INTRODUCTION:

Indonesia has the most enormous biodiversity, one of which is herbal plants, namely basil leaves. People usually use herbal medicines derived from plants that have secondary metabolite compounds. Secondary metabolites in basil plants are phenolic, flavonoids, and tannins that can act as antioxidants¹. Phenolic compounds, flavonoids, and tannins found in plants can prevent cell damage due to oxidation caused by free radicals^2,3. Basil leaves are vegetables that are quickly found daily⁴. The community uses basil leaves as a plant and has pharmacological activities, such as analgesic solid, antipyretic, antiseptic, antibacterial, antifungal, anti-inflammatory, anticancer, anthelmintic, anticoagulant and antiacne^5–10. In certain countries, basil has traditionally been used to treat conditions such as anxiety, leprosy, diabetes, cardiovascular diseases, headaches, nerve pain, and as an anticonvulsant and anti-inflammatory remedy¹¹.

The essential oil of basil was found to contain 34 constituents, with linalool (29.27%), estragole (9%), eucalyptol (13.52%), and cedrelanol (6.83%) as the predominant components¹². According to research by Timotius et al.¹³, the methanol extract of basil leaves showed essential antioxidant properties, marked by an IC₅₀ value of 174.04ppm. The study quantitatively analyzed phytochemicals in the ethanolic extract of Ocimum basilicum L, reporting total phenols (5.02± 0.06 µg GAE/mg), tannins (7.80±0.05µg GAE/mg), and flavonoids (6.00±0.06µg QE/mg). The extract exhibited significant antioxidant activity with a maximum inhibition of 55.12% at 60µg/ml and an IC₅₀ value of 24.81µg/ml, as determined by the DPPH assay¹⁴. The essential oil extracted from O. basilicum L. leaves, containing significant compounds like eugenol (32%) and ethyl iso-allocholate (19%), exhibited potent antioxidant activity against DPPH, ABTS, and OH free radicals, suggesting its potential as a natural source of antioxidants for managing diseases caused by free radicals¹⁵.

Basil leaves can grow in fertile soil, both lowlands and highlands, so the community can quickly cultivate them¹⁶. However, different growing places can affect the composition of the plant's active compounds^17–20. This is because the height difference can affect the photosynthesis process, so the production of secondary metabolites is not the same. Several factors that can affect the secondary metabolite content of plants are place altitude, temperature, humidity, and nutrient content, all of which can affect yield^21,22. Determining differences between samples with similar physicochemical properties between variables is often subjective and difficult to do with general statistical methods. Therefore, an analysis method that can manage multivariate data effectively and accurately is needed. Chemometry is used to manage and handle multivariate data and experimental design²³.

MATERIALS AND METHODS:

Materials:

The material used is basil leaves obtained from various places in Central Java, namely Klaten, Pekalongan, Kudus, Bandungan, Banjarnegara, and Magelang. Other ingredients used are 96% ethanol, ethanol p.a, magnesium metal powder, hydrochloric acid (concentrated HCl), amyl alcohol, NaCl 1%, gelatin 5%, FeCl₃, DPPH (2,2-diphenyl-1-picryhydrazyl), vitamin C, quercetin, AlCl₃ (Aluminium Chloride) 10%, potassium acetate (CH₃COOK) 1M, tannic acid, gallic acid, sodium carbonate, aquadest.

The tools used in this study are Uv-Vis spectrophotometry (Shimadzu), electric scale (Ohaus), rotary evaporator (Heidolph), oven, blender (Maspion), Buchner funnel, vacuum pump set (Rocker 600), analytical scale (Ohaus), moisture balance (Ohaus), pollinator (Fomac), maceration tool set, water bath, micropipette (Socorex), yellow/blue tip, glass tool set (Iwaki Pyrex).

Determination:

The identification of the basil plant (Ocimum basilicum L.) was conducted to confirm the accuracy of the plant selected for the study. This process took place in the Ecology and Biosystematics Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Diponegoro University, Semarang.

