INTRODUCTION:

Medicinal plants, with their diverse secondary metabolite contents frequently connected with their pharmacological activity, make an outstanding contribution to health. Additionally, certain plant secondary metabolites have been effectively turned into modern therapeutic compounds^1,2. Plants produce volatile chemical compounds for self-defense as well as to attract pollinators³. Cananga odorata (Lam.) Hook.f. and Thomson (CO) is a plant species produces essential oils that assist humans in a variety of ways, including health. CO is a species from the Magnoliales ordo and Annonaceae family which currently has International Union for Conservation on Nature (IUCN) status as least concern species (LC) and is reported to have three forma, namely Cananga odorata forma fruticosa (shrub Cananga), Cananga odorata forma macrophylla (Javanese Cananga), and Cananga odorata forma genuina (ylang-ylang)⁴. Both Javanese Cananga and ylang-ylang forma have a tree habitus with a height of up to 30m, larger and thicker leaves, complete yellow-green flowers with a characteristic fragrant perfume, varying flower sizes with a length of up to 7.5cm, and a diameter of 11.5cm⁵. The ylang-ylang has been widely developed and cultivated by numerous entities and has been commercially employed as a raw material in the perfume and cosmetics industries⁶.

The extract and fractions of CO are believed to have some pharmacological activities needed for human health. Antibacterial, antibiofilm, antioxidant, anti-acne, anti-eczema, anti-psoriasis⁷, reducing neuropathic-pain, anti-anxiety, anti-depression, and treating other mood disorders⁸, anti-inflammatory, insect-repellent, antifertility and anti-melanogenesis activities have been reported by both in vitro and in vivo investigations of CO^9,10. These activities are correlated to bioactive phytochemicals include the phenolics, flavonoids, saponins, alkaloids, and essential oils. Plants rich in phenolics and flavonoids are related to antioxidant and antidiabetic activities due to their ability to reduce the oxidative stress responsible for diabetic and other degenerative diseases¹¹.

Due to the safety issues, the exploration of natural antioxidants from plants has grown recently. Natural antioxidants are any bioactive components present in extracts or fractions or pure compounds capable of delaying or inhibiting the oxidation reactions through several mechanisms including radical scavenging activities, lipid peroxidation inhibition, chelating agents, and synergists. Among these mechanisms, the radical scavenging activity is the most reported one due to its simplicity and rapidity¹². In addition, the determination of natural antioxidant capacity is related to the phenolics and total flavonoid contents, therefore, the studies of antioxidant activities are typically correlated to phenolic and flavonoid content¹³.

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by abnormally high levels of blood glucose in the human body¹⁴. In recent studies, some anti-diabetic drugs have been developed and used with some modes of action intended for controlling the levels of blood sugar by inhibiting metabolic enzymes including α-glucosidase and α-amylase, enhancing the uptake of glucose and blocking the DPP-4 enzyme¹⁵. However, undesirable side effects due to the use of anti-diabetic drugs including abdominal distention, liver disorder, diarrhea, nausea flatulence, and poor remedy effectiveness have been reported¹¹. Therefore, some antidiabetic agents from natural plants having less or even without side effects should be continuously explored and developed. One of the natural sources of antidiabetic agents is CO. The purpose of this study was to assess the antioxidant and anti-diabetic actions of three forma of CO namely of shrub Cananga, Javanese Cananga, and ylang-ylang and their relationship with phenolic and flavonoid levels.

MATERIALS AND METHODS:

Sampel Preparation:

In order to reduce environmental influences and the height of the plant's growth place, fresh leaves of shrub Cananga, Javanese Cananga, and ylang-ylang were collected from the same area, Rumah Atsiri Karanganyar in Central Java, Indonesia at an elevation of 766 meters above sea level and a soil pH of 6.43 in March 2024. The number of trees from which leaves were collected for each type of CO was one, with the criteria of having entered the generative phase, as indicated by the presence of flowers, and additional criteria for Javanese Cananga and ylang-ylang with tree habitus being a circumference of the main tree trunk greater than 31.4cm. The leaves collected were a mix of young and old leaves from trees that met the criteria. The leaves were thoroughly washed and dried with a mix of air-drying and oven-drying at 40°C for 2-3 days to produce a dry-herbs with a water content of less than 10% before being powdered with a 40mesh sieve and macerated in 70% ethanol for 72hours with stirring twice daily^16,17. The filtrate was evaporated to yield a dry or thick extract. The three CO samples were identified and determined at National Research and Innovation Agency's Traditional Medicine Raw Materials Standardization Laboratory through E-Layanan Sains-BRIN with the ID numbers 6449-18148212-1, 6449-18148212-2, and 6449-18148212-3 respectively.

