INTRODUCTION:

Since ancient times, medicinal plants have been used to treat various diseases and other beneficial purposes.

The belief among people that natural medicines are much safer than synthetic drugs has led to exceptional growth in the usage of plants and plant products as traditional or folk medicine in primary health care.

Plants have protein, total sugar, fat, Vitamin C, alkaloids, flavonoids, glycosides, phenolics, and terpenoids which are responsible for their pharmacological and medicinal properties¹.

According to WHO, 80% of the World ‘s population is dependent on medicinal plants to treat various diseases, especially for people living in developing countries². However, less than 1% of plants are characterised by bioactive compounds and phytochemical profiling of phytochemicals³.

Pratia begonifolia (Wall.) Lindl. synonym Lobelia nummularia Lam. is a herb belonging to the Campanulaceae family with purple-red berries. These plants are mostly found in India (the Himalayas and the North East States), Nepal, Bhutan, Bangladesh, Sri Lanka, SW China, Myanmar, Thailand, Laos, Vietnam, Malaysia, Philippines, and New Guinea⁴. The ethnic group of Karbi Anglong District, Assam, practised treating headaches by using plant fruit⁵. In Mizoram, within Aizawl, people use whole plant juice to cure bee stings, toothache, diarrhoea, stomach ulcer, and tonsillitis⁶, and as reported, boiled extract of the entire plant is consumed for kidney stones⁷. In Bhutan, aqueous extract of fruits and leaves for Epilepsy, boils and ear pus⁸. In Manipur, the plant is traditionally used to dissolve kidney stones and can be consumed raw and boiled with unrefined sugar for kidney stone-related problems. These plants mainly grow fully mature in the hot season between April and September under the shady foothills and riversides.

Furthermore, plants may contain bioactive compounds used as therapeutic and prophylactic agents, raw materials for drug designs, or pharmacologically active compounds⁹. Still, medicinal plants are traditionally used everywhere around the corner of World without scientific experiments. Therefore, the present study attempts to reveal the phytochemical profiling, antimicrobial and toxicity activity of Pratia begonifolia (Wall.) Lindl. to evaluate the pharmacological action of the plants, which is very much needed.

MATERIALS AND METHODS:

1. Sample collection and identification:

Plants were collected from June to September from the foothills of the Imphal-East district, (24˚37’N and 93˚39’E) Manipur, North Eastern State of India, which lies 2590 feet above sea level, identification was done in Botanical Survey of India, Eastern Regional Centre, Shillong and identification letter no. BSI/ERC/Tech/Identification/2018/20. Plants were washed with distilled water and shade dried for 72hrs.

2. Determination of elemental composition by Graphite Furnace Atomic Absorption Spectroscopy (GF-AAS):

The dried plants were ground into fine powder by using mortar and pestle. The powdered sample (0.5gm) was digested in a Teflon digestion vessel with HNO₃, and with double distilled water, volume made up to 50 ml and analysed using GF-AAS.

3. Determination of elemental composition by Scanning Electron Microscopy Energy Dispersive X-ray (SEM-EDAX):

The dried plants were ground into fine powdered by using mortar and pestle and investigate elemental analysis using Scanning electron microscopy and EDAX (Quanta 200)

4. Determination of functional groups by Fourier Transform Infrared Spectroscopy (FT-IR):

The dried plants were ground and macerated with ethanol for 48hrs, and dried plant extract was mixed with KBr salt using mortar and pestle and compressed into thin pellet; infrared spectra were recorded on Shimadzu FTIR spectrometer.

5. Determination of volatile bioactive compounds by using Gas Chromatography-Mass Spectrometry (GC-MS):

The shade-dried plants (150 gm) were extracted using a Soxhlet extractor with methanol and chloroform solvent, and the extract was vacuum dried using a rotary vacuum evaporator. Analysis was performed using GC-MS Perkin Elmer (USA) in Guwahati Biotech Park, IIT Guwahati campus, and the GC-MS Model was Clarus 680GC and Clarus 600MS. The capillary column(60.0m×250µm) was used, maintaining the initial temperature at 70ºC for 3min, ramp 6ºC/min to 200ºC and hold 3min, ramp 6ºC/min to 300ºC have 10min and maintaining the injection temperature at 280ºC. As a carrier gas, Helium gas was used, and a ratio of 10:1 was used as a split injection, and the solvent delay was 9 min. The transfer and source temperature were maintained at 200ºC and 180ºC, respectively. The mass spectral scan range was at the rate of 40 to 600Da. The compounds were matched with the combinations listed in the National Institute of Standards and Technology (NIST) library.

