and 0.30 mL/kg, respectively [41].Collectively, the yield of our essential oil is similar to those mentioned above, which is consistent with the Rubiaceaecharacteristic yield.The total ion chromatogram (TIC) of S. alata is shown in Figure 1.The retention time (RT), retention index (RI), and percentage (%) of this essential oil are listed in Table 1 according to the elution sequence on the HP-5MS column.A total of 67 compounds were identified, accounting for 95.44% of the essential oil from GC-FID (Supplementary Materials) and GC-MS analysis [42].The main components were palmitic acid (30.74%), linolenic acid (16.13%), phenylheptatriyne (8.07%), hexahydrofarnesyl acetone (4.44%), tetradecanoic acid (3.16%), linalool (3.08%), and caryophyllene oxidate (2.69%).Among them, fatty acids account for 55.03%, sesquiterpenoids for 9.70%, and monoterpenoids for 9.49%.These results indicate that S. alata could be classified as a fatty acid chemotype.As previously reported, essential oils from other Spermacoce plants, such as S. pusilla, contained main compounds including palmitate (25.09%), oleic acid (7.78%),The retention time (RT), retention index (RI), and percentage (%) of this essential oil are listed in Table 1 according to the elution sequence on the HP-5MS column.A total of 67 compounds were identified, accounting for 95.44% of the essential oil from GC-FID (Supplementary Materials) and GC-MS analysis [42].The main components were palmitic acid (30.74%), linolenic acid (16.13%), phenylheptatriyne (8.07%), hexahydrofarnesyl acetone (4.44%), tetradecanoic acid (3.16%), linalool (3.08%), and caryophyllene oxidate (2.69%).Among them, fatty acids account for 55.03%, sesquiterpenoids for 9.70%, and monoterpenoids for 9.49%.These results indicate that S. alata could be classified as a fatty acid chemotype.As previously reported, essential oils from other Spermacoce plants, such as S. pusilla, contained main compounds including palmitate (25.09%), oleic acid (7.78%), humulene (6.19%), and humulene oxide II (6.08%) [41], which also exhibited a notable abundance of fatty acids and esters.However, the main components of S. verticillata essential oil were phytol (56.30%), 1,8-cineole (20.40%), α-pinene (7.10%), and p-cymene (4.00%).This ter-penoid chemotype is different from other species within the genus Spermacoce, suggesting the chemical diversity within the genus [43].One previous study focused on the chloroform extracts of S. alata and identified 35 compounds, including mono (2-ethylhexyl) phthalate (21.64%), isobutyl acetate (14.62%), hexahydrofarnesyl acetone (12.66%), n-hexanal (12.28%), and 2-nitrocthanol (8.09%) [44].These compounds are similar to those of the essential oil of S. alata analyzed in the present study, which is rich in fatty acid and esters compounds.The differences in their compound composition may be attributed to their distinct extraction methods.In previous studies, fatty acid chemotype essential oil was considered to possess antioxidant, anti-acetylcholinesterase, and other biological activities, providing a research direction for further exploration of phytomedicine [45].The predominate component in essential oil, palmitic acid (30.74%), a saturated fatty acid, has been reported to exhibit antibacterial activity by impairing the bacterial cell membrane, causing the leakage of cellular contents and ultimately resulting in bacterial death [46].Additionally, low concentrations of palmitic acid exert a protective antioxidant effect in cardiomyoblasts, suggesting its potential to protect the heart from oxidative stress [47].The second most abundant compound is linolenic acid (16.13%), an unsaturated fatty acid commonly found in nature, which can attenuate Alzheimer's disease pathology, such as tau phosphorylation, bloodbrain barrier disruption, synaptic dysfunction, and cognitive impairment.Furthermore, it can be a therapeutic agent for diabetes mellitus by enhancing mitochondrial biogenesis and modulating insulin signaling [48].The third major component, phenylheptatriyne (8.07%) exhibits significant antibacterial activity against various strains of bacteria, laying a foundation for replacing synthetic fungicides with phenylheptatriyne [49].The compounds in the S. alata mentioned above can treat various diseases, livestock breeding, and the food industry.Therefore, regarding the rich chemical composition, we further conduct experiments on the antioxidant activity, anti-acetylcholinesterase, anti-α-glucosidase, and anti-β-lactamase activities of the essential oil to explore its potential medical value.

