Phytochemical analysis, antioxidant, α-glucosidase inhibitory activity, and toxicity evaluation of Orthosiphon stamineus leaf extract

Ocimum aristatum, commonly known as O. stamineus, has been widely studied for its potential as an herbal medicine candidate. This research aims to compare the efficacy of water and 100% ethanolic extracts of O. stamineus as α-glucosidase inhibitors and antioxidants, as well as toxicity against zebrafish embryos. Based on the study findings, water extract of O. stamineus leaves exhibited superior inhibition activity against α-glucosidase, ABTS, and DPPH, with IC50 values of approximately 43.623 ± 0.039 µg/mL, 27.556 ± 0.125 µg/mL, and 95.047 ± 1.587 µg/mL, respectively. The major active compounds identified in the extract include fatty acid groups and their derivates such as linoleic acid, α-eleostearic acid, stearic acid, oleanolic acid, and corchorifatty acid F. Phenolic groups such as caffeic acid, rosmarinic acid, 3,4-Dihydroxybenzaldehyde, norfenefrine, caftaric acid, and 2-hydroxyphenylalanine and flavonoids and their derivates including 5,7-Dihydroxychromone, 5,7-Dihydroxy-2,6-dimethyl-4H-chromen-4-one, eupatorin, and others were also identified in the extract. Carboxylic acid groups and triterpenoids such as azelaic acid and asiatic acid were also present. This study found that the water extract of O. stamineus is non-toxic to zebrafish embryos and does not affect the development of zebrafish larvae at concentrations lower than 500 µg/mL. These findings highlight the potential of the water extract of O. stamineus as a valuable herbal medicine candidate, particularly for its potent α-glucosidase inhibition and antioxidant properties, and affirm its safety in zebrafish embryos at tested concentrations.

exhibit disease-prevention properties 3,4 .According to Ahda et al. 5 , this plant has various mechanisms of action to lower blood glucose levels, including boosting GLP-1 secretion and blocking α-glucosidase and α-amylase.Additionally, a nuvastatic supplement made from standardized O. stamineus extract for administration in diabetic retinopathy (DR) patients in clinical research has been registered (registration number NCT04552600).
The efficacy and potency of herbal medicines have made them a popular preventive measure against various diseases, with fewer risks of side effects compared to synthetic drugs.It is crucial to conduct toxicity evaluations to ensure the safety of herbal remedies.Improper usage, high dosage, long-term consumption, and inadequate monitoring can lead to increased side effects and potential toxicity [6][7][8] .Therefore, toxicological assessments are necessary to ensure the safety of potent herbal candidates.
Standard toxicity evaluations typically involve clinical studies on humans, as they provide relevant data for assessing the safety of herbal medicines before market authorization 8 .However, prior to human testing, it is essential to conduct preliminary screening tests to assess for toxic herbs.These tests commonly employ animal models such as mice and rabbits.This study aimed to identify the non-toxic potent extracts of O. stamineus leaves through toxicity evaluation using zebrafish embryos.
This method for toxicity evaluation offers several advantages, including large sample size, short-term use, genetic similarity, and cost-effectiveness 9,10 .Previous studies have assessed the toxicity of various herbal extracts on zebrafish embryos, revealing lethal concentrations (LC 50 ) dependent on the type of herbal and solvent used.For instance, the LC 50 values for water-methanol and water-ethanol extracts of Moringa oleifera were found to be 163.87 ± 12.88 mg/mL and 337.48 ± 30.04 mg/mL, respectively 11 .Norazhar et al. 12 demonstrated an LC 50 value of 419.84 μg/mL for the methanolic extract of Christia vespertilionis.
These findings highlight the significant impact of the type of herb and extraction solvent used on final toxicity.Notably, Sajak et al. 13 compared toxicity testing using zebrafish embryos and Wistar rats, finding that a polyphenolic-rich herbal mixture (PRM) had an LC 50 of approximately 487.50 μg/mL in zebrafish embryos, despite no lethal effects being observed in rats at a dose of 2000 mg/kg body weight.Zebrafish embryos exhibited higher sensitivity in toxicity evaluations, allowing for more sensitive detection and for herbal extracts with LC 50 values above 500 μg/mL to be classified as non-toxic.Therefore, this research provides an original viewpoint before approval and use of this herb in future clinical studies and treatment.

