INTRODUCTION:

A rumored African traditional remedy, Kigelia africana DC. is now widely available in India. It is a member of the Bignoniaceae family and has historically been used to treat a variety of illnesses. Among the many components that are utilized, root bark has been shown to have therapeutic qualities for gynecological ailments such as fibroid and uterine cancer, as well as biological activities including antioxidant, antimalarial, and antiprotozoal activity¹.

Plants such as Kigelia africana produce phytochemicals for a variety of purposes, such as defense against insect infestations, disease transmission, etc. Kigelia africana bioactive molecules have been useful in identifying lead compounds for use in the creation of pharmaceuticals to treat human ailments. Plant-based remedies are used by almost half of the world's population in Latin America, Asia, and Africa².

Kigelia africana fruit has a cylindrical shape, measuring 25 to 50cm in length and 7.5 to 15cm in width. The fruits are characterized by their woody texture and take on a greyish brown color when fully ripe. They have the ability to stay attached to the tree for a duration of up to one year. The weight of the fruit can reach a maximum of 3-3.5kg.

The purpose of the study was to thoroughly investigate the biologically active ingredients in Kigelia africana fruit extract using liquid chromatography/mass spectroscopy (LC/MS). It was carried out with both polarities in the ESI ionization mode. There are thirty-eight recognized chemicals in total positive mode. The molecules belong to the class group known as terpenoid, flavonoids, phenols, glycosides, and others. Among these molecules some have biological activities that include anti-inflammatory, antibacterial, antioxidant, and anti-diabetic properties. The Kigelia africana fruit meal exhibited significant antioxidant characteristics.

To the best of our knowledge, despite the root of Kigelia africana having several traditional uses, not much has been published on its phytochemistry or therapeutic usage. The primary aim of this investigation was to employ Liquid Chromatography/Mass Spectroscopy, an effective analytical method, to thoroughly examine the biologically active components present in K. africana fruit bark extract.As a result, this study reveals the phytochemical profile of K. africana fruit, highlighting its potential as a medicine for major human illnesses.

MATERIALS AND METHODS:

Plant Material:

The K. africana fruit was collected from local market of Agra (27.1929°N, 78.0231°E). The plant was taxonomically identified from Raw Material Herbarium and Museum, Delhi, CSIR-NISCAIR as Kigelia africana.The fruit was gathered from the tree. Then it was shade-dried for ten days. crushed, and then ground using a grinder. The dried plant parts were crushed to produce coarse powder, which was kept in polythene airtight containers at room temperature for future use³.

Reagents and Chemicals

All further compounds were analytical grade, unless noted otherwise. Aluminum chloride, Wagner's reagent, Mayer's reagent, Sodium hydroxide, Ferric chloride, Copper sulfate, n-Hexane, Quercetin, Ammonia liquid, Nitric acid, Sulphuric acid, Sodium carbonate, Ninhydrin, Chloroform (Fisher Scientific), Potassium hydroxide, Potassium bromide, Glacial acetic acid, Methanol (Qualigens-Thermo Fisher Scientific), Ethanol (Changshu Hongsheng Fine Chemical Co. Ltd.), Ethyl acetate (Finar Ltd.), DPPH (Sisco Research Laboratories Pvt Ltd., India).

Preparation of Extracts:

The fruit of Kigelia africana was dried in the shade and ground into coarse powders weighing 500g. The powders were then extracted using 500 mL of ethanol by the soxhlet technique, and maceration process. Following a 24 hr. period, the crude extract underwent filtration and was subsequently evaporated to complete dryness using a water bath maintained at a temperature of 70°C. An analysis was conducted on the desiccated residue of the unrefined extract⁴.