Antioxidant Activity Evaluation
Free radical diphenylpicrylhydrazyl (DPPH), 2,20-azinobis (3-ethylbenzothiazoline-6sulfonate) (ABTS), and ferric reducing activity power assay (FRAP) are the most prevalent analytical assays used in antioxidant evaluation [50].In this study, the antioxidant activity of S. alata essential oil was measured by DPPH, ABTS, and FRAP assays.The antioxidant values of the three assays are presented in Table 2. DPPH determination is a simple, effective, and rapid method widely used in studying natural compounds to evaluate antioxidant activity [51].The DPPH radical accepts electrons or hydrogen radicals from the donor compound and exhibits a strong absorption band at 515-520 nm [52].At the highest concentration (10 mg/mL), the antioxidant activity of essential oil was 31.98%,whose potency seems weaker than those obtained from essential oils of other species [45,53,54].
The results in Figure 2 showed that the activities of essential oil and Trolox increased in a sigmoidal dose-dependent manner within the concentration range in the ABTS assay.The IC 50 values of ABTS-scavenging capacity of our essential oil and Trolox were 3.84 ± 2.12 mg/mL and 6.1 ± 1.4 µg/mL, respectively.Prior research has shown the IC 50 values of A. annua and O. vulgare to be at 5.97 ± 0.51 mg/mL and 7.35 ± 0.30 mg/mL, respectively [55,56], suggesting their ABTS •+ free radical-scavenging activities are inferior to our essential oil.Nonetheless, other essential oils, such as S. rhombifolia and T. triquetrum, have demonstrated superior ABTS •+ -scavenging activities with IC 50 values of 1.47 ± 0.01 mg/mL [53] and 2.12 ± 0.05 mg/mL [54], indicating a more potent ability to neutralize free radicals.In the preceding experiments, the ABTS assay consistently demonstrated superior sensitivity in detecting free radical-scavenging activity compared to the DPPH assay.The result suggests that the ABTS assay may offer enhanced precision in antioxidant activity [57].The difference between the DPPH and ABTS assay may be attributable to slightly different mechanisms of action: DPPH is mainly based on the hydrogen atom transfer (HAT) mechanism.In contrast, the ABTS assay is primarily based on the electron transfer (ET) mechanism [58].Furthermore, DPPH radicals are more sensitive to the reaction environment (solvent, pH, and temperature) than ABTS •+ radical cations, resulting in higher variability, which may be why the ABTS assay manifested a stronger ability to scavenge The difference between the DPPH and ABTS assay may be attributable to slightly different mechanisms of action: DPPH is mainly based on the hydrogen atom transfer (HAT) mechanism.In contrast, the ABTS assay is primarily based on the electron transfer (ET) mechanism [58].Furthermore, DPPH radicals are more sensitive to the reaction environment (solvent, pH, and temperature) than ABTS •+ radical cations, resulting in higher variability, which may be why the ABTS assay manifested a stronger ability to scavenge free radicals [59].
The FRAP assay is used to evaluate the total antioxidant capacity of antioxidants to reduce Fe (III)-TPTZ to Fe (II)-TPTZ in the presence of a low pH [60].The absorbance increases with the formation of the Fe (II)-TPTZ complex [61].As shown in Table 2, the essential oil of S. alata exhibited an antioxidant capacity of 87.22 ± 12.22 µM/g measured by the FRAP assay, surpassing other plants such as O. basilicum (47.88 ± 1.08 µM/g) [62], M. coromandelianum (63.24 ± 4.81 µM/g) [63].Since the FRAP assay is based on the single electron transfer (SET) mechanism, it offers a distinct advantage because it is not limited to specific structural groups or compounds.Instead, it evaluates all oxidizable entities that can engage with the assay reagent.Consequently, it provides a quantifiable measure of reducing capacity, distinguishing it from methods focusing on radical-scavenging activity [59].
The antioxidant capacity is generally a multifaceted attribute.It is more worthwhile to select different methods that are not closely related to each other to understand the antioxidant mechanisms in the specific antioxidant [59].
Prior research has demonstrated that molecules capable of scavenging DPPH free radicals feature double bonds, especially those with conjugated double bonds which afford rapid and efficient scavenging activities, such as β-cyclotrienal (0.17%), β-ionic ketone (1.29%), and α-terpinol (0.39%) in this essential oil.Their conjugated double bonds can form a resonance structure with the DPPH free radical, reducing it to hydrazine and forming an antioxidant free radical, thereby terminating the free radical chain reaction [64].Nonetheless, the relatively low abundance of such compounds may account for its diminished DPPH free radical-scavenging activity within our essential oil.In addition, phenolic compounds, such as 4-ethyl-2-methoxyanisole (0.26%) in this essential oil, can provide hydrogen in the hydroxyl group to scavenge superoxide anions, hydroxyl radicals, and other free radicals under in vitro conditions effectively [65,66].Moreover, terpenoids containing enols have unsaturated hydroxyl groups, such as phytol (1.25%) and linalool (3.08%) in this essential oil may also exhibit intense antioxidant activity [67].This could potentially be the principal contributor to the antioxidant ability of this essential oil.