Samples preparation
O. stamineus was planted by the civil society in Yogyakarta, Indonesia (East longitude: 107° 15′ 03″ and East longitude: 107° 29′ 30″; South latitude: 7° 34′ 51″ and 7° 47′ 30″).Time of cultivation: February-March 2021 (Condition: last rainy season to first dry season).Because this plant grows naturally, it can be used freely (no permission required).Taxonomic identification was performed by Hery Setiyawan, M.Si (Department of Biology, Universitas Ahmad Dahlan).Fresh samples (leaf and stem) were washed and dried in an oven at 45 °C for four days.The dried samples were then ground into a powder and separated with a sieve size of 60 mesh.All procedures followed Good Agricultural and Collection Practice (GACP) scientific guidelines for starting materials of herbal origin and legislation.

Extraction process
The leaf powder of O. stamineus was extracted using ultrasonic-assisted extraction.10 g of O. stamineus leaf powder was dissolved in solvent (100% ethanol and water) with a solid-to-solvent ratio of 1:10 (w/v).Both samples were sonicated for 60 min at 50 °C using an ultrasonic batch and were incubated overnight 14 .Filtration was used to obtain the extract solution, which was then evaporated to obtain the dried extract.All extracts were freeze-dried and stored at 4 °C for further testing.

Determination of total phenolic content
Total phenolic content (TPC) was determined using the Folin Ciocalteu method, as described by Ahda et al. 15 .25 mg extract of O. stamineus leaf or stem was dissolved in 25 mL Aquadest and mixed with 1.5 mL Folin Ciocalteu (1:10 in water) for 3 min.The solution was then mixed with 1.2 mL of 7.5% sodium carbonate (w/v) and then left for 60 min.Absorbance was measured at 743 nm using a UV-Vis spectrophotometer (Shimadzu Uv-Vis 1800, Japan).Gallic acid equivalent concentration was used as the standard for total phenolic concentration.Phenolic content was expressed in μg/mg GAE (gallic acid equivalent) of dry weight extract.All measurements were carried out in triplicates.

Determination of total flavonoid content
Total flavonoid content (TFC) was determined by colorimetric method using aluminum chloride (AlCl 3 ) as reported by Chandra et al. 16 with minor modifications.25 mg of ethanolic extracts of O. stamineus leaf or stem was dissolved in 25 mL ethanol. 1 mL of the solution was mixed with 0.5 mL of 10% AlCl 3 and incubated at room temperature for 74 min.The absorbance was measured at 410 nm with a Uv-Vis spectrophotometer (Shimadzu Uv-Vis 1800, Japan).Quercetin standard was measured ranging from 5 to 20 µg/mL as the standard for total flavonoid concentration.TFC was calculated as μg/g quercetin equivalent (QE) of dried extract.All measurements were carried out in triplicates.

Inhibition activity of DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals
25 mg of O. stamineus leaf or stem was dissolved in 25 mL ethanol.The extract samples were diluted to concentrations ranging from 0 to 500 µg/mL 15 . 1 mL of extract solution was mixed with 1 mL of 0.05 mM DPPH solution and vortexed for 1 min.The mixture was kept for one hour.The mixture absorbance was analyzed using a www.nature.com/scientificreports/UV-Vis spectrophotometer at 516 nm (Shimadzu Uv-Vis 1800, Japan).The 50% inhibition concentration (IC 50 ) was calculated following the equation below: where A 0 is the absorbance of the control, A 1 is the absorbance of the samples.