Preliminary Phytochemical Screening:

Estimation of TFC:

A colorimetric test was used to determine the total flavonoid content (TFC). To 4 milliliters of distilled water, 100µL of extract was added. 0.3mL of 5% sodium nitrite was then added. A 10% aluminum chloride solution (0.3mL) was added after 5 minutes. 2 ml of 1 M sodium hydroxide was added to the mixture after 6 minutes. 3.3 CC of distilled water was added right away to dilute the mixture, and it was thoroughly mixed. In comparison to a blank, the absorbance at 510 nm was measured. The calibration curve was based on quercetin as the standard. The extract's total flavonoid concentration was given in mg of quercetin equivalents per gram of sample (mg/g).

By using established procedures, the phytoconstituents in Kigelia africana were screened out of the maceration and soxhlet extract⁵.

FT-IR analysis:

On the Shimadzu IR Affinity-1S FTIR spectrophotometer, FTIR spectra were recorded. With a 1 cm^-1resolution, the scanning range was 4000–400cm^-1.

LCMS analysis:

At Punjab University's Sophisticated Analytical Instrumentation Facility in Chandigarh, the LC-MS separation was carried out utilizing a Water Micro mass Quadrupole-ToF MS. The molecular weight of the structures that were previously known to be present in Kigelia africana fruit was used to identify each molecule from the reference compounds⁶.

DPPH Scavenging Activities:

Five distinct methanol concentrations (2, 4, 8, 16, and 32 mg/mL) were used to synthesize the plant extracts under investigation. They produced the same levels of ascorbic acid, a popular antioxidant. The examined extract solution (2.5mL) and 1mL of methanol were used as the starting point for the blank solutions. For the negative control, 1 mL of methanol and 2.5mL of DPPH solution were used. The absorbance was measured at 517nm following incubation. The tests were done three times. The IC50 value was computed⁷.

RESULTS:

Plants contain a large number of secondary metabolites, according to research conducted using a range of techniques to profile their phytochemical profiles. FTIR, LCMS, and total flavonoid content were used in this work to profile the plants. The DPPH technique was used to measure antioxidant activity.

Extractive value:

Kigelia africana fruits were collected and dried under shade and pulverized in desired size (#40). The results of extractive values of ME and SE was found to be 8 and 7.6%.

Preliminary Phytochemical Screening:

The result of phytochemical screening showed that both the extracts are rich in secondary metabolites like alkaloids, flavonoids, phenols, terpenoids, carbohydrates, saponins and cardiac glycosides.

Total Flavonoid Content:

The TFC of quercetin, which was estimated (Fig. 1) (y = 0.0108x+0.0806 R² = 0.9567) and represented in QAE/g of dry extract weight (Mean±SEM). Comparing maceration extraction (ME) (4.47±0.643) to other extraction methods, the TFC of SE was the greatest at 6.4±0.173.

Figure 1: Standard curve of Quercetin

FTIR interpretation:

The FTIR analysis of maceration and soxhlet extraction methods of Kigelia africana fruit confirmed the presence ofaliphatic primary amines (N-H_(S)), Alkynes (C-H_(b)), ketones,organic Sulphur compounds (C=S_(S)),Amino acids, esters and lactones, mononuclear aromatic hydrocarbon (C-H_(b)), acid halides, carboxylic acid anhydride, lactams (C=O_(S)), alkanes, alkyl (C-H_(S)), aromatic hydrocarbon (C-H_(S)), heteroaromatic (C-H_(S)), silicon compounds (SiO-H_(S)), amines (N-H_(b)), amine salts (N-H_(b)), ester (C-O_(S)), nitro compound (N-O(S)), ether, epoxides, peroxides (C-O_(S)), nitrates (cis isomer N=O_(S)), alkenes, ketones,, cyclic alkanes (C-H_(S)),alcohol and phenols (O-H_(S)), alkynes, sulfonamides (N-H_(S)), Phosphorous compounds (P-C_(s)), (Fig. 2) (Silverstein et al., 2005)

Figure 2: FTIR spectrum of ME and SE

Liquid chromatography-Mass Spectroscopy (LC/MS) Analysis:

The chromatogram detected 38 components, which are shown in Table 1. The identities of the compounds were confirmed by examining the mass/charge [m/z] data and comparing it with previously released information. Various secondary metabolites were identified from the LCMS data which includes the phenolic compounds (balaphonin, caffeic acid, p-coumaric acid, ferulic acid, luteolin, kigeliol, 6-p-coumaroyl-sucrose), steroids (β-sitosteron, stigmasterol), triterpenes (oleanolic acid, phytol), Quinones (pinnatal, norviburtibnal), Iridoids (jiofuran, jioglutolide), Alkanes (n-hentriacontane, tritriacontane), Esters (hydroxyphenyl ethyl ester), Unsaturated Fatty acids. Apart from the thirty-eight identified compounds from the extracts, twelve molecules have reported biological activities (Table 2)

Table 1: Characterization of LC-MS/MS of Extract

S. No.	m/z	Molecular Formula	Mol. wt. g· mol⁻¹	RT (Min.)	Identified Compound	Classification	Extract/ Fraction
1	165.05	C₉H₈O₃	164.16	36.42	p-Coumaric acid	Phenolic Compounds	SE,ME
2	181.04	C₉H₈O₄	180.16	32.83	Caffeic acid	Phenolic Compounds	SE,ME
3	133.06	C₁₀H₁₀O₄	194.18	3.08	Ferulic acid	Phenolic Compounds	SE,ME
4	289.08	C₁₅H₁₀O₆	286.23	23.23	Luteolin	Phenolic Compounds	ME
5	423.23	C₁₂H₂₃O₁₄P	422.28	23.09	6-p-coumaroyl-sucrose	Phenolic Compounds	SE,ME
6	239.09	C₁₂H₁₄O₅	238.24	28.79	Kigeliol	Phenolic Compounds	SE,ME
7	357.22	C₂₀H₂₀O₆	356.40	19.12	Balaphonin	Phenolic Compounds	SE,ME
8	209.08	C₁₁H₁₂O₄	208.21	3.05	8-hydroxy-6, 7-dimethoxy-3-methyl-3, 4-dihydroisocoumarin	Coumarins	SE,ME
9	415.15	C₂₉H₅₀O	414.71	2.70	β-Sitosterol	Sterols	ME
10	413.11	C₂₉H₄₈O	412.70	25.24	Stigmasterol	Sterols	SE,ME
11	457.15	C₃₀H₄₈O₃	456.71	31.93	Oleanolic acid	Triterpenes	SE,ME
12	281.05	C₂₀H₄₀O	296.53	39.87	Phytol	Triterpenes	SE,ME
13	257.26	C₁₉H₃₄O₂	294.50	32.89	(9Z,12Z)-Methyl octadeca-9,12-dienoate	Unsaturated Fatty acids	SE,ME
14	243.07	C₁₅H₁₄O₃	242.27	18.68	Lapachol	Quinones	SE,ME
15	241.06	C₁₅H₁₂O₃	240.30	15.22	Dehydro α-lapachone	Quinones	SE
16	339.24	C₂₀H₁₈O₅	338.11	22.10	Pinnatal	Quinones	SE,ME
17	147.04	C₉H₆O₂	146.14	23.09	Norviburtinal	Quinones	SE,ME
18	243.07	C₁₄H₁₀O₄	242.23	40.01	2-(1-Hydroxyethyl)-naphtho[2,3-b] furan-4,9-quinone	Quinones	SE,ME
19	189.16	C₉H₁₆O₄	188.22	37.75	7-hydroxy-10-deoxyeucommiol	Iridoids	SE,ME
20	173.12	C₉H₁₆O₃	172.22	19.31	10-Deoxyeucommiol	Iridoids	SE,ME
21	185.12	C₉H₁₂O₄	184.00	29.10	Jiofuran	Iridoids	SE
22	189.16	C₉H₁₆O₄	188.22	29.37	3-(2-hydroxyethyl)-5-(2-hydroxypropyl)-4,5-dihydrofuran-2(3H)-one	Iridoids	SE,ME
23	241.06	C₁₁H₁₂O₆	240.21	34.00	7-hydroxyeucommic acid	Iridoids	SE
24	189.16	C₉H₁₆O₄	188.22	31.11	7-hydroxy eucommiol	Iridoids	SE,ME
25	187.14	C₉H₁₄O₄	186.20	27.77	Jioglutolide	Iridoids	SE,ME
26	187.14	C₉H₁₄O₄	186.21	27.89	Des-p-hydroxy benzoyl kisasagenol B	Iridoids	SE,ME
27	511.37	C₂₄H₃₀O₁₂	510.48	20.61	6-Trans-caffeoyl ajugol	Iridoids	SE
28	525.46	C₂₄H₂₈O₁₃	524.54	22.84	Verminoside	Iridoids	SE
29	509.19	C₂₄H₂₈O₁₂	508.50	28.40	Specioside	Iridoids	SE,ME
30	177.05	C₂₅H₃₀O₁₃	538.51	28.34	Minecoside	Iridoids	SE,ME
31	437.2	C₃₁H₆₄	436.85	32.47	n-hentriacontane	Alkanes	ME
32	367.2	C₂₆H₅₄	366.70	31.37	11-(2,2-dimethylpropyl) heneicosane	Alkanes	SE,ME
33	185.12	C₁₃H₂₈	184.36	29.10	4,4-dimethylundecane	Alkanes	SE
34	353.22	C₁₆H₃₃I	352.34	32.78	1-iodohexadecane	Alkanes	SE,ME
35	465.1	C₃₃H₆₈	464.90	34.96	Tritriacontane	Alkanes	SE
36	437.2	C₃₁H₆₄	436.85	32.47	Hentriacontane	Alkanes	ME
37	409.25	C₂₉H₆₀	408.79	25.78	Nonacosane	Alkanes	SE,ME
38	181.08	C₁₀H₁₂O₃	180.27	32.83	2-(4-hydroxyphenyl) ethyl ester	Esters	SE,ME