Anti-Acetylcholinesterase Activity
Acetylcholinesterase inactivates neurotransmitters in cholinergic synapses by hydrolyzing acetylcholine.Therefore, compounds that possess anti-acetylcholinesterase ability are considered promising in the treatment of neurodegenerative diseases such as Alzheimer's disease [68].In this study, we assessed the anti-acetylcholinesterase activity of the essential oil.As detailed in Table 3, the essential oil demonstrated an IC 50 value of 286.0 ± 79.04 µg/mL, indicating a stronger anti-acetylcholinesterase activity than essential oil from C. limon (849.90 ± 11.50 µg/mL) and F. vulgare (1187.7 ± 11.50 µg/mL) [69] but weaker than essential oil from L. nervosa (51.96 ± 14.26 µg/mL) and O. majorana (150.33 ± 2.02 µg/mL) [45,66], suggesting that this essential oil possesses moderate antiacetylcholinesterase activity.Previous studies have shown that the anti-acetylcholinesterase activity of essential oil can be attributed to monoterpenoids or oxygen-containing monoterpenoids, especially the bicyclic monoterpenoids containing allyl methyl groups [66].In another study, linalool is one of the monoterpenoids with the most vigorous acetylcholinesterase inhibitory activity [70].Therefore, we speculated that linalool (3.08%), camphol (0.56%), and nerol (0.33%) may be the sources of anti-acetylcholinesterase activity (Figure 3).terase activity.Previous studies have shown that the anti-acetylcholinesterase activity of essential oil can be attributed to monoterpenoids or oxygen-containing monoterpenoids, especially the bicyclic monoterpenoids containing allyl methyl groups [66].In another study, linalool is one of the monoterpenoids with the most vigorous acetylcholinesterase inhibitory activity [70].Therefore, we speculated that linalool (3.08%), camphol (0.56%), and nerol (0.33%) may be the sources of anti-acetylcholinesterase activity (Figure 3).

Anti-α-Glucosidase Activity
Diabetes mellitus is a chronic metabolic disease characterized by hyperglycemia.Studies have shown that α-glucosidase is vital in glucose suitable for intestinal absorption.Consequently, the inhibition of α-glucosidase can significantly assist individuals with diabetes mellitus in maintaining tighter control over their blood glucose levels [71].In this

