INTRODUCTION:

Plants and microorganisms in particular provide an infinite source of unique molecules that may be used to create brand-new drugs. Since ancient times, medicinal herbs in particular have been utilized in traditional medicine to promote overall wellness and to treat infectious diseases both preventatively and therapeutically.¹Numerous secondary metabolites, including alkaloids, terpenoids, tannins,and flavonoids, which are known to have immunomodulatory, antidiabetic, antibacterial, antioxidant, and anticancer activities, are abundant in medicinal plants.²

Given their potential health advantages, phenolic compounds are a promising target in the search for phytochemicals that are good for humans.

One of the many-purpose forest tree species is M. longifoliaJ.Koenig. belonging to family Sapotaceae has a distinct function in medicine thus it is grown for that purpose.³Mahua's phytochemical analysis reveals that it contains a variety of compounds that have pharmacological effects including analgesic⁴, anti-inflammatory⁵, antioxidant⁶, hepatoprotective⁷, anticancer, spasmolytic, anthelmintic, anticonvulsant, antitumor, antiulcer, neuropharmacological, and dermatological activities. Mahua is also high in proteins, vitamins, alkaloids, sugars, flavonoids, tannins, steroids.⁸

The bioactive compounds, mainly phenolics, are plentiful in vegetables, fruits, spices, herbs, and other therapeutic plants. They are categorized as secondary metabolites in phytochemistry. They are believed to possess health-improving qualities. Recent studies have examined and identified the important role that these metabolites play in disease prevention and health promotion. Food specialists and nutritionists are interested in polyphenolic compounds because of their possible health advantages, notably flavonoids and phenolic acids.⁹In order to increase the shelf life of lipid-rich meals, polyphenols are increasingly being targeted due to their potential as antioxidants.¹⁰

Vitamins, carotenoids, and phenolic compound such tannins, lignans, flavonoids, stilbenes, and phenolic acids are among the bioactive substances. They may reduce the likelihood of developing cancers and heart disorders.^1,11

Aim of the current study was to characterized and quantify important phyto-constituents from the leaves of Madhuca longifolia J. Koenig. For this, phenolic compounds were separated, identified, and described in terms of their possible bioactivities using LC-MS/MS-quadrupole-time of flight (QTOF). When compared to commercial medications, substances derived from natural sources will have less negative effects.¹²These substances give researchers studying cancer insight for developing new drugs.

MATERIALS AND METHODS:

2.1 Collection of plant and authentication:

Leave parts of the plantMadhuca longifolia J.Koenig (ML) were collected from the area of Aurangabad, India. Plant was authenticated by BSI, Pune (Specimen No.- NKML-1 (Madhuca longifolia (J.Koenig ex.L.) J.E. Macbr.Var. longifolia). After being thoroughly cleaned three to four times, Madhuca longifolia leaves are dried for two weeks in the shade. To achieve a small particle size, leaves are crushed using mechanical grinder and then sieved using sieve no 36 (420micron) for the uniform particle size. The plant material was then used for further investigation.

2.2 Preparation of extracts:

Hydro-alcoholic (1:1) extraction using cold maceration (soak 500gm powder sample in Distilled water for 24 hrs with occasional stirring). After 24hrs, the extract was filtered through muslin cloth and Whatman's filter paper number 1 and then filtrates were subsequently concentrated using a rotary evaporator at reduced pressure. Aqueous suspension was gradually divided into butanol, chloroform, and ethyl acetate in order to disperse its polar and non-polar compounds. The resulting fractions were dried in a rotary evaporator. Until they were required, each fraction was stored at 4 °C.

2.3 HR-LCMS analysis of Madhuca longifolia:

The ethyl acetate fraction was examined using the Agilent (6550 iFunnel Q-TOFs) system, which contains a column component, hip sampler, and electrospray ion generation (ESI) with Agilent Jet Stream (AJS). A Q-TOF with a dual ion source and a binary pump were used to separate chromatograms. 5μl of ethyl acetate fraction were injected using a needle wash into an Agilent UHPLC system that was equipped with a Hypersil Gold column (C18 100 × 2.1mm-3 MICRON). At a flow rate of 0.3ml/min, 0.1 percent formic acid in water (A) and acetonitrile (B) served as the elution solvent.In 50minutes, the gradient system's composition went from 95% A: 5% B to 5% A: 95% B, then it went back to the original composition for 10 minutes before holding onto it for 5 more. The ESI's positive and negative ionization modes were used to conduct the MS analysis. Capillary voltage of 3500 V, nebulizer pressure of 35 psi, gas temperature of 250°C, and drying gas flow of 13L/min were employed as the MS source parameters. Agilent Mass Hunter software was used for the mass spectrometric analysis and Q-TOF data acquisition.^13-15