Table 2: Reported biological activities of identified molecules.

S. no	Predicted Compounds	Biological Activity	References
1	p-Coumaric acid	Antimicrobial activities, antioxidant and anti-inflammatory	8
2	Caffeic acid	Anti-inflammatory and anticancer activities	9
3	Ferulic acid	Antioxidant, anticarcinogenic, anti-inflammatory, hepatoprotective, antimicrobial, and antiviral activity	10
4	Luteolin	Antioxidant, anti-inflammatory, antimicrobial, and anticancer activities	11
5	β-Sitosterol	Anticancer effect	12
6	Stigmasterol	Anticancer, anti-inflammatory, anti-arthritis, and anti-allergy	13
7	Oleanolic acid	Antioxidant, anticancer, hepatoprotective, anti-inflammatory, and anti-asthmatic activities	14
8	Phytol	Anti-bacterial	15
9	Lapachol	Immunosuppressive, contraceptive, antimicrobial, antiulcer, anticancer, antimalarial, anti-inflammatory, trypanocidal, leishmanicidal, molluscicidal, and antifungal activities	16
10	Pinnatal	Potent oxidative radical scavenging and antioxidant activity against DPPH free radicals	17
11	Verminoside	Sensitizes cisplatin-resistant cancer cells and suppresses metastatic growth of human breast cancer	18
12	Specioside	Anti-inflammatory	19

Antioxidant activity by DPPH Method:

The plant extracts were evaluated for their antioxidant activity by measuring their capacity to decrease the stable DPPH free radical.The extracts were diluted in a series to obtain concentrations of 2, 4, 8, 16 and 32mg/mL. A 100µL portion of a methanol solution containing 0.16 mM DPPH was combined with 200µL of different doses of each extract. A control solution was made without including any plant material. The spectrophotometer was used to measure the absorbance at a specific wavelength of 515nm. The measurements were conducted three times and then averaged.The ability of the extracts to scavenge DPPH radical was calculated (Table no. 3, Fig. 3).