Phenolic and flavonoid content evaluation:

The total phenolic content of the extract was determined using the Folin Ciocalteau reaction^18,19. The extract was produced at 1mg/mL and homogenized with a sonicator for 15minutes. To prepare the extract, 40µL was mixed with 360µL of distilled water (Ikapharmindo, Indonesia) and 10µL of 10% Folin-Ciocalteo reagent (Sigma-Aldrich, USA). The mixture was homogenized and left for 2minutes before adding 500µL of 5% sodium carbonate (E- Merck, Germany) and incubating at 40° for 20minutes. 150µL of sample solution was pipetted into a microplate. A UV-vis spectrophotometer measured absorbance at λ 732, and phenolic contents were determined as mg gallic acid equivalent per gram of sample (µg GAE/g extract). The standard curve equation for gallic acid in determining phenolic content obtained in this study is Y=0.0352X-0.0223. (R2=0.9985).

The total flavonoid content was obtained using quercetin as a reference¹⁹. Samples were taken by diluting extract of 10mg with ethanol of 10mL and homogenizing with a vortex and sonicator for 10minutes. The blank solution was made up of 350µL distilled water, 250µL acetate buffer, and 1 mL ethanol. To create the test solution, 350 µL of distilled water, 250µL of acetate buffer, 150µL of AlCl₃, and 1 mL of ethanol were combined. Next, the blank solution and sample of 150µL each was pipetted into a microplate, and the absorbance was read using a UV-vis spectrophotometer at λ 421nm. The total flavonoid concentration was reported in terms of quercetin equivalents (mg QE/g sample). The standard curve equation for quercetin in determining flavonoid content derived in the current study is Y=0.0231X-0.0006 (R2=0.9991).

DPPH radical scavenging activity:

The DPPH radical scavenging activity was measured following Sharma and Bhat (2009)²⁰. The sample solution contains a final volume of 1.0mL, an extract concentration of 0-100µg/mL, and a DPPH concentration of 70µM. Absorption measurements were measured at 515nm shortly after 25-60 minutes (depending on the operation time optimization). The inhibitory activity percentage was estimated by multiplying [(A₀-A₁)/A₀] by 100, in which A₀ is the control absorbance and A₁ is the absorbance of the test extract or standard. The antiradical activity was measured in IC₅₀ using L-(+)-ascorbic acid at a concentration of 0-5µg/mL as the positive control.

Ferric reducing antioxidant power

The test was performed using the approach described by Benzie and Strain (1996) with slight adjustments²¹. The FRAP reagent was prepared by mixing a 10mM TPTZ solution in 40mM HCl, 300mM acetate buffer pH 3.6, and 20mM FeCl3.6H2O solution in a volume proportion of 1:10:1. The vacuum curve was created with FeSO4.7H2O at a final concentration of 100-1,000 µM/mL. Fill a 2 mL microtube with 150µL of the test extract or antioxidant standard solution (quercetin, L-(+)-ascorbic acid) and 1,350µL of FRAP reagent. The test solution was next incubated for 30minutes at 30°C before being measured at a 595nm wavelength. The FRAP values were expressed as mM Fe2+/g sample.

Dipeptidyl peptidase 4 inhibition activities:

The protocol from the Elabscience DPP-4 inhibitor screening assay Elisa kit (Elabscience E-BC-D007), using sitagliptin as a standard, is used to determine DPP-4 inhibitory activity.