6. Determination of non-volatile bioactive compounds by using High-Resolution Liquid Chromatograph Mass Spectrometer (HR-LCMS):

Plants were extracted with methanol solvent using a Soxhlet extractor, and the extract was vacuum dried using a rotary vacuum evaporator. HR-LCMS analysis was carried out at SAIF, IIT Bombay. Analysis was performed both in dual positive and negative mode using instrument Model-C6550A with 0.01% mass resolution. The acquisition method set up the minimum range of 50(m/z) and a maximum of 1000 Dalton, and for a Hip sampler, the G4226A Model was used with speed 100µl/min, ejection speed 100µl/min and flush out factor 5µl and 8µl injection volume. After that chromatogram, retention volume was studied.

RESULT AND DISCUSSION:

Figure 1: Determination of elemental composition of Pratia begonifolia (Wall.) Lindl. by using GF-AAS

The elemental analysis of Pratia begonifolia (Wall.) Lindl. by using GF-AAS revealed the presence of 9 elements which are Chromium (1.46±0.001 ppm), Calcium (0.86±0.01ppm), Magnesium (0.63±0.002 ppm), Potassium (0.62±0.02ppm), Sodium (0.59±0.006 ppm), Zinc (0.27±0.03ppm), Iron (0.13±0.02ppm) and Cobalt (0.08±0.003 ppm). Chromium is found to be the highest.

SEM-EDAX analysis, revealed the presence of 5 elements viz. Phosphorous, Iron, Calcium, Magnesium and Potassium and Potassium are the highest weight percentage concentrations. As reported, these plants are traditionally used for kidney stones related problems, some elements have an inhibitory effect, and Magnesium is one of the inhibitors of kidney stones. Low concentration in the system has a high risk of for kidney stone formation. It also helps to reduce the concentration of oxalate¹⁰ and Zinc also has an inhibitory effect on calcium oxalate stone¹¹.

Figure 2: Determination of elemental composition Pratia begonifolia (Wall.)Lindl. by using SEM-EDAX

FTIR is one technique that helps identify the chemical constituents and elucidate the structural compounds¹². FTIR results were in Table 1, and 9 peak values were found and identified functional groups matched to peak value by using with IR standard table, which helps to detect biomolecular composition. The FTIR analysis revealed the presence of phenols, alkanes, alkenes, aldehydes, amines, nitro compounds and aromatics; all these compounds belong to secondary metabolites¹³.

Table 1: Determination of Peak Value for Pratia begonifolia (Wall.) Lindl. by using FTIR

Peak Value	Functional groups
3439.19	O-H stretch-H-bonded, phenols
2924.18	(C-H-stretch alkanes)
1666.48	(C=C stretch alkenes
1734.06	(C=O stretch aldehydes, saturated aliphatic)
1527.87	N-O asymmetric stretch nitro compounds
1413.87	C-C stretch (in ring) aromatics
1361.79	C-H rock alkanes
1246.06	C-N stretch aliphatic amines
1070.53	C-N stretch aliphatic amines

GC-MS is a sophisticated techniques for analysing of individual components from complex mixture using a temperature-controlled capillary column¹⁴. The unknown compounds from a complex mixture can be studied by matching the spectra with the standard spectra¹⁵. GCMS analysis of methanol extract (Table 2) of Pratia begonifolia (Wall.) Lindl. found 12 compounds namely Cyclotrisiloxane, Hexaphenyl; Methyl 11,14-Octadecadienoate; Phytol; Methyl 11,14,17-Eicosatrienoate; D-fructose,1,3,6-trideoxy-3,6-epithio-; Methyl 10-methyl-undecanoate; Beta. -d-lyxofuranoside, o-nonyl-; Ascaridole epoxide; Z, Z-6,28-Heptatriactontadien-2-One; 2,4,4-Trimethyl-1-Pentanol; Beta. -D-Mannofuranoside, 1-O-(10-Undecenyl)-; Chloroacetic Acid, 2,2-Dimethylpropyl Ester.