Table 3: Free radical scavenging activity of extracts

S. No	Conc. (mg/mL)	Ascorbic acid	SE	ME
1	2	16.01±0.27	13.19±0.31	09.51±0.32
2	4	23.03±0.39	18.58±0.34	11.14±0.06
3	8	37.75±0.12	25.55±0.24	19.62±0.41
4	16	70.68±0.56	44.23±0.18	36.12±0.44
5	32	89.91±0.26	67.66±0.32	49.42±0.06
IC₅₀ value	14.1582 mg/mL

Figure 3: DPPH scavenging activity

DISCUSSION:

This study used maceration and soxhlet extraction to extract the fruit of Kigelia africana. Every extract was given an extractive value (% w/w), with the Soxhlet extraction having the greatest extractive value (8 %). Because the soxhlet process recycles fresh solvent and operates at high temperatures, it enhances the mass transfer rate. Sonolysis, cell membrane disruption, and the extraction of intracellular secondary metabolites are the results of these forces. Using a variety of conventional techniques, the preliminary phytochemical screening of the maceration extract and soxhlet extraction was also carried out. The results indicate the existence of several secondary metabolites, including cardiac glycosides, terpenoids, flavonoids, and alkaloids. The maximum flavonoid content was found in the SE fraction (6.4±0.173mg GAE/g) according to the TFC result, followed by the SE fraction (4.47±0.643 GAE/g). As per the TFC results, K.africana fruit is a fruit that is rich in flavonoid compounds. These compounds will be used in future pharmacological investigations related to diseases including Alzheimer's, Parkinson's, cardiovascular, and numerous inflammatory and lifestyle disorders caused by oxidative stress.