2.4 FT-IR analysis of Madhuca longifolia:

The different peaks and their functional groups were identified using FTIR analysis utilizing a Perkin Elmer Spectrophotometer system with a transmittance range of 400–4000cm-1. The FTIR's peak values were noted. The outcomes of each analysis were double-checked.^13,16

3. RESULT AND DISCUSSION:

3.1 High Resolution-Liquid Chromatography-Mass spectrometry analysis (HR-LCMS) analysis of Madhuca longifolia:

On a chromatogram, Figure 1 depicts the approximations of the various component concentrations that are existing in Madhuca longifolia and that are eluted in as perwith retention time. The height of the peak was used to calculate relative concentration of bioactive substances originate in plants. The compounds which were eluted at different times are examined by the mass spectrometer to ascertain the make-up and structure of components. These mass spectra indicate the unique fingerprint of the molecule in the data store.

24compounds (11 unknown and 13 known) were identified in the Madhuca longifolia ethyl acetate fraction based on mass, retention time, and molecular formula, as shown in Table 1. Principal substances identified related to a number of secondary metabolite groups, such as Flavonoids, Flavonoid glycosides, Coumarins, Alkaloids and triterpenoid glycoside, based on the study of HR-LCMS and comprehensive literature search. The distinctive mass spectra of the Madhuca longifolia bioactives that have been isolated are shown in Figure 2.

Figure 1. HR-LCMS [(a) +ESI and (b) -ESI] chromatogram of ethyl acetate fraction of Madhuca longifolia

Table 1: HR-LCMS isolation of different bioactive compounds from ethyl acetate fraction of Madhuca longifolia

SN.	Name	Mol. formula	RT	Mass	m/z	Chemical nature
1.	Myricetin	C15H10O8	6.399	318.0376	319.0448	Flavonoids
2.	Myricetin-7-rhamnoside	C21H20O12	6.173	464.0879	463.0806	Flavonoids glycoside
3.	Capillarisin	C16H12O7	7.73	316.0583	317.0656	Coumarins
4.	Isoquinoline	C9H7N	8.68	129.058	130.0652	Alkaloids (benzopyridines)
5.	Goyaglycoside C	C38H62O9	19.874	662.4458	663.4532	Triterpenoid glycoside
6.	Hexyl-2-furoate	C11H16O3	7.424	196.1102	197.1175	Flavouring agent
7.	Kaempferol 3-O-β-Dgalactoside	C21H20O11	7.172	448.0969	447.0895	Flavonoid
8.	Sanggenon C	C40H36O12	17.178	708.2031	709.2104	Flavonone flavonoid

Figure 2. Mass fragmentation ofisolated bioactives a) Myricetin, b) Myricetin-7-rhamnoside, c) Capillarisin, d) Isoquinoline, e) Goyaglycoside C, f) Hexyl-2-furoate, g) Kaempferol 3-O-β-D-galactoside

3.2 FT-IR analysis Madhuca longifolia:

The bioactive chemicals that were hypothesized by HR LCMS testing were verified by FTIR analysis. Numerous FTIR spectroscopic peaks were used to identify the functional groups contained in Madhuca longifolia ethyl acetate fraction, as illustrated in Figure 3. Using the FTIR spectrum and the peak value in the infrared radiation band, the functional group of the bioactive components was identified. Ethylacetate fraction ofMadhuca longifolia shows presence of different bioactive compound like alcohols, phenols, aliphatic primary amine, alkanes, alkyne, alkenes, aldehyde, allene, carboxylic acid, alkyl aryl ether, α, and β-D-glucopyranose as shown in Table 2.