Anti-α-Glucosidase Activity
Diabetes mellitus is a chronic metabolic disease characterized by hyperglycemia.Studies have shown that α-glucosidase is vital in glucose suitable for intestinal absorption.Consequently, the inhibition of α-glucosidase can significantly assist individuals with diabetes mellitus in maintaining tighter control over their blood glucose levels [71].In this study, we assessed the anti-α-glucosidase activity of the essential oil.The findings, as presented in Table 3, revealed an IC 50 value of 174.70 ± 13.12 µg/mL for our essential oil, significantly exceeding the activities of essential oils from C. sativum (6.24 ± 0.86 mg/mL) and C. carvi (6.83 ± 0.76 mg/mL) [68], suggesting the essential oil exhibits a relatively stronger inhibitory effect against α-glucosidase, which may contribute to the management of diabetes mellitus.Prior research has demonstrated that essential oils from sesquiterpenerich plants, such as P. nissolii, exhibit notable anti-α-glucosidase activity [72,73].Possessing this characteristic, essential oil encompasses a diverse array of 10 sesquiterpenoid species totaling 9.70%.Notably, hexahydrofarnesyl acetone (4.44%) and caryophyllene oxide (2.69%) are postulated to be the primary constituents within our essential oil that exert the anti-α-glucosidase effect (Figure 4).betes mellitus.Prior research has demonstrated that essential oils from sesquiterpene-rich plants, such as P. nissolii, exhibit notable anti-α-glucosidase activity [72,73].Possessing this characteristic, essential oil encompasses a diverse array of 10 sesquiterpenoid species totaling 9.70%.Notably, hexahydrofarnesyl acetone (4.44%) and caryophyllene oxide (2.69%) are postulated to be the primary constituents within our essential oil that exert the anti-α-glucosidase effect (Figure 4).

Anti-β-Lactamase Activity
β-Lactamase, which hydrolyzes β-lactam antibiotics, poses a significant threat to the efficacy of antibacterials.However, drug discovery and development have led to the introduction of β-lactamase inhibitors, providing a novel strategy to surmount this significant clinical challenge [74].In the experiment, we evaluated the anti-β-lactamase activity of essential oil.The IC50 value in Table 3 was 37.56 ± 3.48 µg/mL, indicating that the essential oil had significantly robust anti-β-lactamase activity.However, the record of anti-βlactamase activity in vitro is currently unavailable.From previous evaluations of the antibacterial activity of bacteria that can produce β-lactamase, we know that monoterpenoids such as citral, laurene, menthol, and camphor have good antibacterial activity.Therefore, we speculate that the abundant terpenoids in the essential oil could be instrumental in its pronounced anti-β-lactamase activity [75,76].In addition, phenylheptatriyne showed significant selective antibacterial activity against gram-positive bacteria in vitro [49].Within the composition of this essential oil, phenylheptatriyne (8.07%) is identified as the third most prevalent compound, which may be the primary factor contributing to the essential oil's potent resistance to β-lactamase (Figure 5).

Anti-β-Lactamase Activity
β-Lactamase, which hydrolyzes β-lactam antibiotics, poses a significant threat to the efficacy of antibacterials.However, drug discovery and development have led to the introduction of β-lactamase inhibitors, providing a novel strategy to surmount this significant clinical challenge [74].In the experiment, we evaluated the anti-β-lactamase activity of essential oil.The IC 50 value in Table 3 was 37.56 ± 3.48 µg/mL, indicating that the essential oil had significantly robust anti-β-lactamase activity.However, the record of anti-β-lactamase activity in vitro is currently unavailable.From previous evaluations of the antibacterial activity of bacteria that can produce β-lactamase, we know that monoterpenoids such as citral, laurene, menthol, and camphor have good antibacterial activity.Therefore, we speculate that the abundant terpenoids in the essential oil could be instrumental in its pronounced anti-β-lactamase activity [75,76].In addition, phenylheptatriyne showed significant selective antibacterial activity against gram-positive bacteria in vitro [49].Within the composition of this essential oil, phenylheptatriyne (8.07%) is identified as the third most prevalent compound, which may be the primary factor contributing to the essential oil's potent resistance to β-lactamase (Figure 5).

Plant Materials
The sample of the S. alata was collected from Pingnan County (23 • 16 ′ 23.78 ′′ N, 110 • 30 ′ 41.92 ′′ E), Guigang City, the Guangxi Zhuang Autonomous Region, China.After being identified by Professor Hong Zhao, the sample was deposited in the Center for Bioscience Analysis and Testing, Shandong University, Weihai, China.The registration number is EO2304.

