INTRODUCTION:

Reactive oxygen species (ROS) are highly reactive chemicals derived molecular oxygen that easily reacts with other molecules in a cell¹. Human body can scavenge free radicals in the normal circumstances; however, in the disequilibrium condition between the excessive level of free radicals and the capability of the cellular biological system to remove them cause DNA damage and lead to oxidative stress².

The high level of ROS in the body is directly related to the developing metabolic diseases like cardiovascular disorders, metabolic syndromes, and cancer^3,4,5.

Antioxidants scavenge free radicals via single electron transfer and hydrogen atom transfer. Assessing antioxidant scavenging activity of natural product using various chemical and enzymatic assays has become the focus of many studies. Some of antioxidant assays use stable radical chromogen compounds as probes to quantify free radical scavenging activity. Widely known assays such as 2,2-azinobis (3-ethyl-benzothiazoline-6-sulphonic acid) (ABTS)⁶, ferric reducing antioxidant power (FRAP)⁷, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) are performed to measure antioxidant activity by different investigators. These methods have different mechanisms for scavenging free radicals. While ABTS estimates the transferability of a single electron⁸, DPPH evaluates hydrogen-donating potential compounds based on the electron transfer^9,10,11. FRAP test values represent the appropriate concentration of electron-donating antioxidants with the reduction in the ferric iron (Fe³⁺) to the ferrous ion (Fe²⁺), ABTS, FRAP and DPPH are methods that measure the single electron transfer^12,13.

Hemeoxygenase (HO-1) is an enzyme that plays an important role in oxidative stress. This enzyme catalyzesheme degradation to produce carbon monoxide, iron, and biliverdin. Biliverdin is converted to bilirubin, a robust antioxidant agent, which is then transferred back to biliverdin through ROS interaction¹⁴. Upregulation of heme oxygenase-1 (HO-1), a phase II detoxifying enzyme in endothelial cells, is thought to be helpful in cardiovascular disease. Understanding heme protein structure and heme binding environment provide valuable guidelines in the development of novel antioxidants action¹⁵. Docking studies on HO-1 (PDB code: 1N3U) were performed on all compounds to correlate the isolated compounds with the indicated activity by determining the interactions of these compounds in the HO-1 active site¹⁶. Co-crystallized ligands/compounds (Heme) are flexibly re-tethered to verify docking protocols. The intermolecular interactions between the ligand/compound and the target receptor were evaluated In recent years, interest in the potential effect of herbal remedies as a source of natural antioxidant has increased due to their efficacy and safety. In Indonesia, some plants have been traditionally used to treat diseases; however, no scientific reports regarding this medical application. The therapeutic capacities of medicinal plants are dependent on their extensive range of phytochemical molecules. Cassia alata, well known as Yellow Candle bush, is a native plant distributed in tropical countries from Latin America, Northern Australia, and Southeast Asia. It is also known as ringworm bush, candelabra bush, and ketepengbadak in different countries around the world and has been used for a decade as an alternative traditional medicine¹⁷. Each part of C. alata, including roots, barks, leaves, and seeds, display biological activities, including antioxidant¹⁸, anti-inflammatory^18,19, antibacterial^20,21, and antidiabetic^22,23. The major constituents in C. alata are compounds in the classes of alkaloids, saponins, flavonoids, tannins, and anthraquinones²⁴. Among ingredients compounds mainly found in C. alata, polyphenols are thought to responsible for their potent antioxidant capacity^25,26.

In the present study, C. alata metabolite profiling was performed using Liquid Chromatography combined with Tandem Mass Spectrometry (LC-QTOF-MS/MS)²⁷. In addition, the antioxidant capacity of investigated extract and fractions was determined through various radical scavenging assays. Molecular docking against HO-1 was also performed to examine the potential antioxidant capacity of bioactive compound in C. alata extract.

MATERIALS AND METHODS:

Materials and chemicals:

Cassia alata leaves were collected from Banjar – South Kalimantan and determined in Botany Herbarium Research Institute, Cibinong, Indonesia (920/IPH.1.01/1f.07/IX/2020). The chemical used in the study were 70% Ethanol, Ethyl Acetate, n-Heksane, Buthanol, Methanol, Trolox, ABTS, Potassium persulfate, Folin-Ciocalteau, Gallic acid, purchased from Merck All (Darmstadt, Germany). DPPH and quercetin were purchased from Sigma - Aldrich Chemicals (St. Louis, MO, USA).