Preparation of 96% ethanol extract of basil leaves (EEBL):

Fresh basil leaves are washed using running water, and then sorting is carried out to separate the basil leaves from the dirt that sticks. The next step involves drying the material in an oven at a temperature of approximately 60°C until the moisture content of the simplicia is reduced to less than 10%. Then, extraction was carried out using the maceration method for five days.

Phytochemical Screening:

a) Phenolic Compounds:

EEBL was weighed 10mg and dissolved using 96% ethanol as much as 5mL; then, a few drops of FeCl₃ solution were added until a color change occurred. Extracts are considered positive if the presence of phenolic compounds is indicated by the formation of blue, purple, green, red, or black colors²⁴.

b) Flavonoid Compounds:

A total of 50mg of EEBL was measured and dissolved in 10mL of absolute ethanol., then filtered and put into two tubes as much as then one of the tubes was added 0.1g of Mg powder and ten drops of hydrochloric acid (concentrated HCl) through the tube wall. The solution was left for a while, and a color change was observed. Compare the two test tubes; if there is a change in color from yellow to orange, it indicates the presence of flavonoid content in the sample²⁴.

c) Tannin Compounds:

A quantity of 50mg of EEBL was measured, dissolved in 10mL of hot distilled water, and then filtered. The solution is divided into 4 test tubes: Tube 1 as blank, tube 2 added 1% NaCl, tube 3 plus 1% NaCl solution and 5% gelatin solution, tube 4 plus FeCl₃ solution. The formation of precipitate in tube 3 indicates the presence of tannin compounds, and positive results after adding FeCl₃ strengthen the results²⁴.

Antioxidant activity test:

Antioxidant activity testing begins by identifying the maximum wavelength and optimal reaction time for DPPH. The antioxidant activity assessment was conducted using DPPH solutions at concentrations of 1, 2, 3, 4, and 5μg/mL, and EEBL solutions at concentrations of 40, 80, 120, 160, and 200μg/mL. Each solution (1mL) was mixed with 4mL of DPPH solution. The mixtures were then kept in a dark environment for 30 minutes, after which their absorbance was measured using a UV-visible spectrophotometer at a wavelength of 516.5nm²⁴.

Determination of phenolic content:

Determining total phenolic content begins with determining gallic acid’s maximum wavelength and operating time. The following process determines the standard curve of the gallic acid and the total phenolic content of EEBL. The gallic acid solution (50, 100, 150, 200, 250, and 300) and the EEBL solution were taken as much as 200mL each, and then 400mL of gallic acid (50, 100, 150, 200, 250, and 300) were added. Folin Ciocalteu leads 4mL 7% N Na₂CO₃. The mixture was left for 125 minutes and read at a wavelength of 743 nm^24,25.

Determination of flavonoid content:

Determining total flavonoid levels begins with determining the maximum wavelength and duration of quercetin operation. The following process is determining the quercetin standard curve and the total flavonoid level of EEBL. Quercetin solution (2, 4, 6, 8, 10, and 12μg/mL) and EEBL solution were taken 1 ml each and reacted with 200μL of 10% AlCl and 200μL CH₃COOK 1M. The mixture was left for 30minutes and read at a wavelength of 431.5nm²⁴.

Determination of tannin content:

Determining tannin levels begins with determining tannic acid's maximum wavelength and operating time. The following process determines the raw curve of tannic acid and the total tannin content of EEBL. The tannic acid solution (40, 80, 120, 160, and 200μg/mL) was pipetted as much as 0.5ml each with 7.5ml of water and 0.5ml of acetic acid. Folin Ciocalteu reagent is left for 5 minutes. 1.5mL of solution is added 20% Na₂CO₃ 20% each. The solution was left for 70 minutes in the dark and read at a wavelength of 745nm. A 200μL aliquot of the EEBL solution was mixed with 200μL of Folin-Ciocalteu reagent and allowed to sit for 5minutes. Then, 100μL of 20% Na₂CO₃ solution was added, followed by distilled water to a final volume of 5mL. The mixture was left for 70 minutes and its absorbance was measured at a wavelength of 745.5nm^24,26.