Inhibition activity of ABTS (2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radicals
The inhibition activity of ABTS radicals was analyzed using a slightly modified method previously described by Byun et al. 17 .The reagent was incubated for 24 h at 37 °C after being mixed with 7.4 mM ABTS and 2.45 mM potassium persulfate solution in a 1:1 (v/v) ratio.The ABTS working solution was ready for use when absorbance value = 0.70 ± 0.02 at 734 nm.Briefly, 1 mL of ABTS solution was incubated with 1 mL of extract for 74 min.The solution used as a controlled standard is quercetin.ABTS radical scavenging activity was calculated using the following equation: where A is the absorbance of the control, B is the absorbance of the test sample.

Determination of inhibition activity against α-glucosidase enzyme
Inhibition activity of O. stamineus extract was determined following slightly modified methods described by 18 .α-glucosidase enzyme was prepared in a sodium phosphate buffer with a pH of 6.8 (15 U/100 mL).O. stamineus extract (final concentration ranging betwen 10-100 µg/mL) was reacted with α-glucosidase for 15 min.After that, the solution was incubated for 20 min with 5 mM p-nitrophenyl α-glucopyranoside (pNPG).The final composition ratio of α-glucosidase: pNPG extract was 200 µL:200 µL:200 µL.Finally, 1 mL of 0.2 M Na 2 CO 3 was added to break up the reaction.All solutions were analysed using a UV-Vis spectrophotometer (Shimadzu Uv-Vis 1800, Japan) at 400 nm.Percentage inhibition was determined by the equation: where As : Absorbance of sample, Ab: Absorbance of blank (without enzyme), Ac: Absorbance of control (DMSO + enzyme + PNPG).

Chemical analysis of O. stamineus leaf extract using high-resolution mass spectrometry (HRMS)
Samples were prepared following Windarsih et al. 19 with slight modifications.Analysis was performed by adding LC-MS-grade methanol into O. stamineus leaf extracts (water and ethanol extract).The mixture was vortexed for 2 min before being subjected to 30 min of ultrasonication.The pellet and the supernatant were separated by centrifuging at 5000 rpm for 5 min.The supernatant was transferred to a 2 mL HPLC vial after being filtered with a 0.22 m PTFE filter.
Analysis was performed using a Thermo Scientific Vanquish UHPLC system with a binary pump coupled with high-resolution mass spectrometry Q-Exactive Orbitrap.Separation was performed on a Thermo Scientific™ Acclaim™ VANQUISH™ C18 stationary phase column with the particle size in column dimensions (150 mm 2.1 mm ID 2.2 m), LC-MS-grade water (Merck) containing 0.1% formic acid (A) and LC-MS-grade methanol (Merck) containing 0.1% formic acid (B) as mobile phase.10 µL of the sample was injected into the column with a flow gradient of 0.30 mL/min from 5 to 90% B in 20 min and maintained at 95% A for 5 min.For mass spectrometric conditions, the sheath gas flow rate was set at 32 arbitrary units (AU), while the auxiliary and sweep gas flow rates were set at 8 and 4 AU, respectively.Scanning was carried out in both MS1 and MS2, with MS1 having a resolution of 70,000 and MS2 having a resolution of 17,500.Analysis was carried out concurrently in positive and negative ionization modes, with the collision energy set at 10 eV and the analytes scanned in the range of 66.7-1000 m/z.The chemical compositions of untargeted and targeted metabolites were identified using Compound Discoverer 3.2 software.The compounds were then examined for peak extraction using MzCloud and ChemSpider databases, with annotated masses ranging from -5 ppm to 5 ppm.Only chemicals with a complete MzCloud and ChemSpider match were selected for analysis.Peak intensities were modified to represent the overall spectrum intensity.