Extraction and fractionation:

The extraction methods²⁸of C alata leave extract was conducted based on a previous study with slightly modification²⁹. Dried leaves of C. alata (1kg) were crushed into powder and extracted with ethanol (2.5 L) at room temperature. A pressurized rotary evaporator was used to concentrate the filtrate. The extraction process resulted in an extract of 225.7g (CAE; yield 22.57%) of crude ethanolic leaf extract. The crude ethanolic extract was then fractionated with various organic solvents, which are ethyl acetate, hexane, butanol, and water, to yield the hexane fraction (CAH; 4.2g; 7%), ethyl acetate fraction (CAEA; 7.7g; 13%), butanol fraction (CAB; 7.1g; 12%) and water fraction (CAW; 31.5g; 53%) The extract and fractions were kept at 4°C for further use.

LC-QTOF-MS/MS Analysis:

LCQTOFMS/MS analysis was performed on an AcquityUltraPerformance LC system connected to XevoG2-XS QT of (Waters, MA, USA). Filtered samples were injected in a volume of 1mL at 40°C into a C18 column (HSS T3 100mm x 2.1mm, 1.7mm) and separated by the UPLC system. The mobile phase was composed of formic acid in water (A; 0.1% v/v) and formic acid in acetonitrile (B;0.1% v/v). Chromatographic separation was performed at a 0.3 mL/min flow rate with the following gradient profile: 95% A followed by gradually degrading B to 5% for 16 minutes. Mass spectroscopy measurements were performed on a quadrupole/ time-of-flight (Q-TOF). The following MS conditions were set: electrospray ionization in positive ion mode (ESI(+)); scanned range, m/z 100 – 1200; capillary voltage, 0.8kV; cone voltage, 30kV; desolvation gas, 1000L/h at 500°C; cone gas, 50 L/h at 120°C. UNIFIÔ was used for data acquisition. The semi-quantitative analysis determined major compounds based on the LC-MS- graph’s high-intensity.

Determination of antioxidant activity:

This study refers to Three antioxidant tests, namely DPPH, ABTS, and FRAP, were conducted in this present study^30,31. Those procedures have been widely used to determine the antioxidant capacities of plant extracts as they require relatively standard equipment and deliver fast and reproducible results. Researcher²⁹had done an interlaboratory comparison of methods used to assess antioxidant potentials. Out of six assays (DPPH, ABTS, TBA, DCFH, PCL, and lipid assays), DPPH and ABTS assays display the easiest method to implement and yield the most reproducible results. Positive antioxidant controls, for instance quercetin and trolox, are essential to compare the results of our samples with those obtained from a standard compound with high antioxidant activity.

DPPH assay:

The DPPH assay was carried out using previous method³². Briefly, 4 mg of sample was dissolved in ethanol. Then, various concentrations of samples were treated with DPPH solution (1mM) in methanol. The mixture was stored in dark room temperature for 30min, and then the absorbance was recorded by using a UV-spectrometerat 517nm. The antioxidant capacity of the sample was expressed as IC_50.Quercetin was employed as reference compound.

ABTS assay:

Radical ABTS³³ was prepared by mixing ABTS (7mM) and potassium persulfate (140mM) and stored at room temperature in the dark for 18 hours. The ABTS reagent was diluted in water (1:3,v/v) until it reached an absorbance value of 0.7 at 734nm. For the measurement of inhibitory activity against ABTS radical, ABTS mixture was added with sample (1:1,v/v) and stored for 6minutes at room temperature. Absorbance was measured at 734nm. The antioxidant (abts) capacity of the sample was expressed as IC_50.. Trolox was used as reference compound

FRAP assay:

FRAP radical scavenging assay is based on the ability of antioxidants to reduce ferric iron (Fe³⁺) to ferrous iron (Fe²⁺) at acidic pH, through an electron transfer mechanism. FRAP assay was done based on a previously described protocol³⁴.The absorption of mixtures was recorded from measured 593nm. FRAP calculation based on the Trolox standard calibration curve. The reducing power was determined as milligram units of Trolox per grams of dried sample.