Data analysis:

The data obtained were IC₅₀, phenolic, flavonoid, and tannins. The data is expressed as an average ± standard deviation and is processed using Microsoft Excel (Microsoft Inc., USA). Additionally, chemometric analysis was performed using Principal Component Analysis (PCA) and Cluster Analysis (CA) with Minitab version 19 (Minitab Inc., USA) to categorize the samples based on variables such as antioxidant activity, total flavonoid content, and tannin content²⁴.

RESULT:

Determination of Basil Plant:

The results of plant determination obtained are in the form of the following determination keys: 1b-2b-3b-4b-6b-7b-9b-10b-11b-12b-13b-14b-16a- (Group 10. Single-leafed, placed face face) -239b-243b-244b-248b-249b-249b-250b-266b-267b-268b 271b- (Fam 110. Labiatae) -1a-2b-4b-6b-7b-(Genus: Ocimum)-(Species: Ocimum basilicum L).

Preparation of 96% ethanol extract of basil leaves (EEBL):

The results of basil leaf extraction are presented in Table 1.

Table 1. Extraction Results of Ethanol Extract of 96% Basil Leaves

No	Region	Powder Weight (gram)	Extract (gram)	Moisture content (%)	Yield (%)
1	Klaten	110	18.3	5.4	16.63
2	Kudus	280	51.1	6.4	18.25
3	Pekalongan	185	25.7	5.3	13.52
4	Bandungan	400	69.5	5.1	17.37
5	Banjarnegara	230	31.8	5.3	13.82
6	Magelang	250	34.9	6.3	13.96

Phytochemical Screening:

Phytochemical screening of phenolic compounds, flavonoids, and tannins is presented in Table 2.

Table 2. Phytochemical Screening Results for Phenolics, Flavonoids, and Tannins

No.	Region	Phenolics	Flavonoids	Tannins
1	Klaten	+	+	+
2	Kudus	+	+	+
3	Pekalongan	+	+	+
4	Bandungan	+	+	+
5	Banjarnegara	+	+	+
6	Magelang	+	+	+

Determination of antioxidant activity, total phenolic, tannins and flavonoid content:

The results of antioxidant activity, total phenolics, tannins, and flavonoids from EEBL is presented in Table 3.

Data analysis:

The antioxidant activity (IC₅₀), total phenolic content (TPC), total flavonoid content (TFC), and total tannin content (TTC) results were analyzed using chemometric methods, including Principal Component Analysis (PCA) and Cluster Analysis (CA), with Minitab software version 19.1.

Figure 1. Eigenanalysis and Scree Plot Graphs from TPC, TFC, TTC and IC₅₀

Their grouping of samples from their score plot results can be seen visually by looking at the proximity between their points, showing similarities between samples 24,27. The closer the dots, the greater the similarity. The result of the score plot can be seen in Figure 2.

Figure 2. Score Plot Graph

Loading plots are used to see the correlation between variables. Their correlation can be seen from the size of their angle formed between the variables. The two variables are considered positively correlated if the angle between the two vectors is less than 90°. If the angle is approximately 90°, the variables are uncorrelated. Conversely, when the angle exceeds 90° or is around 180°, the variables exhibit a negative correlation¹⁸. The result of the loading plot is shown in Figure 3.

Figure 3. Graph Loading plot

Dendrogram chart is a visual representation of a hierarchical clustering analysis. It is a tree-like diagram that shows the arrangement of clusters based on their similarity or dissimilarity. The result of the diagram chart can be seen in Figure 4.

Figure 4. Dendrogram Chart. 1 = Klaten; 2 = Kudus; 3 = Pekalongan; 4 = Bandungan; 5 = Banjarnegara; 6 = Magelang

DISCUSSION:

Their determination of the moisture content of basil leaves has met the simple quality requirement of <10%16. The basil leaf extraction in this study uses the maceration extraction method. The maceration method was chosen in this study because the tools used are simple and do not use high temperatures, which can risk damaging the contents, and are not designed to withstand heating. The extraction process utilizes 96% ethanol due to its universality, polarity, ease of obtainment, and accessibility to the sample cell wall, resulting in a more concentrated extract.