Table 2: Determination of volatile bioactive compounds by using GC-MS for Pratia begonifolia (Wall.) Lindl.methanol extract

AREA %	Compound	RT	Molecular Formula	Molecular Weight
4.800	Cyclotrisiloxane, Hexaphenyl	8.033	C₃₆H₃₀O₃Si₃	512
2.534	Methyl 11,14-Octadecadienoate	42.247	C₁₉H₃₄O₂	294
2.127	Phytol	34.484	C₂₀H₄₀O	296
2.012	Methyl 11,14,17-Eicosatrienoate	42.687	C₂₁H₃₆O₂	320
1.567	D-Fructose, 1,3,6-Trideoxy-3,6-Epithio-	32.438	C₆H₁₀O₃S	162
1.207	Methyl 10-Methyl-Undecanoate	38.021	C₁₃H₂₆O₂	214
1.192	Beta. -D-Lyxofuranoside, O-Nonyl-	32.563	C₁₄H₂₈O₅	276
1.127	Ascaridole Epoxide	26.591	C₁₀H₁₆O₃	184
0.769	Z, Z-6,28-Heptatriactontadien-2-One	35.725	C₃₇H₇₀O	530
0.582	2,4,4-Trimethyl-1-Pentanol	32.838	C₈H₁₈O	130
0.539	Beta. -D-Mannofuranoside, 1-O-(10-Undecenyl)-	33.159	C₁₇H₃₂O₆	332
0.431	Chloroacetic Acid, 2,2-Dimethylpropyl Ester	33.259	C₇H₁₃O₂Cl	164

GCMS analysis of chloroform extract of Pratia begonifolia (Wall.) Lindl. found 17 compounds namely Pentacosane; Eicosane, 2-Methyl-; Hexacosane; Pentatriacontane; Dotriacontane; Phytol; Z, Z-6,28-Heptatriactontadien-2-One; Dotriacontane; Squalene; Triacontane; Eicosane; Hentriacontane; Octadecane, 2,6,10,14-Tetramethyl; Tritetracontane; Octatriacontane, 1,38-Dibromo-; 1 Octadecanoic Acid, 2-Oxo-, Methyl Ester; Eicosane, 2,6,10,14,18-Pentamethyl-. Eicosane and heptadecane shows antibacterial activity¹⁶. As reported, Squalene has anti-bacterial, antioxidant, antitumor, anti-inflammatory, immunostimulant, triterpene natural antioxidants, prevention of cell deterioration, anti-senescence and improved immunity and sexual function ¹⁷, reduction of blood levels of cholesterol and triglycerides.

Table 3: Determination of volatile bioactive compounds by using GCMS of Pratia begonifolia (Wall.)Lindl.forChloroform extract

AREA %	Compound	RT	Molecular formula	Molecular Weight
5.280	Pentacosane	43.518	C₂₅H₅₂	450
4.722	Eicosane, 2-Methyl-	55.472	C₂₁H₄₄	296
4.041	Hexacosane	45.138	C₂₆H₅₄	366
4.041	Pentatriacontane	45.138	C₃₅H₇₂	492
3.885	Dotriacontane	46.659	C₃₂H₆₆	450
3.818	Phytol	34.364	C₂₀H₄₀O	296
3.818	Z, Z-6,28-Heptatriactontadien-2-One	34.364	C₃₇H₇₀O	530
3.415	Dotriacontane	41.767	C₃₂H₆₆	450
3.038	Squalene	52.976	C₃₀H₅₀	410
2.580	Triacontane	48.094	C₃₀H₆₂	422
2.278	Eicosane	35.324	C₂₀H₄₂	282
2.276	Hentriacontane	37.710	C₃₁H₆₄	436
1.839	Octadecane, 2,6,10,14-Tetramethyl	30.162	C₂₂H₄₆	310
1.417	Tritetracontane	32.743	C₄₃H₈₈	604
1.258	Octatriacontane, 1,38-Dibromo-	50.836	C₃₈H₇₆Br₂	690
1.016	1 Octadecanoic Acid, 2-Oxo-, Methyl Ester	53.732	C₁₉H₃₆O₃	312
0.519	Eicosane, 2,6,10,14,18-Pentamethyl-	20.022	C₂₅H₅₂	352

Table 4: HR-LCMS for Pratia begonifolia (Wall.) Lindl. (Negative ion)