Total phenolic content (TPC):

A previously described protocol was followed to determine total phenolic content (TPC) of C. alataleaf extract^26,35. In concise, a mixture was prepared by mixing leaf extract (1mL) and distilled water (7.5mL). A freshly Folin– Ciocalteu reagent (0.5mL) was added and mixed thoroughly for 8 min, followed by the addition of sodium carbonate (20% w/v; 1.5mL). After incubating for 60minutes in the dark, the absorbance was measured at 765nm. Standard reference calibration curve was established using gallic acid with a range concentration of 0.005 to 0.2mg/ml as a standard reference plotted. TPC was expressed as milligrams of gallic acid equivalent (GAE) per gram of the dry weight.

Total flavonoid contents (TFC):

Total flavonoid content (TFC) was examined by aluminium chloride (AlCl₃) colorimetric assays based and expressed as quercetin equivalents (QE), in milligrams per gram of the extract. The absorbance was determined at measured 415nm^33,36.

Molecular docking:

The compounds identified in the crude ethanolic extract of C. alataleaf by LC-QTOF-MS/MS analysis were considered as potential compounds contributing to the antiradical activities. The identified compounds docked to the active site of the HO-1 crystal structure. Figure 1 shows the 3D crystal structure of the HemeOxygenase enzyme (PDB ID: 1N3U), which was obtained in PDB format from a protein data bank (http://www.rcsb.org//pdb).

Figure 1: Crystallographic model of 1N3U protein 3D structure.

The 2D structure of the ligand was drawn in ChemDraw version 12.0 and conerted to 3D format. Discovery Studio Visualizer 4.0 and AutoDock 4.2 were used for molecular docking analysis and visualization. The hydrogen bond network was refined through the optimize option. The docking grid box was at the center of the crystallized ligand binding site, with the dimension of -0.812, 52,353, and 23,137 (x, y, and z, respectively). The molecular docking method was validated by re-docking the native ligand on the targeted protein with its native ligand removed using the AutoDock 4.2 application with the RMSD parameter. The smaller the Root Mean Square Deviation (RMSD) value obtained (close to 0), the more similar the structure pair to the native ligand.

Statistical analysis:

The assays experiment was conducted in triplicate and data were presented as mean±standard deviation (SD). Statistical analysis was determined by one-way analysis of variance (ANOVA) using SPSS version 16(SPSS Inc, IL, USA).

RESULT:

Determination of TPC (Total Phenolic Compound), TFC (Total Flavonoid Compound), and radical scavenging activity

Table 1: Determination of TPC and TFC of C. alata leaves

Sample	TPC (mg GAE/g extract)	TFC (mg QE/g extract)
CAE	14.966 ± 0.4354	6.359 ± 0.0855
CAH	1.697 ± 0.3989	4.026 ± 0.8803
CAEA	40.778 ± 1.3539	34.034 ± 0.1026
CAB	22.544 ± 2.2963	9.538 ± 0.1197
CAW	5.351 ± 0.1152	1.761 ± 0.0006

CAE-Ethanol extract of C. alata leaves; CAH- Hexane extract of C. alata leaves; CAEA- Ethyl acetate extract of C. alata leaves; CAB- Buthanol extract of C. alata leaves; CAW- Water extract of C. alata leaves; GAE-Gallic Acid Eqiuvalent/gram, QE/g-Quarcetin Eqiuvalen/gram *Values are the mean of ± standard deviation (n = 3).

TPC of C. alata crude ethanolic extract and resultant fractions ranged from 1.697±0.3989 to 40.778±1.3539 mg GAE/g of dry weight, while TFC ranged from 1.761 ±0.0006 to 34.034±0.1026mg QE/g of dried extract. The amount of TPC and TFC in the leaves of C. alataare given in Table 1. It is shown that ethyl acetate fraction exhibited the highest TPC and TFC with 40.778±1.3539 and 34.034±0.1026mg/g dried sample for GAE and QE values, respectively. This sequence corresponds to the order the TPC value of the solvent (CAEA>CAB>CAE >CAW>CAH). TFC also showed a similar trend as TPC.