Phytochemical Screening:

EEBL from various regions in Central Java showed that positive EEBL contained phenolic compounds characterized by a colour change to blackishness. The color change occurs due to the reaction between phenols and Fe³⁺ ions, resulting in the formation of complex compounds. EEBL also showed that positive contained flavonoid compounds, characterized by a colour change from the initial colour of light green to orange after adding Mg powder, concentrated HCl, and amyl alcohol. Amyl alcohol serves to form two layers. The orange discoloration is attributed to the interaction between magnesium and HCl powder, which reduces the benzopyron nuclei in flavonoid structures, leading to the formation of red or orange flavilium salts in the amyl alcohol layer²⁴. The results of the phytochemical screening of basil leaves were positive for tannin compounds, marked by the formation of precipitates when added to gelatin. Moreover, it strengthens the suspicion of the existence of phenolic compounds. This is demonstrated by the formation of complex compounds between tannins and FeCl3, which is indicated by a change in color^24,30.

Determination of antioxidant activity, total phenolic, tannins and flavonoid content:

The antioxidant activity was tested using the DPPH method, which measures the ability to capture free radicals. When the purple DPPH solution interacts with an electron-donating substance, it gets reduced, causing the purple color to fade and be replaced by the yellow color of the acrylic group. The color change intensity reflects the antioxidant capacity. DPPH reagents react with antioxidants, shifting from purple to yellow, with the degree of color change being directly proportional to the antioxidant activity in neutralizing free radicals. The higher the concentration, the fading of the colour³¹. Basil leaf samples were taken from 2 different regions, namely lowland and highland areas. The lowland areas are Kudus, Klaten, and Pekalongan, while the highlands are Bandungan, Magelang, and Banjarnegara. The data obtained showed that the IC₅₀ value was better in the highlands than in the lowlands. This is because the height of the growing place can affect the antioxidant activity in basil leaf plants¹⁷. The results of antioxidant activity from EEBL can be seen in Table 3.

The phenolic content in this study is determined using Folin-Ciocalteu reagent and 7% Na₂CO₃. Galic acid is a stable phenolic compound that can react with Folin-Ciocalteu and 7% Na₂CO₃ to form a blue molybdenum-tungsten complex. The complex formation reaction occurs in alkaline conditions, dissociating protons in phenolic compounds into phenolic ions that will react with Folin-Ciocalteu. The complex formed from the reaction has a blue colour that can be read for absorption using UV-Vis spectrophotometry^24,26.

The study results revealed that the Banjarnegara area had the highest phenolic content in EEDK, measuring 106.068mgGAE/g, whereas the Kudus area had the lowest phenolic content at 69.336mgGAE/g. This difference can be caused by several factors, one of which is the height of the place where it grows. In addition to the height of the place where it grows, environmental factors also significantly affect the levels of flavonoids in plants. According to Qaderi, et al.¹⁷, environmental factors that can affect the content of secondary metabolites in plants are temperature, humidity, light intensity, rainfall, content in different soils, and harvest time.

In this study, the total flavonoid content was determined using a colorimetric method with UV-Vis spectrophotometry, employing AlCl₃ and CH₃COOK reagents. The principle behind this method involves the formation of a complex between AlCl₃ and the keto groups at the C-4 position, as well as the hydroxyl groups at the C-3 and C-5 positions³². The addition of CH₃COOK solution helps stabilize and maintain the wavelength in the visible area³³. The comparator used for determining flavonoid levels is quercetin. Quercetin was chosen because it belongs to the flavonoid class of flavonoids that can form a complex colour with AlCl₃. The quercetin flavonoid structure contains a keto group at the C-4 position and hydroxyl groups at the adjacent C-3 and C-5 positions³⁴.

The data obtained determined that the levels of flavonoids from the highlands were higher than the levels of flavonoids from the lowlands. This difference can be caused by several factors, one of which is the height of the place where it grows. In addition to the height of the place where it grows, environmental factors also significantly affect the levels of flavonoids in plants¹⁸.