Essential Oil Hydrodistillation
The dried leaves and stems (1 kg) were crushed into powder by a grinder and then put into a 5 L round-bottom flask with 2.0 L ultrapure water (Milli-Q Reference, Millipore, Billerica, MA, USA).The essential oil was extracted from the plant materials by hydrodistillation in a Clevenger-type apparatus for about 4 h.The essential oil was separated from the water layer using ether, and the resulting essential oil was then dried by nitrogen (Termovap sample concentrator, MD200-1, Shanghai Huyi Technology Co., Ltd, Shanghai, China) and anhydrous sodium sulfate to obtain the essential oil.The obtained essential oil was stored at a low temperature (−4 • C) for further analysis.

GC-MS and GC-FID Analysis
Agilent gas chromatographic-mass spectrometer (7890-5975C, Agilent, Santa Clara, CA, USA) was used for the GC-MS analysis of the essential oil, equipped with HP-5MS type fused quartz string (30 m × 0.25 mm × 0.25 µm, Agilent, Santa Clara, CA, USA).The gas chromatographic conditions were set as follows: Interface temperature: 280 Nitrogen was used as the carrier gas at a flow rate of 1.1 mL/min [78].Identifying these compounds in the essential oil was based on comparing mass spectrometry data with the NIST/EPA/NIH 2020 Mass Spectral Database and Kovat's retention indices associated with retention times.Kovat's retention indices were calculated by the retention time of a series of n-alkanes (C 8 -C 30 ) [79].

DPPH Method
The DPPH radical-scavenging abilities of essential oil were determined according to the procedure in previous studies [80,81].6-Hydroxy-2,5,7,8-tetramethylchrome-Roman-2carboxylic acid (Trolox) was used as the positive control.An amount of 200 µL prepared 0.17 mmol/L DPPH (2,2-diphenyl-1-picroyl-hydrazine hydrate) solution with 50 µL ethanol was added to the microplate as a control.The sample blank was prepared with 50 µL ethanolic essential oil solution and 200 µL ethanol.Then, 50 µL ethanolic essential oil solutions (50, 25, 10, 5, 2.5, and 1 mg/mL) were added to 200 µL DPPH solution in a microplate.After incubating in darkness for 30 min, the microplate reader (Epoch, Biotech company, Minneapolis, MN, USA) was used to measure the absorbance at 516 nm.Microplate Manager software Gen5 (Version 2.09) was used to record the reading of each sample.The absorbance was tested three times to obtain the mean value.Finally, the free radical-scavenging activity (RSA%) was calculated according to the following formula: where, A Sample is the absorbance of the sample under different concentrations, A Control is the absorbance of the ethanol solution containing DPPH, and A Sample Blank is the absorbance of the ethanol solution without DPPH.

ABTS Method
In the experiment, 2,2-azolium-(3-ethylbenzothiazole-6-sulfonic acid) diammonium salt (ABTS, 7.4 mmol/L) was mixed with potassium persulfate (K 2 S 2 O 8 , 2.6 mmol/L) to produce ABTS •+ free radicals.The mixture is placed in a dark environment for 12 h to ensure a full reaction.The resulting ABTS •+ was diluted in anhydrous ethanol to obtain the working solution with an absorbance of 0.7 at 34 nm.Then, 200 µL diluted ABTS •+ solution was mixed with 50 µL gradient-diluted ethanol solutions (50, 25, 10, 5, 2.5, and 1 mg/mL) in a 96-well microplate.An amount of 50 µL ethanolic essential oil solution and 200 µL ethanol were mixed as a sample blank.After incubation for 6 min, the absorbance was measured at 734 nm [81,82].The experiment was repeated three times.The inhibition percentage (inhibition%) of the measured essential oil is calculated as follows: where A 0 represents the absorbance of 200 µL ABTS •+ diluted solution mixed with 50 µL ethanol, and A represents the absorbance of 200 µL ABTS •+ diluted solution mixed with 50 µL sample solution.