In the FRAP assay, the presence of a reducing agent lead to the reduction of Fe³⁺ to Fe²⁺, forming a colored TPTZ complex with maximum absorption peak at 593 nm. Table 2 expresses the evaluation of antioxidant activity by FRAP test. The most excellent reducing power was 27, 376TE, obtained in the ethyl acetate fraction. This sequence corresponds to the order the Fe³⁺ reduction to Fe²⁺of the solvent (CAEA > CAW > CAE > CAH > CAB). It was observed that the antioxidant activity of various samples determined by FRAP was positively correlated to the flavonoid content (Figure 2), explaining the positive correlation between flavonoids and it antioxidant potential.

Table 2: Antioxidant capacities measured by DPPH radical scavenging, ABTS and FRAP assays.

Sample	IC₅₀(µg/mL)
Sample	DPPH radical scavenging assay	ABTS radical scavenging assay	FRAP assay
Positive control	4.45 ± 0.0055	4.26 ± 0.00
CAE	93.040 ± 0.035	27.504 ± 0.11	7.743 ± 0.01
CAH	>2000	50.799 ± 0.93	6.591 ±0.06
CAEA	18.540 ± 0.0076	11.294 ± 0.01	27.376 ± 0.26
CAB	84.690 ± 0.0143	4.286 ± 0.26	4.391 ±0.11
CAW	407.331 ± 0.0060	12.108 ± 0.45	9.466 ± 0.05

CAE-Ethanol extract of C. alata leaves; CAH-Hexane extract of C. alata leaves; CAEA- Ethyl acetate extract of C. alata leaves; CAB- Butanol extract of C. alata leaves; CAW- Water extract of C. alata leaves

*Values are the mean of triplicate ± SD (n = 3). Quercetin is the positive control for DPPH, Trolox is the positive control for ABTS and FRAP

Figure 2: A significant correlation between TFC and FRAP values.

LC-QTOF-MS/MS analysis of fractions of C. alata leaves extract:

C. alataethanolic leaves extract were fractionated using various organic solvents to give the respective fractions. Table 3 presents the LC-QTOF-MS/MS analysis results of fractions found in C. alata leaves extract. Compound identification was done by matching their retention time (RT), fragmentation spectra and accurate mass to the LC-MS/MS library.

Table 3: LC-QTOF-MS/MS analysis of C. alataleaves.

Compound No.	Constituent	Source of fraction	Formula	RT (min)	*Molecular Weight (m/z)*
1	3-Tert-butyl-4-methoxyphenol	Hexane	C₁₁H₁₆O₂	9.71	181.1224
2	Coclaurine	Hexane, Ethanol, Ethyl acetate	C₁₇H₁₉NO₃	9.83	286.1442
3	Piperolein B	Hexane	C₂₁H₂₉NO₃	10.02	344.2224
4	3-Hydroxy-7-methoxy baicalein	Ethyl acetate	C₁₆H₁₂O₆	9.23	301.0710
5	Kaempferol-3-O-β-D-glucopyranoside	Ethyl acetate, Butanol, Ethanol	C₂₁H₂₀O₁₁	6.64	449.1078
6	Quercetin	Ethyl acetate	C₁₅H₁₀O₇	8.23	303.0503
7	3′,5-Dihydroxy-7,4'-dimethoxy flavone	Butanol	C₁₇H₁₄O₆	7.44	315.0867
8	Kaempferol-3,7-diglucoside	Butanol, Ethanol, Water	C₂₇H₃₀O₁₆	5.86	611.1620
9	Methyl 11α-hydroxytormentate	Butanol	C₃₁H₅₀O₆	10.55	519.3663
10	5,7,2',5'-Tetrahydroxy-flavone	Ethanol	C₁₅H₁₀O₆	9.22	286.04774
11	1β,3α,9β-Trihydroxyeudesma-5,11(13)-dien-12-oic acid	Water	C₁₅H₂₂O₅	1.52	305.1344
12	3-O-β-D-Galacopyanosylquercetin	Water	C₂₁H₂₂O₁₂	4.72	489.1003

Table 4: Molecular docking results analysis of analysis compounds from C. alataleaves extract against HO-1 (PDB ID: 1N3U).