α-Glucosidase inhibition activity:

The extract's α-glucosidase inhibitory activity was assessed using the working instructions specified in the α-glucosidase inhibition activity Elisa kit (BioVision K938-100), with acarbose as the standard

Data analysis:

The data were examined using a chemometric approaches, specifically Principal Component Analysis (PCA) using the 18^th version of Minitab software (Minitab LLC Corp., State College, Pennsylvania, USA). The score plot of PCA was used to classify CO forma employing DPPH, FRAP, DPP-4, α-glucosidase, flavonoid, and phenolic contents variables, whereas the loading plot of PCA was utilized to determine the relationship between the variables.

RESULT:

Shrub Cananga, Javanese Cananga, and ylang-ylang:

The current research examined multiple variables coming from three forma of CO, namely shrub Cananga, Javanese Cananga, and ylang-ylang (Figure 1) obtained in Karanganyar Regency, Central Java, Indonesia, as Indonesia is one of the finest growing areas²², involving antidiabetic activity through inhibiting α-glucosidase and DPP-4, antioxidants by DPPH radical scavenging and ferric-reducing antioxidant capacity mechanisms, and figuring out the total amount of phenolic and flavonoid compounds of the extract. According to the extraction results, ylang-ylang exhibited the highest extract yield of 24.18%, followed by Javanese Cananga and shrub Cananga in order of 23.45 and 14.48%. Javanese Cananga and ylang-ylang have a tree-like habit, whilst shrub Cananga is shrubby. Figure 1 shows that each forma has distinct leaf and flower shapes, sizes, and colours. Javanese Cananga and ylang-ylang leaves are larger and thicker. The shrub Cananga flower stalks are longer than ylang-ylang.

Figure 1. Ylang-ylang (a), Javanese Cananga (b) and shrub Cananga (c) plants, leaves, and flowers (the picture is the author's property, taken first-hand in the Karanganyar Region, Central Java, Indonesia)

α-Glucosidase and DPP-4 Relative Inhibition (RI) activity of CO extracts

Table 1. α-Glucosidase and DPP-4 Relative Inhibition (RI) activity of ylang-ylang, Javanese Cananga, and shrub Cananga extracts

Sample	α-glucosidase RI activity		DPP-4 RI activity
	% RI	Mean of % RI ± SD	% RI	Mean of % RI ± SD
Ylang-ylang	23.95	22.28 ± 2.445	48.68	47.76 ± 1.26
	19.47		48.28
	23.42		46.31
Javanese Cananga	26.32	22.72 ± 5.344	63.42	60.32 ± 3.90
	25.26		61.58
	16.58		55.94
Shrub Cananga	12.37	10.53 ± 1.987	60.94	57.07 ± 4.22
	10.79		57.69
	8.42		52.57
Standard	97.11	97.63 ± 0.456 (Acarbose)	95.68	95.64 ± 0.04 (sitagliptin)
	97.89		95.6
	97.89		95.64

The extracts were investigated for antidiabetic efficacy in vitro inhibitory techniques for α-glucosidase and DPP-4, followed the procedures of the α-glucosidase inhibitor activity and Elisa screening kit for DPP-4 inhibitors with acarbose and sitagliptin as a standard. The study found that shrub Cananga had the lowest α-glucosidase inhibitory activity, followed by ylang-ylang and Javanese Cananga in that order. Meanwhile, ylang-ylang showed the lowest DPP-4 inhibitory activity, followed by shrub Cananga and Javanese cananga for as of 47.76; 57.07; and 60.32% respectively as stated on the Table 1.

Antioxidant activity of CO extracts:

Table 2. Ferric reducing activity power (FRAP) and 2,2- diphenyl-1-picrylhydrazyl (DPPH) radical scavenging data of ylang-ylang, Javanese Cananga, and shrub Cananga extracts

Sample	FRAP (mM Fe (II)/g extract)	FRAP mean ± SD	DPPH IC₅₀ (μg extract /mL)	DPPH IC₅₀ mean ± SD
Ylang-ylang	512.89	526.19 ± 12.78	59.67	60.53 ± 0.93
	527.29		60.41
	538.39		61.52
Javanese Cananga	718.1	724.30 ± 5.38	46.98	47.93 ± 0.99
	727.03		48.97
	727.77		47.84
Shrub Cananga	393.85	402.11 ± 7.31	82.07	83.09 ± 1.18
	404.76		84.38
	407.73		82.81

The antioxidant activity measurement employs the DPPH radical scavenging method and the reducing power of the Fe³⁺ complex to the Fe²⁺ complex revealed that Javanese Cananga had the most significant FRAP value and the lowest DPPH IC₅₀ compared to the other two forma, with values 724.30±5.38mM Fe (II)/g extract and 47.93±0.99μg extract/mL, respectively. Conversely, shrub Cananga had the lowest FRAP value and the highest DPPH IC₅₀ value for 402.11±7.31mM Fe (II)/g extract and 83.09±1.18μg extract/mL (Table 2).