Tannin content was measured using UV-Vis spectrophotometry with the Folin-Ciocalteu reagent. This reagent is utilized because phenolic compounds react with it to form a colored solution, the absorption of which can be measured. The addition of the Folin-Ciocalteu reagent helps maintain the wavelength within the visible spectrum. The principle of the Folin-Ciocalteu method is the formation of blue complex compounds³⁵. The phenolic compound reacts with Folin-Ciocalteu in an alkaline atmosphere so that a Folin-Ciocalteu reduction reaction occurs by the hydroxyl group (OH) of the polyphenol in the sample, which will form a blue molybdenum-tungsten complex. To create alkaline atmospheric conditions, saturated Na₂CO₃ is used³⁶. The results of tannin levels in each reigion differ from the data obtained. This can be due to the height of the growing place and the harvest time. The height of the ever increasing place can cause changes in temperature and climatic conditions; it will cause a change in the content of secondary metabolites contained in the plant¹⁸.

Data analysis:

The eigenanalysis values obtained show that PC1, PC2, and PC3 contribute to the variance of 86%, 11.1%, and 0.3%, respectively. Figure 1 illustrates the relationship between each PC and the eigenanalysis value. A score plot is the output of a Minitab that describes the similarity between samples based on PC1 and PC2, which can be marked by points that are close to each other. Based on the results of the plot score, the sample was grouped into three groups. Group 1 is Klaten, Kudus, and Pekalongan; group 2, namely Bandungan and Magelang; group 3 is Banjarnegara (Figure 2).

The correlation between TPC and IC₅₀, TTC and IC₅₀, and TFC and IC₅₀, which form a wide angle close to 180°, shows the value of the negative correlation coefficient and a negative correlation relationship (Figure 3). A negative correlation indicates that the higher the value of variable 1, the lower the variable 2. The higher the phenolic values, flavonoids, and tannins, the lower the IC₅₀. This is because phenolic compounds, flavonoids, and tannins have the potential to be antioxidants. The correlation between TTC and TPC and TPC and TFC, which forms an angle of slightly less than 90°, shows a positive correlation, meaning that the higher the variable 1, the higher the variable 2. This is because phenolic compounds, flavonoids, and tannins are in the same secondary metabolic group.

The score plot and dendrogram (Figure 4) graph show that the six basil leaf samples produced two groups. Group 1 is Klaten, Kudus, and Pekalongan; group 2, namely Bandungan and Magelang; group 3 is Banjarnegara. Research conducted by Widyastuti et al. showed that the content of flavonoids was negatively correlated with IC₅₀, which means that the higher the phenolic and flavonoid content in Temulawak, the lower the value. Based on this study, it was found that the total phenolic and flavonoid content in basil leaves had a negative correlation with IC₅₀, which means that the high phenolic and flavonoid content in basil leaves will affect a lower IC₅₀ value. Tannin compounds correlated with IC₅₀ showed that tannins affected the antioxidant activity of the samples. This can be caused by tannins, which are phenolic compounds. Phenolic compounds and flavonoids are compounds that have the potential to ward off free radicals.

CONCLUSION:

The ethanol extract of basil leaves from the Banjarnegara area has the highest antioxidant activity and flavonoid, phenolic, and tannin content. Based on chemometrics, samples can be divided into three groups.

CONFLICT OF INTEREST:

The authors have declared no conflicts of interest.

ACKNOWLEDGMENTS:

We extend our gratitude to everyone who assisted us in reaching our research objectives and to the Faculty of Pharmacy at Universitas Wahid Hasyim Semarang for their support of the study.

REFERENCES:

1. Harianja M, Rahman H, Wigati S. Invitro: Evaluasi Aktifitas Peluruhan Batu Ginjal Ekstrak Daun Kemangi (Ocimum basilicum) Menggunakan Spektrofotometer Serapan Atom. Jurnal Sains dan Kesehatan. 2021; Jun 30; 3(3): 451–7. DOI: 10.25026/jsk.v3i3.359

2. Antarti AN, Lisnasari R. Uji Aktivitas Antioksidan Ektrak Ethanol Daun Family Solanum Menggunakan Metode Reduksi Radikal Bebas DPPH. JPSCR : Journal of Pharmaceutical Science and Clinical Research. 2018; Oct 9; 3(2): 62. DOI: 10.20961/jpscr.v3i2.15378