Toxicity evaluation using zebrafish embryos
Toxicity testing was performed following the OECD test guideline (TG) 236 described by Nipun et al. 20 with slight modifications.This procedure has been approved by the IIUM ethics committee, namely the IIUM Animal Care and Use Committee (I-ACUC) with register number: IACUC 2022-018.Zebrafish AB strain eggs (age < 5 h) were used in this study.100% ethanolic and water extracts of O. stamineus, negative control (E3 medium), positive control (4 mg/L of 3,4-dichloroaniline in E3 medium), and solvent control (1% dimethyl sulfoxide in E3 medium) were used in this investigation.Each group contained 20 eggs for each test concentration and 4 eggs as the internal plate control for each plate.Each well contained 300 μL of the solution consisting of 150 μL of E3 medium and 150 μL of the sample in 1% dimethylsulfoxide (DMSO).Zebrafish deaths were counted at intervals of 24 h, 48 h, 72 h, and 96 h.LC 50 was calculated via zebrafish mortality.Besides toxicity, teratogenicity criteria

Results and discussion
Yields, total phenolic content (TPC), and total flavonoids content (TFC) of O.

stamineus extracts
The extraction process of herbs is an important factor in the industry, due to the need for producing high yields while retaining bioactivity.Extracted herbs typically yield around 33.69 wt%, 6.05%, 4.42%, and 3.08% using various solvents such as water, ethyl acetate, ethanol, and n-hexane, respectively 21 .The results of this study show that different solvents used in extraction affect final yields.This is consistent with the findings of Ghasemzadeh et al. 22 , who report that increasing solvent polarity tends to increase yields.Extraction using 100% ethanol had no significant effect on yields between the leaf and stem of O. stamineus, whereas water extraction of O. stamineus leaves produced higher yields compared to stems.Furthermore, O. stamineus water extract contained more TPC than other extracts.The highest concentration of TFC was found in 100% ethanolic extract of O. stamineus leaves (See Table 1).According to Ibrahim and Jaafar 23 , O. stamineus leaves contained around 3.11 ± 0.27 mg/g and 1.47 ± 0.21 mg/g of TPC and TFC, respectively.Meanwhile, O. stamineus stems contained less TPC and TFC than its leaves.Therefore, the antioxidant activity of O. stamineus leaves is predicted to be greater than its stem.Hence, the assessments of α-glucosidase inhibitory activity, antioxidant properties, and toxicity on zebrafish embryos were performed using 100% ethanolic and water extracts of O. stamineus leaves.
Table 2 shows that 100% ethanolic extract had α-glucosidase inhibition activity compared to the water extract.According to a previous study, IC50 of the methanolic and ethanolic extracts of O. stamineus leaves were in the upper range of 1250 ppm 27 .Due to its ability to inhibit ABTS and DPPH radicals, the O. stamineus leaf water extract can be used as an antioxidant.The antioxidant capacity of this herb measured using Oxygen Radical  23 .The active components present in the O. stamineus leaf, including sinensetin, contributes to the extract's potency 25 .According to Yam et al. 28 , increasing the polarity of the solvent used reduces sinensetin, 3′hydroxy-5,6,7,4′-tetramethoxyflavone, and Eupatorin content in the extract.However, the water extract of O. stamineus leaves has high potency as an α-glucosidase inhibitor and antioxidant agent, as reported in Table 2.The difference in biological activities between the two extracts may be influenced by other active compounds present.Therefore, this study uses HR-MS to identify chemical compounds that are active in these extracts.

Toxicity evaluation of O. stamineus extracts using zebrafish embryos
Herbal remedies are currently preferred in the medical sector.However, their effectiveness, efficiency, and safety are crucial consideration factors before use in treatment.Zebrafish embryos are a frequently used model to test for the toxicity of herbs.Their use in pre-clinical studies provides the link between in-vitro and in-vivo studies 29 .Zebrafish embryo toxicity (ZFET) testing offers a number of advantages, including the large sample size, low cost, and simple handling 9,30 .Therefore, this toxicity evaluation model was used in this study.
According to Table 3, 100% ethanolic extract of O. stamineus leaves had a worse effect on Zebrafish embryo development than water extract when concentration was below 100 g/mL.The survival rate of zebrafish larvae exposed to 100% ethanolic O. stamineus leaf extract at 22.5 µg/mL was less than 50%, while the survival rate of zebrafish larvae contacted by water extract at 800 µg/mL was still higher than 90% (Fig. 1).Less pigmentation, delayed hatching, yolk edema, heart edema, and crooked backbone were among the physical defects present in numerous zebrafish embryos (see Fig. 2).
Lethal concentration 50 (LC 50 ) of the 100% ethanolic extract and aqueous extracts of O. stamineus leaves was 21.623 µg/mL and > 800 µg/mL, respectively.According to prior work by Ismail et al. 31 , the water extract of O. stamineus has an LC 50 of 1685 µg/mL, therefore the results reported here are consistent with previous findings.According to the recommendations of the Organization for Economic Cooperation and Development, compounds with LC 50 values of between 400 and 1000 µg/mL are classified as non-toxic 12,32 .In light of this research, O. stamineus leaf water extract is classified as a non-toxic herbal preparation.