The flavonoid and phenolic content of CO extracts

Table 3. The flavonoid content of ylang-ylang, Javanese Cananga, and shrub Cananga extracts

Sample	Blank (B) absorbance		Sample (S) absorbance		S-B	X value	Flavonoid (µg QEq/mL)	Mean±SD
Sample	1	2	1	2
Ylang-ylang	0.1221	0.1162	0.1333	0.1303	0.0127	0.5736	143.398	15.57 ± 1.19
	0.1022	0.1027	0.1183	0.1163	0.0149	0.6688	167.208
	0.1018	0.1024	0.119	0.1129	0.0139	0.6255	156.385
Javanese Cananga	0.0994	0.1004	0.1077	0.1061	0.007	0.329	82.251	9.13 ± 1.47
	0.1051	0.1045	0.1157	0.1127	0.0094	0.4329	108.225
	0.1059	0.1044	0.1127	0.1118	0.0071	0.3333	83.333
Shrub Cananga	0.0992	0.0992	0.1073	0.115	0.012	0.5433	135.823	14.30 ± 0.90
	0.1004	0.1034	0.1125	0.1184	0.0136	0.6126	153.139
	0.1028	0.1072	0.1149	0.1198	0.0124	0.5606	140.152

Table 4. The phenolic content of ylang-ylang, Javanese Cananga, and shrub Cananga extracts

Sample	Blank (B) absorbance		Sample (S) absorbance		S-B	Value		Phenolic (mg GAE/g)	Mean ± SD
	Blank (B) absorbance		Sample (S) absorbance		S-B	Value
	1	2	1	2		Y	X
Ylang-ylang	0.0833	0.083	12.905	12.911	12.077	12.077	336.747	84.19	83.75 ± 0.42
	0.0836	0.0868	13.036	12.691	12.012	12.012	334.901	83.73
	0.0823	0.0854	12.909	12.683	11.958	11.958	333.366	83.34
Javanese Cananga	0.0839	0.0853	14.792	14.548	13.824	13.824	386.392	96.6	96.35 ± 0.24
	0.0859	0.0832	14.549	14.711	13.785	13.785	385.270	96.32
	0.0836	0.0835	14.732	14.453	13.757	13.757	384.489	96.12
Shrub Cananga	0.0867	0.0855	0.9499	0.9475	0.8626	0.8626	238.722	59.68	59.37 ± 0.27
	0.0841	0.1027	0.9522	0.9462	0.8558	0.8558	236.790	59.2
	0.0998	0.088	0.9839	0.9166	0.8564	0.8564	236.946	59.24

The secondary metabolite content of the three forma was determined from the total phenol and flavonoid content. Table 3 demonstrates that ylang-ylang has the highest flavonoid concentration of 15.57±1.19µg QEq/mL, whereas Javanese Cananga has the highest total phenolic content of 96.35±0.24mg GAE/g, using gallic acid as a standard (Table 4). Shrub Cananga exhibited the lowest amount of both total flavonoids and total phenols.