Compound No	Constituent	ΔG (kcal/mol)	Ki (µM)	H- bonds	Non-H-bonds
1	3-Tert-butyl-4-methoxyphenol	-6.44	18.91	2; Glu353, Leu387	Pro324, Leu387, Ile326
2	Coclaurine	-7.94	1.5	1; Glu353	Ile424, His524, Met421, Leu387, Ala350, Leu391, Leu346, Met 343
3	Piperolein B	-8.33	0.779	NIL	Leu387, Leu349, Phe404, Ala350, Leu525, Trp383, Leu428, Leu346, Met388, Ile424, Met343, Met 421, Thr347, Asp351
4	3-Hydroxy-7-methoxy baicalein	-7.32	4.3	1; Gly521	Ile424, Met 421, Leu349, Leu387, Ala350, Leu346, Leu384, Leu525, Met343, Phe404, His524
5	Kaempferol-3-O-β-D-glucopyranoside	-8.95	0.273	5; Arg394, Glu353, Gly521, His524, Thr347	Leu391, Leu384, Ala350, Met388, Leu387, Leu525
6	Quercetin	-7.66	2.44	4; Gly521, Arg394, Leu387, Glu353	Leu525, Ile424, Leu391, Ala350, Met388, Phe404
7	3′,5-Dihydroxy-7,4'-dimethoxy flavone	-7.94	1.38	1; Gly521	Met388, Leu387, Ala350, Leu349, Leu525, His524, Glu353, Leu346, Leu391, Ile424
8	Kaempferol-3,7-diglucoside	-3.72	1890	1; Asp351	Ala350, Leu346, Leu925, Thr347, Met343
9	Methyl 11α-hydroxytormentate	-5.48	96.96	2; Asp352, Leu536	Leu525, Trp383, Pro535, Glu380
10	5,7,2',5'-Tetrahydroxy-flavone	-8.19	0.991	3; Gly521, Glu353, Arg394	Ile424, Leu346, Leu391, Leu387, Ala350, Phe404, His524
11	1β,3α,9β-Trihydroxyeudesma-5,11(13)-dien-12-oic acid	-5.37	114.94	1; Gly521	Leu346, Ala350, Ile424, Leu525, Met388
12	3-O-β-D-Galacopyanosylquercetin	-8.69	0.428	5; Thr347, Asp351, Leu346, Arg394, Glu353	Ala350, Met421, Ile424, Met343, His524, Leu525

Evaluation of Molecular docking:

To explore more detail on the interaction between phytochemicals constituent present in C. alata extract, including its fractions and targeting antiradical properties, molecular docking was conducted to simulate the potential inhibitors of plant extract against HO-1 protein that encode human heme oxygenase-1, which is responsible for redox homeostasis leading to a relevant decrease of oxidative damage. This enzyme has a critical anti-inflammation, antioxidant and immunomodulatory effects in the vascular cell ¹⁰. Molecular docking studies were carried out on the chemical compounds in the C. alataextract against the HO-1 receptor. HO-1 receptor has a native HEM ligand with an atomic number of 3666 and a total structural weight of about 55.14 KDa. By inhibiting this enzyme, some phytochemical constituents might be suggested as potential antioxidant agents. The molecular docking result is presented in Table 4.

Table 4 serves the binding free energy (ΔG) and protein-ligand interaction for C. alata constituents docked at HO-1. The lower the (ΔG) consequences, the more significant the interaction between the receptor and the ligands with antioxidant ability. Kaempferol-3-O-β-D-glucopyranoside identified in C. alataexhibited the best binding affinity compared to other ligands with high docking score of -8.95 kcal/mol, followed by coclaurine (-7.94 kcal/mol), quercetin (-7.66 kcal/mol) and 3-Hydroxy-7-methoxy baicalein (-7.32 kcal/mol). Interestingly, those constituents were found in the ethyl acetate fraction of C. alata, based on LC-QTOF-MS/MS.

Figure 3 displays the interaction of C. alata constituents with the HO-1 receptor. Compared to other tested molecules, the higher binding affinity of Kaempferol-3-O-β-D-glucopyranoside is attributed to the conventional hydrogen formed with residues. Kaempferol-3-O-β-D-glucopyranoside forms five hydrogen bonds with Arg394, Glu353, Gly521, His524, and Thr347. In addition, it also has Pi-alkyl interaction with Leu391, Leu384, and Ala350, as well as Pi-sigma interaction with Leu387 and Leu525. Similar conventional hydrogen interactions with Gly521 also are shown for both 3-Hydroxy-7-methoxy baicalein and quercetin, but not for coclaurine, since the latter has a unique conventional hydrogen bonding with Glu535. A similar interaction with Leu387 and Ala350 also are shown for all previously mentioned ligands. The similar ligand-receptor interactions and types of bonds influence the binding affinity and free energy between each ligand, indicating an excellent antioxidant activity of tested molecules.