3. Tong Z, He W, Fan X, Guo A. Biological Function of Plant Tannin and Its Application in Animal Health. Frontiers in Veterinary Science. 2022; Jan 10; 8. DOI: 10.3389/fvets.2021.803657

4. Arrayyan MA, Dwiloka B, Susanti DS. The Effect of Vegetable Fat Enfleurage Concentration to Antioxidant Activity and Physical Characteristic of Basil Essential Oil (Ocimum americanum L.). Jurnal Teknologi Pangan. 2019; 3(2): 221–7. DOI: Https://doi.org/10.14710/jtp.2019.23828

5. Nuzulia R, Santoso O. Pengaruh Ekstrak Daun kemangi (Ocimum basilicum Linn) Pada Berbagai Konsentrasi Terhadap Viabilitas Bakteri Streptococcus mutans. Diponegoro Medical Journal (Jurnal Kedokteran Diponegoro). 2020; 6(4): 1565–71.

6. Satpute KL, Kalyankar TM. Investigation of Antiacne activity of some Medicinal Plants against Pathogenic Bacteria. Research Journal of Pharmacy and Technology. 2018; 11(7): 2776. DOI: 10.5958/0974-360X.2018.00513.9

7. Purushothaman B, Suganthi N, Jothi A, Shanmugam K. Molecular Docking Studies of potential anticancer agents from Ocimum basilicum L. against human colorectal cancer regulating genes: An insilico approach. Research Journal of Pharmacy and Technology. 2019; 12(7): 3423. DOI: 10.5958/0974-360X.2019.00579.1

8. Nadimohamed N, Mahmoud B, Abdel-Reheim ES, Abdel-Moneim A. Olive and basil leave extracts protect against ibuprofen induced nephrotoxicity in albino rats. Research Journal of Pharmacy and Technology. 2020; 13(9): 4190. DOI: 10.5958/0974-360X.2020.00740.4

9. Shagana S, Navenaa S, Vijay J, Vishali S, Rajendran V. Investigation of in vitro Anthelmintic activity of Ocimum basilicum Linn. (Lamiaceae). RESEARCH JOURNAL OF PHARMACY AND TECHNOLOGY. 2021; 14(1): 52–4. DOI: 10.5958/0974-360X.2021.00010.X

10. Masoudi R, Aldiab D, Hasan N. Study of the Anticoagulant effect of Ocimum basilicum extract. Research Journal of Pharmacy and Technology. 2024; Jul 24; 3339–45. DOI: 10.52711/0974-360X.2024.00522

11. Pundir S, Kumari R, Chauhan B. A Review on Treatment of Leprosy by using Medicinal Plants. Research Journal of Pharmacognosy and Phytochemistry. 2024; May 28; 130–2. DOI: 10.52711/0975-4385.2024.00025

12. Mahcene Z, Mahcene Z, Bireche K, Serdouk F. Socioeconomic valorization and development of a bio-fungicide from essential oils of four Algerian aromatic and medicinal plants: Artemisia herba alba Asso, Mentha pulegium L, Rosmarinus officinalis L and Ocimum basilicum L. Asian Journal Of Research in Chemistry. 2020; 13(6): 473–84. DOI: 10.5958/0974-4150.2020.00084.X

13. Timotius T, Limanan D, Ferdinal F. Uji Toksisitas, Aktivitas Antioksidan dan Kadar Metabolit Sekunder Daun Kemangi (Ocimum X Africanum lour). Jurnal Muara Medika dan Psikologi Klinis. 2022; Mar 24; 1(2): 139. DOI: 10.24912/jmmpk.v1i2.15308

14. Kumar Pandey A, Prasad Tiwari S, Biswas D, Patel Y, M. Jajda H, S. Dave G. Evaluation of Phytochemicals, Antioxidant and Anti-inflammatory properties of leaves of Ocimum basilicum L. Research Journal of Pharmacy and Technology. 2023; Apr 29; 1981–6. DOI: 10.52711/0974-360X.2023.00325