Figure 3: FTIR Spectrum of ethyl acetate fraction of Madhuca longifolia

Table 2: Peak values of FTIR Spectrum of Madhuca longifolia

Peak Value	Functional Group	Spectroscopic Assignments
3289.05	Alcohols, phenols	O-H stretch, H–bonded
2923.18	Carboxylic acids	O-H stretching
2856.68	Alkane	C-H stretch
2673.58	Alcohol, carboxylic acid	O-H stretch
1709.10	Carboxylic acid	C=O stretching
1649.81	Alkenes/Aromatics	C=C-H stretch
1601.85	Cyclic alkene	C=C Stretching
1507.50	Nitro compound	N-O Stretching
1452.38	Alkenes	C-H bend stretching
1355.12	Nitro compound	N-O stretching
1293.56	Amines	C-N bending
1258.15	Alcohol, ether, ester	C-O stretching
1193.16	Alkyl halides	-CH2Xstretching
1081.94	Aliphatic amines	C-N stretching
1010.67	Alcohol, Ether	C-O stretching
864.48	Primary, secondary amine	N-H stretching
789.76	Halogen compound	C-X Stretch
691.56	Aromatic compound	C-Hbending
673.61	Alkenes	C=C bending

DISCUSSION:

The prevention of oxidative stress-related illnesses, including numerous malignancies, cardiovascular disorders, and neurological diseases, has been extensively researched for phenol and flavonoids.^17,18 The phenolic acids, flavonoids, stilbenes, carotenoids, and vitamins found in medicinal plants may be obtained sustainably and in plenty. These plants are also attractive prospects for the creation of novel medicinal medicines due to the availability of bioactive substances with diverse characteristics.¹⁹ It has been demonstrated that flavonoids have a range of anticancer properties.^20,21 In this work, 11 novel and 13 recognized chemicals were found during phytochemical screening of the ethyl acetate fraction of Madhuca longifolia.Flavonoids identified using HR-LCMS such as myricetin, myricetin-7-rhamnoside, Kaempferol 3-O-β-Dgalactoside and sanggenon C prominently induces apoptosis in various type of cancers.

By focusing on and controlling the expression of several molecular targets implicated in inflammation, angiogenesis, cell proliferation, invasion, apoptosis, and metastasis, myricetin demonstrates its therapeutic promise. Myricetin slows the growth of tumors by activating and deactivating a number of signaling pathways, triggering apoptosis through both intrinsic and extrinsic routes, and reactivating a number of tumor suppressor genes.²²Myricetin induces apoptosis in a variety of cancer cell types, including A2780 and OVCAR3 ovarian cancer cells, HepG2, A431 human skin cancer cells, PC 3 and DU 145 prostate cancer cells, and MCF-7 human breast cancer cells, through altering a number of pathways.^23-25Sanggenon C inhibits NO synthesis, iNOS expression, and ROS activation of the mitochondrial pathway to cause apoptosis in colon cancer cells.²⁶ In the human NCI-H460 non-small cell lung cancer cell line, kaempferol3-O-β-Dgalactosidepromotes apoptosis via extrinsic and intrinsic mechanisms.²⁷

The bioactive compounds identified using HR-LCMS being responsible for the pharmacological activity in plants. This modern hyphenated technique of analysis opens the new horizon for detecting more and more bioactives precisely.^28,29The results of mass spectroscopy showed that the loss of an electron from a molecule caused the highest mass to charge ratio in the mass spectrum. It is therefore concluded that after functionalization, molecular weight increased due to the attachment of functional groups (OH, C=O), which confirms the functionalization.³⁰ According to IR measurements, oxygen-containing groups (OH, C=O) were found.³¹

CONCLUSION:

The use of secondary metabolites produced from natural sources is a successful strategy for discovering and developing novel medications. According to the results of the current study phytochemical investigation, Madhuca longifolia may be a source of beneficial substances. A total of 24 phenolic compounds (11 unknown and 13 recognized) were identified in an attempt to confirm their fragmentation pattern using MS/MS.It was revealed that Madhuca longifolia has therapeutically significant bioactive substances includingFlavonoids, Flavonoid glycosides, Coumarins, Alkaloids and triterpenoid glycoside by HR-LCMS and FTIR analysis. The results of this study indicate that Madhuca longifolia are an excellent source of biogenic compounds with both structural and biological activity. The identification of many bioactive chemicals in this investigation supports Madhuca longifolia's historical use for number of diseases. Isolating substances and scrutinizing their pharmacological effects are currently the subjects of investigation.

CONFLICT OF INTEREST:

Authors did not report any conflicts of interest.

ACKNOWLEDGMENTS:

Authors are thankful to the Indian Institute of Technology, Bombay (IIT Bombay), Sophisticated Analytical Instrument Facility (SAIF), performed an HR-LCMS analysis.

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Received on 01.01.2023 Modified on 07.04.2023

Research J. Pharm. and Tech 2024; 17(4):1435-1440.

DOI: 10.52711/0974-360X.2024.00227