CONCLUSION:

In this present study, a number of compounds have been identified from C. alata crude ethanolic Kaempferol-3-O-β-D-glucopyranoside identified in ethyl acetate fraction of C. alata exhibited the best binding affinity compared to other ligands based on in silico molecular docking analysis. The rich phenolic compounds in ethyl acetate fraction could be one of reasons this fraction possessed a better antioxidant activity than the other fractions. These results indicate that the free radical scavenging capacity of C. alataleaves extract and fractions are due to a synergic effect of identified compounds inside the extract. Nevertheless, further meticulous studies should be conducted to isolate and characterize the bioactive compounds from C. alata responsible for this antioxidant activity.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

This work was supported by The Indonesia Endowment Fund for Education and Ministry of Research and Technology/ National Research and Innovation Agency of Indonesia (Grant number: 4/FI/P-KCOVID-19.2B3/X/2020).

REFERENCES:

1. Lobo V, Patil A, Phatak A, Chandra N. Free Radicals, Antioxidants and Functional Foods: Impact on Human Health. J.Pharmacogn. Rev. 2010; 4, 118-126.

2. Pizzimenti S,Toaldo C, Pettazzoni P, Dianzani MU, Barrera G. The “two-faced” Effects of Reactive Oxygen Species and The Lipid Peroxidation Product 4-hydroxynonenal in The Hallmarks of Cancer.Cancers. 2010; 2: 338-363.

3. Asmat U, Abad K, Ismail K. Diabetes Mellitus and Oxidative Stress-A Concise Review. J. Saudi.Pharm. 2016; 24: 547-553.

4. Deng L, DuC, Song P. The Role of Oxidative Stress and Antioxidants in Diabetic Wound Healing. Oxid. Med. Cell Longev. 2021; 1:1-11.

5. Ghanshyam B, Jadhav, Ravindranath B. Saudagar. Free radical Scavenging and Antioxidant Activity of Punicagranatum Linn. Asian J. Res. Pharm. Sci. 2014; 2:51-54.

6. Leong LP, Shui G. An investigation of Antioxidant Capacity of Fruits in Singapore markets. J. Food. Chem. 2002; 76: 69-75.

7. Guo C, Yang J, Wei J, Li Y, Xu J, Jiang Y. Antioxidant Activities of Peel, Pulp and Seed Fractions of Common Fruits as Determined by FRAP Assay. J. Nutr. Res. 2003; 23: 1719-1726.

8. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant Activity Applying an Improved ABTS Radical Cation Decolorization Assay. J. Free. Radic. Biol. Med. 1999; 26: 1231-1237.

9. Gulcin I. Antioxidants and antioxidant methods-An updated overview. Archives of Toxicology 2020; 94: 651-715.

10. Kondragunta K, Karuppuraj V, Perumal K. Antioxidant activity and Folic acid content in indigenous isolates of Ganodermalucidum. Asian J. Pharm. Ana. 2016; 6(4): 213-215.

11. Valli G, Jeyalakshmi M. Preliminary Phytochemical and Antioxidant Study of Odina woodier Leaf Extract. Asian J. Pharm. Res.2012; 2(4): 153-155.

12. Sultana K, Kizhekkedath J, Pandey MC. Evaluation of Antioxidant Activity, Radical Scavenging, and Reducing Power of Clove Oil and Clove Oleoresin in Comparison with Natural and Synthetic Antioxidants in Chevon (Capra aegagrus hircus) and Chicken Meat. J.Def. Life. Sci. 2017; 3: 51-58.

13. Muthukumaran P, Salomi S, Umamaheshwari R. In vitro Antioxidant Activity of Premnaserratifolia Linn. Asian J. Res. Pharm. Sci. 2013; 3(1): 15-18

14. AraujoJ, Zhang M, Yin F. Heme Oxygenase-1, Oxidation, Inflammation, and Atherosclerosis. J. Front. Pharmacol. 2012; 3: 1-17.

15. Choi AM, Alam J. Heme Oxygenase-1: Function, Regulation, and Implication of a Novel Stress-inducible Protein in Oxidant-induced Lung Injury. Respir. Cell. Mol. Biol. 1996; 1: 9-19.