Score and loading plot of CO extracts using chemometric approach

Figure 2. Score plot analysis of ylang-ylang (G1-G3), Javanese Cananga (M1-M3), and shrub Cananga (F1-F3) based on DPPH, FRAP, α-glucosidase, DPP-4, flavonoid and phenolic contents parameters

Figure 3. Loading plot from PCA analysis of DPPH, FRAP, α-glucosidase, DPP-4, flavonoid (TFC), and phenolic contents (TPC) parameters correlation of ylang-ylang, Javanese Cananga, and shrub Cananga extracts

Chemometric approaches with unsupervised pattern recognition using principal component analysis was conducted to figure out the clustering and correlation among variables. Figure 2 depicts the area plot of each CO forma depending on the variables analyzed, which include alpha glucosidase inhibitory activity, DPP-4, DPPH radical scavenging, ferric-reducing antioxidant capacity, the extract's total phenol and flavonoid content. Figure 3 shows PC1 and PC2 of analyzed samples based on DPPH, FRAP, DPP-4, α-glucosidase, flavonoid, and phenolic contents. PC1 accounted for 69.3% of the variance, whereas PC2 contributed 23.9%; hence, the first two PCs represented 93.3%. PC1 was dominated by FRAP, DPPH, and phenolic contents, whereas PC2 was driven by α-glucosidase and flavonoid contents.

DISCUSSION:

The medicinal properties of the CO flower have been widely reported, and has even been used commercially in the cosmetics and perfume industries; however, research on the CO leaves were still quite limited. This current research captures the antidiabetic activity, antioxidant properties, total phenolic, flavonoid content and its correlation among three forma of CO namely ylang-ylang, Javanese Cananga, and shrub Cananga specifically for their leaves part. Forma is a taxonomic category that classifies plants with morphological distinctions from other varieties, such as leaf colour and size or stem shape, but no substantial genetic differences and occurs irregularly in random populations²³. Aside from the tree habitus of ylang-ylang and Javanese Cananga and the shrub habitus of shrub Cananga, the three forma of CO differ in the shape and size of their leaves and flowers, with ylang-ylang having thicker leaves primarily arranged along a plane²⁴ and shorter flower petals than the other two. This is following previous studies that revealed that Javanese Cananga has larger leaf and flower sizes than ylang-ylang, and the flower petals of shrub Cananga are smaller but have greater numbers²⁵. Ylang-ylang is considered to offer various advantages over other forma, including better quality essential oils, faster flower growth, and an average tree height of 10-20meters, making it commonly cultivated for commercial purposes^26,27. Cananga oil of Javanese Cananga remains inferior in quality to the ylang-ylang essential oil extracted from ylang-ylang^4,28. The odorous component produced with various grades increases significantly as the flower matures as well as the distillation time^29,30.

The experimental results revealed that the three forma of CO exhibited antidiabetic efficacy, with the Javanese Cananga showing the strongest activity toward both α-glucosidase with acarbose as a standard and DPP-4 inhibition with sitagliptin as standard compared to the other two forma. Shrub Cananga has the least inhibitory effect for both antidiabetic activity inhibition methods. α-Glucosidase inhibitor prevents the conversion of oligosaccharides into glucose in the intestinal lumen as a result of food intake, preventing an increase in blood glucose levels in the body³¹. While DPP-4 inhibitors block the DPP-4 enzyme, an enzyme released by the small intestine that naturally degrades glucagon-like peptide-1, an enzyme responsible for stimulating insulin secretion from the pancreas^32,33. Javanese Cananga was discovered to have β-caryophyllene, caryophyllene oxide, benzyl benzoate, E-E farnesol, trans-β-guanine, benzyl alcohol, and geraniol³⁴. In agreement with the aforementioned, β-caryophyllene has been found to be among the constituent components of Ayurvedic formulations that have been accountable for their antidiabetic properties³⁵.

The antioxidant activity experiment yielded data consistent with antihyperglycemic activity, with Javanese Cananga having the greatest FRAP value and the lowest IC₅₀ DPPH radical scavenging compared to ylang-ylang and shrub Cananga. Likewise, shrub Cananga had the least FRAP value and the highest IC₅₀ for DPPH radical scavenging. The IC₅₀ value indicates the concentration of extract that delivers 50% of the DPPH radical scavenging inhibitory action. The lower the IC₅₀value, the higher the antioxidant activity of the extract. The FRAP value describes the extract's ability to reduce the Fe³⁺ complex into a yellow Fe²⁺ complex³⁶. The extract's antioxidant activity improves as the FRAP value increases. Furthermore, as compared to the latest literature, our findings seem to be consistent with prior studies, notably for the DPPH and FRAP tests. In this regard, Mrani et al. (2024)⁷ reported CO DPPH IC₅₀ and FRAP EC₅₀ values of 1.57-3.51 and 0.17mg/mL, respectively. These findings imply that COs' antioxidant effectiveness is reliable and consistent throughout investigations.