15. Desta A, Chaithanya K K, Kumar A.D N, Cheekurumelli P, Dogulas Palleti J, Rai S, et al. Physicochemical Characteristics, Chemical composition and In vitro Antioxidant activities of Essential oil from Ocimum basilicum L. Leaves. Research Journal of Pharmacy and Technology. 2024; May 15; 2352–8. DOI: 10.52711/0974-360X.2024.00368

16. Nurfitriyah R, Wurjani W, Augustien K N. Respon Pertumbuhan Dan Hasil Tanaman Kemangi (Ocimum basilicum L.) Pada Pemberian Berbagai Dosis Pupuk Nitrogen. Jurnal Agrium. 2022; Sep 16; 19(3):257. DOI: 10.29103/agrium.v19i3.8754

17. Qaderi MM, Martel AB, Strugnell CA. Environmental Factors Regulate Plant Secondary Metabolites. Plants. 2023; Jan 18; 12(3): 447. DOI: 10.3390/plants12030447

18. Widyastuti I, Luthfah HZ, Hartono YI, Islamadina R, Can AT, Rohman A. Antioxidant Activity of Temulawak (Curcuma xanthorrhiza Roxb.) and its Classification with Chemometrics. Indonesian Journal of Chemometrics and Pharmaceutical Analysis. 2020; Jul 19; 29. DOI: 10.22146/ijcpa.507

19. Raharjo A, Elida T, Prajitno D. Studi Kesesuaian Lahan terhadap Sukun (Artocarpus sp.) di Kota Tarakan, Kalimantan Utara. Jurnal Agribisnis Terpadu. 2020; Jun 1; 13(1): 120. DOI: 10.33512/jat.v13i1.7350

20. Meenakumari K, Vasanth S, Buelah JJ, Bupesh G. GC-MS and HPTLC Analysis of Ocimum basilicum Ethanolic extract obtained from five locations of Tamilnadu. Research Journal of Pharmacy and Technology. 2023; Nov 30; 5167–72. DOI: 10.52711/0974-360X.2023.00837

21. Yanti NLMYI, Arpiwi NL, Yulihastuti DA. Minyak Atsiri Daun Kemangi (Ocimum × africanum Lour.) dan Efektivitasnya Sebagai Lotion Antinyamuk terhadap Aedes aegypti (Linnaeus, 1762). Metamorfosa: Journal of Biological Sciences. 2020; Sep 29; 7(2): 105. DOI: 10.24843/metamorfosa.2020.v07.i02.p14

22. Katuuk RHH, Wanget SA, Tumewu P. Pengaruh perbedaan ketinggian tempat terhadap kandungan metabolit sekunder pada gulma babadotan (Ageratum conyzoides L.). Jurnal COCOS. 2019; 1(4): 6.

23. Rohman A, Can AT, Irnawati, Rafi M, Lukitaningsih E, Fadzilah NA. Principal Component Analysis of Antioxidant Activities, Total Phenolic contents, and Total Flavonoid contents of Turmeric (Curcuma longa L.). International Journal of Pharmaceutical Research. 2020; Nov 2; 12(sp2). DOI: 10.31838/ijpr/2020.SP2.354

24. Yumni GG, Primitasari R, Afifah NN. Chemometric Analysis of Ethanol Extract of Breadfruit Leaves (Artocarpus altilis) from Various Regions in Central Java. Jurnal Kefarmasian Indonesia. 2024;14(August): 167–75.

25. Pérez M, Dominguez-López I, Lamuela-Raventós RM. The Chemistry Behind the Folin–Ciocalteu Method for the Estimation of (Poly)phenol Content in Food: Total Phenolic Intake in a Mediterranean Dietary Pattern. Journal of Agricultural and Food Chemistry. 2023; Nov 22; 71(46): 17543–53. DOI: 10.1021/acs.jafc.3c04022

26. Nofita D, Dewangga R. Optimasi Perbandingan Pelarut Etanol Air Terhadap Kadar Tanin pada Daun Matoa (Pometia pinnata J.R and G. Forst) Secara Spektrofotometri. Chimica et Natura Acta. 2022; 9(3): 102–6.