RT	Mass	Name	Formula
5.001	338.1069	2-Isoprenylemodin	C₂₀H₁₈O₅
8.053	272.0259	Quinalizarin	C₁₄ H₈O₆
9.001	286.0403	Luteolin	C₁₅ H₁₀ O₆
9.749	206.0542	Isoeugenitol	C₁₁H₁₀O₄
11.828	310.1655	O-Desmethylquinidine	C₁₉H₂₂N₂O₂
12.258	310.1296	Descarboethoxyloratadine	C₁₉H₁₉CLN₂

(Table 4, 5.) For HR-LCMS analysis of Pratia begonifolia (Wall.) Lindl. performed both positive and negative ion, and 6 compounds were revealed for negative ion namely2-Isoprenylemodin, Quinalizarin, Luteoline, Isoeugenitol, O-Desmethylquinidine, Descarboethoxyloratadine. Luteolin has biological activities like anti-inflammatory and anti-cancer¹⁸. Isoeugenitol has anti-inflammatory property as it inhibited COX-1 activity¹⁹.

Table 5: HR-LCMS analysis of Pratia.begonifolia (Wall.) Lindl. (Positive ion)

RT	MASS	NAME	FORMULA
5.765	431.2694	Hydroxysalmeterol	C₂₅H₃₇N O₅
6.239	402.1283	2’,4’-Dihydroxychalcone	C₂₁H₂₂O₈
6.538	478.2753	Ipecac (Methylpsychotrine)	C₂₉H₃₈N₂O₄
6.943	578.1597	Rhoifolin	C₂₇H₃₀ O₁₄
8.299	284.0661	Genkwanin	C₁₆H₁₂ O₅
9.117	253.1526	Anabasamine	C₁₆H₁₉N₃
10.855	221.1866	Cuscohygrine	C₁₃H₂₄N₂O
11.574	342.2855	Eicosanedioic Acid	C₂₀H₃₈O₄
13.257	370.3167	Docosanedioic Acid	C₂₂H₄₂O₄
18.027	281.2696	Oleamide	C₁₈H₃₅NO
18.329	570.2826	Khayanthone	C₃₂H₄₂O₉

11 compounds namely Hydroxysalmeterol, 2’,4’-dihydroxychalcone, Ipecac (methylpsychotrine), Rhoifolin, Genkwanin, Anabasamine, Cuscohygrine, Eicosanedioic acid, Docosanedioic acid, Oleamide, Khayanthone were found in methanol extract as positive ion. Oleamide is reported as chemopreventive agent against Alzheimer’s disease ²⁰. Oleamide has a role in anti-allergic reaction by attenuating the secretion of histamine and beta hexosaminidase and the production of allergic-related cytokines such as IL-4 and TNF-a ²¹ and Oleamide acts as a potent inhibitor of microcystis aeruginosa a cyanobacterium which produces lethal toxin44. Rhiofolin, a flavone glycoside possesses antioxidant, anti-inflammatory, antimicrobial, hepatoprotective, anti-cancer ²², and besides, 13% inhibition of COX sackievirus B3 infection against E. coli²³ and is an ideal antitumor agent²⁴. Genkwanin has pharmacological effects like anti-bacterial, antioxidant, anti-inflammatory, anti-cancer properties²⁵_.

Generally, flavanoids are known as anti-bacterial agents against different microorganisms and the flavonoids like Luteoline, Rhiofolin, Genkwanin are found in Pratia begonifolia(Wall.) Lindl. extract²⁶_.

CONCLUSION:

It may conclude that above study reveals the presence of elements and bioactive compounds related to kidney-related problems and other diseases in Pratia begonifolia (Wall.) Lindl. Bioactive compounds with anti-bacterial, antioxidant, anti-inflammatory, anti-allergic, hepatoprotective and anti-tumour bioactivities have presence in Pratia begonifolia (Wall.) Lindl. Elements which have inhibitory properties in kidney stone formation also present. The properties of these compounds on the pharmacological activity should be further evaluated for drug design and others beneficial purposes.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors are grateful to Head, SAIF, NEHU, Shillong, Department of Physics Manipur University, Department of Chemistry Manipur University, Guwahati Biotech Park, IIT Guwahati Campus, Head SAIF IIT Bombay for allowing to used GF-AAS, SEM-EDAX, FTIR, GC-MS and HR-LCMS respectively.

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Received on 03.01.2022 Modified on 14.07.2022

Research J. Pharm. and Tech 2023; 16(4):1556-1560.

DOI: 10.52711/0974-360X.2023.00254