FRAP Method
A standard solution of Trolox was used as the positive control, and the blank sample was prepared with distilled water.To obtain the FRAP working reagent, 0.3 M pH 3.6 acetic acid buffer solution, 10 mmol/L TPTZ solution, and 20 mmol/L Fe (III) solution were mixed at the ratio of 10:1:1.50 µL diluted essential oil solution (5000, 2500, 1000, 500, 250, 100, 50, and 25 µg/mL) and 0.25 mg/mL Trolox solution (2.5, 5, 10, 15, 20, 25, and 50 µL) were mixed with 200 µL FRAP working reagent in a 96-well microplate, followed by incubation in a water bath at 37 • C for 40 min.After 40 min, the absorbance of the tested sample at 593 nm was measured using the microplate reader (Epoch, Biotech company, Minneapolis, MN, USA).All tests were performed in triplicate.The standard curve was constructed using Trolox, and the FRAP value was determined using Trolox as the standard.The absorbance values of the samples at known concentrations are substituted into the standard curve to obtain the equivalent value of Trolox, which serves as the standard for antioxidant capacity [81,83].

Anti-Acetylcholinesterase Activity Test
Ethanolic essential oil solution was diluted with pH 8.0 phosphate-buffered saline (PBS) solution to 2.5, 1.0, 0.5, 0.25, 0.10, and 0.05 mg/mL.Galantamine was used as a positive control.An amount of 145 µL PBS solution (0.1 M, pH = 8.0), 20 µL test sample solution, and 15 µL 0.11 U/mL acetylcholinesterase solution were mixed and then stored at 4 • C for 20 min.After that, 10 µL 2 mM 5,5-dithiobis-(2-nitrobenzoic acid) DTNB and 10 µL 15 mM acetylthiocholine iodide (ATCI) were added.The absorbance of each solution at 412 nm was measured every 1 min for 6 min [10].The experiments were performed in triplicate.The acetylcholinesterase inhibitory rate was calculated as where K E is the initial reaction rate of inhibited acetylcholinesterase, and K S is the initial reaction rate of uninhibited acetylcholinesterase. IC 50 was calculated using nonlinear regression.

Anti-α-Glucosidase Capacity Test
Ethanolic essential oil solution was diluted with pH 7.0 PBS solution to 5.0, 2.5, 1, 0.5, 0.25, 0.1, 0.05, and 0.025 mg/mL.Acarbose solution was used as the positive control.An amount of 80 µL 100 mM pH 6.8 PBS solution, 20 µL test sample solution, and 40 µL 0.25 U/mL α-glucosidase solution were mixed in a microplate and incubated at 30 • C for 10 min.Then, 20 µL 3.0 mg/mL of 4-nitrophenyl-α-D-glucopyranoside (pNPG) solution was added, homogenized, and incubated for 4 min.The absorbance was measured at 410 nm and recorded every 1 min for 6 min [84].All tests were performed in triplicate.The α-glucosidase inhibitory rate was calculated as where K E is the initial reaction rate of the uninhibited enzyme, and K S is the initial reaction rate of the inhibited enzyme.The IC 50 value was calculated using nonlinear regression.

Anti-β-Lactamase Capacity Test
Ethanolic essential oil solution was diluted with pH 7.0 PBS solution.Clavulanate Potassium solution was used as the positive control.An amount of 20 µL Test sample solution, 100 µL 1000 U/mL β-lactamase solution, and 30 µL PBS solution (50 mM, pH = 7.0) were added to the microplate.The mixture is incubated at 30 • C for 10 min.Then, 50 µL of Nitrocefin (0.1 mg/mL) was added, and the mixture was further incubated at 30 • C for another 10 min [85,86].The absorbance was measured at 489 nm.The tests were carried out in triplicate.The β-lactamase inhibitory rate is shown below: where A S is the absorbance of the sample containing essential oil, A sb is the blank reaction absorbance of the sample, A e is the enzymatic determination absorbance, and A b is the blank reaction absorbance.The IC 50 value was calculated using nonlinear regression.