27. Amin A. Determinasi dan Analisis Finger Print Daun Miana (Coleus scutellarioides Linn.) Sebagai Bahan Baku Obat Tradisional. Jf Fik Uinam. 2016; 4(2): 58–64.

28. Yumni GG, Sumantri S, Nuraini I, Nafis IJ. Profil Antioksidan dan Kadar Flavonoid Total Fraksi Air dan Etil Asetat Ekstrak Etanol Bunga Telang (Clitoria ternatea L.). Cendekia Eksakta. 2022; May 25; 7(1): 12–7. DOI: 10.31942/ce.v7i1.6547

29. Wendersteyt NV, Wewengkang DS, Abdullah SS. Uji Aktivitas Antimikroba dari Ekstrak dan Fraksi Ascidian herdmania momus dari Perairan Pulau Bangka Likupang terhadap Pertumbuhan Mikroba Staphylococcus aureus, Salmonella typhimurium dan Candida albicans. PHARMACON. 2021; Feb 24; 10(1): 706. DOI: 10.35799/pha.10.2021.32758

30. Harborne JB. Metode Fitokimia: Penuntun Cara Modern Menganalisis Tumbuhan. Bandung: ITB Press; 1987.

31. Fatmawati IS, Haeruddin, Mulyana WO. Uji Aktivitas Antioksidan Ekstrak Etil Asetat Daun Belimbing Wuluh (Aveerrhoa bilimbi L.) dengan Metode DPPH. SAINS: Jurnal Kimia dan Pendidikan Kimia. 2023; 12(1): 41–9.

32. Azizah DN, Kumolowati E, Faramayuda F. Penetapan Kadar Flavonoid Metode AlCl3 pada Ekstrak Metano Kulit Buah Kakao (Theobroma cacao L.). Kartika Jurnal Ilmiah Farmasi. 2014; Dec 9; 2(2). DOI: 10.26874/kjif.v2i2.14

33. Lindawati NY, Ma’ruf SH. Penetapan Kadar Total Flavonoid Ekstrak Etanol Kacang Merah (Phaseolus vulgaris L.) Secara Spektrofotometri Visibel. Jurnal Ilmiah Manuntung. 2020; Jun 30; 6(1): 83–91. DOI: 10.51352/jim.v6i1.312

34. Sari AK, Ayuchecaria N. Penetapan Kadar Fenolik Total dan Flavonoid Total Ekstrak Beras Hitam (Oryza sativa L) dari Kalimantan Selatan. Jurnal Ilmiah Ibnu Sina. 2017; 2(2): 327–35.

35. Listiana L, Wahlanto P, Ramadhani SS, Ismail R. Penetapan Kadar Tanin Dalam Daun Mangkokan (Nothopanax scutellarium Merr) Perasan dan Rebusan dengan Spektrofotometer UV-Vis. Pharmacy Genius. 2022; Oct 20; 1(1): 62–73. DOI: 10.56359/pharmgen.v1i01.152

36. Noviyanty Y, Hepiyansori, Agustian Y. Identifikasi dan Penetapan Kadar Senyawa Tanin Pada Ekstrak Daun Biduri (Calotropis gigantea) metode Spektrofotometri UV-Vis. Jurnal Ilmiah Manuntung. 2020; 6(1): 57–64.

Received on 12.12.2024 Revised on 15.04.2025

Accepted on 17.06.2025 Published on 01.12.2025

Available online from December 06, 2025

Research J. Pharmacy and Technology. 2025;18(12):6028-6034.

DOI: 10.52711/0974-360X.2025.00871

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

No	Region	IC₅₀ (ppm)	TPC (mgGAE/g)	TFC (mgQE/g)	TTC (mgTAE/g)
1	Klaten	149.85	77.023	3.58	23.45
2	Kudus	165.36	69.336	6.93	17.06
3	Pekalongan	128.53	73.301	9.5	23.44
4	Bandungan	61.6	82.767	16.16	26.5
5	Banjarnegara	56.26	106.068	18.56	42.75
6	Magelang	70.19	80.583	11.14	23.41