Chemical compound profiling of O. stamineus leaf extract
The identification of active compounds in herbs is an important aspect of quality control.Storage condition is an important factor in maintaining consistent herb quality.This study employs high-resolution mass spectrometry (HR-MS) to identify the active compounds in O. stamineus leaf extracts.This analytical method predicts the active compounds in complex samples, especially herbal plants.He et al. 33 , utilised HR-MS to identify 167 illegal medicines found in herbal tea.Additionally, 68 compounds from B. intermedia and 81 compounds from S. marginata have been detected using HR-MS 34 .HR-MS combined with chemometrics is more efficient for investigating herbs used in Traditional Chinese Medicine based on quality markers 35 .
The HR-MS chromatograms show that water extract and 100% ethanolic extract of O. stamineus leaves contain different putative active compounds.According to Fig. 3, the water extract of O. stamineus leaves has dominant active compounds at retention times ranging between of 1-1.5 min and 20.5-23.5 min.These chemical compounds are grouped into fatty acid groups, triterpenoids, flavonoids and their derivates, quinones, hexoses compounds, phenolic compounds, and carboxylic acid groups and their derivates (Table 4).These compounds may explain the biological activities observed, including α-glucosidase inhibition and antioxidant activity.The extract also contains fatty acids such as stearic acid, α-eleostearic acid, linoleic acid, and others.Meanwhile, the 100% ethanolic extract contains carboxylic acid groups, flavonoid methyl ester groups, fatty acid groups, phenolic compound groups, and acyl groups.Table 4 lists the other active compounds present in this extract.Various compounds present in both water and ethanolic extracts of O. stamineus leaves have the ability to prevent oxidation processes, including caffeic acid, rosmarinic acid, etc. (Table 4).Caffeic acid is a member of the phenolic family with good antioxidant properties and works synergistically with other compounds to improve its action; however, it can occasionally act as a prooxidant when consumed in excessive amounts 36 .
Polyphenol caffeic acid (CA), derived from hydroxycinnamic acid, has been claimed as a remedy for many kinds of illnesses, including diabetes 37 .It can reduce blood glucose levels through the inhibition of α-glucosidase and α-amylase.Oboh et al. 38 discovered that caffeic acid had a superior ability to inhibit α-amylase and α-glucosidase with IC 50 values of 3.68 µg/mL and 4.98 µg/mL, respectively, and that its activity was better than chlorogenic acid (IC 50 values for α-amylase and α-glucosidase were 9.10 µg/mL and 9.24 µg/mL, respectively).This is a brief justification of the scientific data supporting the potency of both extracts as antioxidants and α-glucosidase inhibitors.
Although, both extracts contain many compounds or metabolites which can potentially protect against inflammation, as previously reported.For example, kukoamine A is a reported anti-inflammation compound, and is present in both water and 100% ethanolic extracts of O. stamineus leaves.This compound inhibits reactive oxygen species (ROS), nitric oxide (NO), prostaglandin E2, cyclooxygenase-2 activity, tumor necrosis factor-α, and interleukin-1 (IL-1), and IL-6 production, according to Wang et al. 39 .The extracts also contain polyunsaturated fatty acids, which are responsible for anti-inflammatory activities.However, the 100% ethanolic extract