16. Gabr SK, Bakr RO, Mostafa ES, El-Fishawy AM, El-Alfy TS. Antioxidant Activity and Molecular Docking Study of Erythrina × Neillii Polyphenolics. South African Journal of Botany. 2019; 121: 470-477

17. Shrivastava A, Parveen N, Dewangan G, Verma VS, Bhattacharya A, Sharma M. Evaluation of Anti-Pyretic Potential of Cassia alata Leaves. Asian J. Pharm. Res. 2014; 4(4): 195-197.

18. Sagnia B, Fedeli D, Casetti R, Montesano C, Falcioni G, Colizzi V. Antioxidant and Anti-Inflammatory Activities of Extracts from Cassia alata, Eleusine indica, Eremomastax Speciosa, Carica Papaya and Polyscias Fulva Medicinal Plants Collected in Cameroon. PLoS One 2014; 9.

19. Lewis A, Levy A. Anti-inflammatory Activities of Cassia Alata Leaf Extract in Complete Freund’s Adjuvant Arthritis in Rats. J. West. Indian. Med. 2011; 60: 615-621.

20. Saito ST, Trentin D, Macedo AJ, Pungartnik C, Gosmann G, Silveira J, Guecheva TN, Hendriques JAP, Brendel M. Bioguided Fractionation Shows Cassia alata Extract to Inhibit Staphylococcus Epidermidis and Pseudomonas Aeruginosa Growth and Biofilm Formation. Evidence-Based Complement Altern. Med. 2012; 867103: 1-13

21. Somchit MN, Reezal I, Nur IE, Mutalib AR. In Vitro Antimicrobial Activity of Ethanol and Water Extracts of Cassia Alata. J. Ethnopharmacol. 2003; 84: 1-4.

22. Uwazie JN, Yakubu MT, Ashafa AOT, Ajiboye TO. Identification and Characterization of Anti-diabetic Principle in Senna Alata (Linn.) Flower Using Alloxan-Induced Diabetic Male Wistar Rats. J. Ethnopharmacol. 2020; 261: 1-15.

23. Varghese GK, Bose LV, HabtemariamS. Antidiabetic Components of Cassia Alata Leaves: Identification Through α-Glucosidase Inhibition Studies. Pharm. Biol. 2013; 51: 345-349.

24. Sarkar B, Khodre S, Patel P, Mandaniya M. HPLC Analysis And Antioxidant Potential Of Plant Extract Of Cassia Alata J. Asian. Pharm. Sci. Technol. 2014; 4: 4-7.

25. Pamulaparthi A, Prathap VR, Banala M, Nanna RS. Total Phenolic, Flavonoid Contents and Antioxidant Assays in Leaf Extracts of Senna Alata (L.) Roxb. J. Pharm. Sci & Res. 2015; 8: 981-985.

26. Kaur C, Kapoor HC. Anti-oxidant Activity and Total Phenolic Content of Some Asian Vegetables. IntJ. Food. Sci. Technol. 2002; 37: 153-161.

27. Hemalatha K, Girija K. Evaluation of Drug Candidature of some Benzimidazole Derivatives as Biotin Carboxylase Inhibitors: Molecular docking and Insilico studies. Asian J. Res. Pharm. Sci. 2016; 6(1): 15-20. DOI: 10.5958/2231-5659.2016.00002.3.

28. Zhang Q, Lin L, Ye W. Techniques for Extraction and Isolation of Natural Products: a Comprehensive review. Chin. Med. 2018; 3: 1-26

29. Angelina M, Mardhiyah A, Dewi RT, Fajriah S, Muthiah N, Ekapratiwi Y, Dewijanti IID, Sukirno, Jamilah, Hartati S. Physicochemical and Phytochemical Standardization, and Antibacterial Evaluation of Cassia Alata Leaves from Different Locations in Indonesia. Pharmacia, 2021; 68: 947–956.

30. Buenger J, Ackermann H, Jentzsch A, Mehling A, Pfitzner I, Reiffen KA. An interlaboratory Comparison of Methods used to Assess Antioxidant Potentials. Inter.J. Cosmet.Sci. 2006; 28: 135-146.