FTIR analysis revealed that the -OH and C=O functional groups seen in the extracts were consistently present. The SE revealed peaks at 2973, 3337, 1362, 1735, 1743, and 879 cm-1, which were linked to lactams, alkanes, heteroaromatic, aromatic hydrocarbons, alcohol and phenols, epoxy, lactones, acid halides, and carboxylic acid anhydride. It was discovered that the FTIR interpretation and the LCMS data were associated. Liquid chromatography Using mass spectroscopy, one may identify, measure, and perform a mass analysis of a variety of inorganic or organic molecules that are either non-volatile or semi-volatile in a mixture. Another useful technique for examining complex plant extracts is the combination of mass spectrometry and liquid chromatography (LC/MS). Among hyphenated approaches, it is an extremely powerful and sophisticated instrument for identifying low and high molecular weight research. One major advantage of ESI is its ability to provide mild ionization in LCMS. The hydro-ethanolic extract's LCMS chromatogram is shown in Fig. The degree of similarity between the molecular mass and molecular weight was used to identify the extracted and fractionated molecules. There were 38 chemicals found in all. Ep-Coumaric acid, or phenolic compound (m/z 165.05; 36.42min), was the first compound to elute.Similarly, luteolin (m/z 289.08), 6-p-coumaroyl-sucrose (m/z 423.23), kigeliol (m/z 239.09), balaphonin (m/z 357.22), and caffeic acid (m/z 181.04) are other cardiac glycosides. Other compounds such as coumarin 8-hydroxy-6, 7-dimethoxy-3-methyl-3, and 4-dihydroisocoumarin (m/z 209.08) were also eluted at 3.05 minutes, in that order. Additionally, additional substances such as β-Sitosterol (m/z 415.15) and Stigmasterol (m/z 413.11) were also eluted at 2.70 and 25.24 minutes. Additionally, additional molecules such as phytonolic acid (m/z 281.05) and oleanolic acid (m/z 457.15) were also eluted from triterpenes at 31.93 and 39.87 minutes. At 32.89 minutes, 9Z,12Z)-Methyl octadeca-9,12-dienoate (m/z 257.26), an unsaturated fatty acid, was eluted. Additionally, several Quinones were also eluted, including 2-(1-Hydroxyethyl)-naphtho[2,3-b] furan-4,9-quinone (m/z 243.07), Dehydro α-lapachone (m/z 241,06), Pinnatal (m/z339.24), and Norviburtinal (m/z147.04) at 18.68, 15.22, 22.10, 23.09, and 40.01 minutes. In addition, various iridoids, such as 7-hydroxy10-Deoxyeucommiol (m/z 189.16), Jiofuran (m/z 185.12), and 10-Deoxyeucommiol (m/z 173.12) 3-(Ethyl-2-hydroxy)2-hydroxypropyl)-5-2-(3H)-one, or -4,5-dihydrofuran (m/z 189.16), Des-p-hydroxy benzoyl kisasagenol B (m/z 187.14), Jioglutolide (m/z 187.14), 7-hydroxyeucommic acid (m/z 241.06), and 7-hydroxy eucommiol (m/z 189.16), Sixth, trans-caffeoyl ajugol (m/z 511.37), verminoside (m/z 525.46), specioside (m/z 509.19), and minecoside (m/z 177.05) at 37.75, 19.31, 29.10, 29.37, 34.00, 31.11, 27.77, 27.89, 20.61, 22.84, 28.40, and 28.34 minutes. Additionally, a few other alkanes were also eluded, including n-hentriacontane (m/z 437.0), 11-(2,2-dimethylpropyl) heneicosane (m/z 367.20), 4,4-dimethylundecane (m/z 185.12), 1-iodohexadecane (m/z 353.22), Tritriacontane (m/z 465.10), Hentriacontane (m/z 437.20), and Nonacosane (m/z 409.25) at 32.47,31.37,29.10,32.78,34.96,32.47, 25.78 mint. Additionally, one ester (the 2-(4-hydroxyphenyl) ethyl ester; m/z 181.08) is eluted at 32.83 minutes.

The yield and composition of bioactive chemicals, such as flavonoids and phenolic compounds, derived from plant sources are significantly influenced by the extraction technique used. Researches have demonstrated that the antioxidant activity of these compounds can be strongly impacted by the extraction solvent used. Antioxidant molecules from plant materials are frequently extracted using polar and medium-polar solvents, such as water, ethanol, methanol, and their aqueous mixes. Comparing soxhlet extraction to maceration, a more straightforward extraction procedure, it has been found that the former produces greater concentrations of flavonoids and phenolic chemicals due to its repeated cycles of solvent evaporation and condensation. The Soxhlet method's superior solubilization and recovery of these bioactive chemicals accounts for the observed difference in extraction efficiency. Plant extracts exhibit a clear correlation between their quantity of flavonoids and phenolic components and their antioxidant properties. Because Soxhlet extracts contain more of these chemicals than maceration extracts, it has been discovered that Soxhlet extracts have a stronger antioxidant capacity.

CONCLUSIONS:

Research interest in K. africana has increased due to its acknowledged medicinal potential. Many of its traditional medical use, however, have not yet been the subject of scientific investigation. The present study provided valuable insights into the antioxidant potential of Kigelia Africana fruit extracts. The results indicate that the extract exhibited potent antioxidant activity, which could be attributed to the presence of bioactive compounds such as polyphenols. More investigation into the existential studies on its pharmacological action is advised in future.

ACKNOWLEDGEMENT:

The authors are thankful to the Director, Bhagwant Global University, Kotdwar for providing the necessary facilities to carryout this research work.

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Received on 11.09.2024 Revised on 10.01.2025

Accepted on 21.03.2025 Published on 02.08.2025

Available online from August 08, 2025

Research J. Pharmacy and Technology. 2025;18(8):4032-4037.

DOI: 10.52711/0974-360X.2025.00579

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