Surprisingly, the study's findings showed that the total flavonoid concentration was significantly distinct from the antidiabetic and antioxidant activity of Javanese Cananga extract of CO, which was 9.13µg QEq/mL. This value was the lowest compared with ylang-ylang and shrub Cananga, which possessed flavonoid contents of 15.57 and 14.30µg QEq/mL, respectively. Indeed, samples of CO of the same forma obtained from various locations exhibit genetic changes and chemical content that are considered to be influenced by the environment and temperature during the distillation process²⁴.

In contrast with the flavonoid content of the extract, the phenolic content utilizing standard gallic acid generated findings that were comparable to the antidiabetic and antioxidant activity, whereas Javanese Cananga had the greatest levels of phenolic compounds compared to the other two forma with a value of 96.35mg GAE/g. This is confirmed by earlier investigations. According to Allouache et al (2024)³⁷, high phenolic content in Diospyros kaki L., is implicated in the greater antioxidant properties. Irshad et al (2025)³⁸ and Shukla et al (2021)³⁹ both reported the same issue. This positive association is related to phenolic compounds' hydroxyl groups' contributions by donating their hydrogen atoms, which react with oxygen-free radicals and replace them with hydroxyls (OH), inhibiting or even stopping the radical chain reaction³⁹.

In this present study, we applied chemometric approaches with unsupervised pattern recognition using principal component analysis to investigate the three-forma categorization and correlation with evaluated variables. The score plot successfully provided an overview of the clustering of the three forma of CO, with each form dispersed at various ordinates. A Loading plot with loading ranges between -1 and 1 with PCA analysis was used to assess each variable's contribution to the principal component and the correlation between experimental variables (DPPH, FRAP, DPP-4, α-glucosidase, flavonoid, and phenolic contents) in three forma of CO. The variable's contribution to the component increases as the loading approaches -1 or 1. The loading variable goes near zero, indicating a weak contribution to the primary component⁴⁰. Phenolic content, DPPH, and FRAP have greater contributions among the first component. Analytical results demonstrate a positive association between phenolic content, DPPH, FRAP, DPP-4, and α-glucosidase, with the most significant correlation occurring between phenolic contents and DPPH of shrub Cananga, Javanese Cananga, and ylang-ylang. The study's limitation is that the evaluation of antidiabetic and antioxidant capacity is still limited to only two approaches: inhibition of α-glucosidase and DPP4 for antidiabetic, and DPPH radical scavenging and FRAP for antioxidant. Further study on shrub Cananga, Javanese Cananga, and ylang-ylang antidiabetic and antioxidant activities employing many other methods and other pharmacological activities need to be carried out.

CONCLUSION:

The study's findings highlighted the antidiabetic and antioxidant properties along with the flavonoid and phenolics content of three forma of CO (shrub Cananga, Javanese Cananga, and ylang-ylang). Javanese Cananga was shown to be more effective in inhibiting α-glucosidase and DPP-4, as well as DPPH radical scavenging and ferric-reducing capacity effects. PCA analysis revealed a positive correlation between phenolic content against DPPH, FRAP, and α-glucosidase activities among three forma of CO. Hence, three forma of CO leaves can potentially be used as an antioxidants and anti-diabetic agent to avert and regulate oxidative stress and glucose levels. The research's results are expected to be beneficial to several stakeholders in regards to conducting further study on three forma of CO efficacy, formulation, and extract standardization, as well as selecting appropriate CO forma based on the study's goals and revealing up opportunities for utilizing the CO leaf part besides the flower as a raw material by industries.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank Rumah Atsiri Karanganyar, Indonesia and all parties who have given permission to collect CO leaf samples and National Research and Innovation Agency Indonesia for facilitating Rumah Program 2024.

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Received on 23.04.2025 Revised on 27.09.2025

Accepted on 31.12.2025 Published on 20.05.2026

Available online from May 25, 2026

Research J. Pharmacy and Technology. 2026;19(5):2031-2038.

DOI: 10.52711/0974-360X.2026.00291

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