Conclusions
The present study found that the essential oil of S. alata is a fatty acid chemotype containing major volatile compounds such as palmitic acid, linoleic acid, phenylheptatriyne, hexahydrofernesyl acetone, and tetradecanoic acid.The essential oil demonstrated moderate DPPH radical-scavenging ability (IC 50 > 10 mg/mL), ABTS •+ radical-scavenging ability (IC 50 = 3.84 ± 2.12 mg/mL), and FRAP total antioxidant capacity (87.22 ± 12.22 µM/g).In addition, the essential oil showed notable acetylcholinesterase inhibitory activity, with an IC 50 value of 286.0 ± 79.04 µg/mL.This study also revealed significant anti-α-glucosidase and anti-β-lactamase activities (IC 50 = 174.7 ± 13.12 µg/mL and 37.56 ± 3.48 µg/mL, respectively).Our results suggest that the essential oil may possess medicinal value in treating diseases such as Alzheimer's disease and diabetes mellitus.Additionally, it could be used in combination with antibiotics to enhance their antibacterial effects.However, further research involving in silico analysis, such as molecular docking and molecular dynamics simulations, as well as in vivo experiments, is necessary for the development and application of S. alata essential oil in the future.

Figure 1 .
Figure 1.The total ion chromatogram of S. alata essential oil derived from GC-MS.

Figure 1 .
Figure 1.The total ion chromatogram of S. alata essential oil derived from GC-MS.

Molecules 2024 , 17 Figure 2 .
Figure 2. Variation in ABTS radical−scavenging percentage with varying concentrations for S. alata essential oil (a) and Trolox (b).The data were calculated from three parallel experiments.

Figure 2 .
Figure 2. Variation in ABTS radical−scavenging percentage with varying concentrations for S. alata essential oil (a) and Trolox (b).The data were calculated from three parallel experiments.

Figure 3 .
Figure 3.The concentration−dependent anti−acetylcholinesterase activity of S. alata essential oil (a) and Galantamine (b).The data were calculated from three parallel experiments.

Figure 3 .
Figure 3.The concentration−dependent anti−acetylcholinesterase activity of S. alata essential oil (a) and Galantamine (b).The data were calculated from three parallel experiments.

Figure 4 .
Figure 4.The concentration−dependent anti−α−glucosidase activity of S. alata essential oil (a) and Acarbose (b).The data were calculated from three parallel experiments.

Figure 4 .
Figure 4.The concentration−dependent anti−α−glucosidase activity of S. alata essential oil (a) and Acarbose (b).The data were calculated from three parallel experiments.

Molecules 2024 , 17 Figure 5 .
Figure 5.The concentration−dependent anti−β−lactamase activity of S. alata essential oil (a) and Clavulanate Potassium (b).The data were calculated from three parallel experiments.

Figure 5 .
Figure 5.The concentration−dependent anti−β−lactamase activity of S. alata essential oil (a) and Clavulanate Potassium (b).The data were calculated from three parallel experiments.
• C; Injector temperature: 260 • C; Carrier gas: He; Flow rate: 1.0 mL/min; Heating program setting: initial temperature of 50 • C for 4 min, 6 • C/min to 280 • C, and held for 3 min.The mass spectrum conditions were as follows: EI: 70 eV; Scanning range: 25-500 amu; Scan rate: 4.0 scan/s; Quadrupole temperature: 150 • C; Sample size: 0.3 µL [77].GC-FID analysis was performed using a PerkinElmer gas chromatograph (Clarus 500, Shelton, CT, USA) with an HP-5 fused silica capillary column (30 m × 0.25 mm, film thickness of 0.25 µm, Agilent, Santa Clara, CA, USA).The injector temperature was 260 • C, and the detector temperature was 305 • C. The oven temperature was initially set at 50 • C and held for 4 min, then raised from 50 • C to 280 • C at a rate of 6 • C/min, and maintained steady for 3 min.

Table 1 .
Chemical composition of essential oil distilled from S. alata.
Concentration is calculated from the total ion chromatogram; RI Calc : Calculated retention index.RI lib : Retention index obtained from the mass spectral database.RRI: Relative retention indices calculated against n-alkanes; Identification method based on the relative retention indices (RRI) of authentic compounds on the HP-5MS column; MS, identified based on computer matching of the mass spectra with NIST/EPA/NIH 2020 Mass Spectral Database and comparison with literature data.

Table 2 .
Antioxidant activities expressed as IC 50 values for DPPH, ABTS, and the antioxidant capacity of FRAP assays.