31. Roy A, Bhoumik D, Sahu RK, Dwivedi J. Phytochemical Screening and Antioxidant Activity of Sesbaniagrandiflora Leaves Extracts. Asian J. Res. Pharm. Sci. 214; 4(1): 16-21

32. Kosalec I, Bakmaz M, Pepeljnjak S, Vladimir-Knezević S. Quantitative analysis of the flavonoids in raw propolis from northern Croatia. Acta. Pharm. 2004; 54: 65-72.

33. Kulkarni K, Govindaiah. Evaluation of Anti-oxidant properties of some medicinal plant products by ABTS Radical Scavenging Assay. Research Journal of Science and Technology. 2022; 14(4): 213-8.

34. Ahmed D, Zara S, Baig H. In Vitro Analysis of Antioxidant Activities of Oxalis Corniculata Linn. Fractions in Various Solvents. J. Afr. Tradit. Complement. Altern. Med. 2012; 10: 158-165.

35. Rangasamy P, Hansiya VS, Maheswari PU, Suman T, Geetha N. Phytochemical Analysis and Evaluation of In vitro Antioxidant and Anti-urolithiatic Potential of various fractions of Clitoriaternatea L. Blue Flowered Leaves. Asian J. Pharm. Ana. 2019; 9(2): 67-76. DOI: 10.5958/2231-5675.2019.00014.0.

36. Abriyani E, Fikayuniar L. Screening Phytochemical, Antioxidant Activity and Vitamin C Assay from Bungoperak-perak (Begonia versicolar Irmsch) leaves. Asian J. Pharm. Res. 2020; 10(3):183-187. DOI: 10.5958/2231-5691.2020.00032.5.

37. Sadeghi Z, Valizadeh J, Shermeh OA, Akaberi M. Antioxidant Activity and Total Phenolic Content of Boerhavia Elegans (choisy) Grown in Baluchestan, Iran. J. Avicenna. Phytomedicine. 2015; 5: 1-9.

38. Loizzo MR, Pugliese A, Bonesi M, Menichini F, Tundis R. Evaluation of Chemical Profile and Antioxidant Activity of Twenty Cultivars from Capsicum Annuum, Capsicum Baccatum, Capsicum Chacoense and Capsicum Chinense: A comparison Between Fresh and Processed Peppers. LWT – Food. Sci. Technol 2015; 64: 623-631.

Received on 06.02.2023 Modified on 31.07.2023

Research J. Pharm. and Tech 2024; 17(4):1599-1605.

DOI: 10.52711/0974-360X.2024.00253

Materials and chemicals:

Extraction and fractionation:

LC-QTOF-MS/MS Analysis:

DPPH assay:

ABTS assay:

Total phenolic content (TPC):

Total flavonoid contents (TFC):

Total flavonoid content (TFC) was examined by aluminium chloride (AlCl3) colorimetric assays based and expressed as quercetin equivalents (QE), in milligrams per gram of the extract. The absorbance was determined at measured 415nm33,36.

Determination of TPC (Total Phenolic Compound), TFC (Total Flavonoid Compound), and radical scavenging activity

Table 2: Antioxidant capacities measured by DPPH radical scavenging, ABTS and FRAP assays.

Sample

IC50 (µg/mL)

DPPH radical scavenging assay

ABTS radical scavenging assay

FRAP assay

Positive control

4.45 ± 0.0055

4.26 ± 0.00

CAE

93.040 ± 0.035

27.504 ± 0.11

7.743 ± 0.01

CAH

>2000

50.799 ± 0.93

6.591 ±0.06

CAEA

18.540 ± 0.0076

11.294 ± 0.01

27.376 ± 0.26

CAB

84.690 ± 0.0143

4.286 ± 0.26

4.391 ±0.11

CAW

407.331 ± 0.0060

12.108 ± 0.45

9.466 ± 0.05

Figure 2: A significant correlation between TFC and FRAP values.

LC-QTOF-MS/MS analysis of fractions of C. alata leaves extract:

Table 4: Molecular docking results analysis of analysis compounds from C. alataleaves extract against HO-1 (PDB ID: 1N3U).

DISCUSSION:

Evaluation of Molecular docking:

Total flavonoid content (TFC) was examined by aluminium chloride (AlCl₃) colorimetric assays based and expressed as quercetin equivalents (QE), in milligrams per gram of the extract. The absorbance was determined at measured 415nm^33,36.

IC₅₀(µg/mL)