INTRODUCTION:

Natural products have been a cornerstone of medicine for thousands of years. Many cultures have utilized plants, minerals, and animal products for the treatment of different illnesses long before the advent of modern medicine. Several products have demonstrated pharmacological action, and these drugs are approved for use in humans¹due to increased public understanding of the harmful effects of synthetic drugs, the advancement of natural products as remedies for diseases has expanded. This led scientists and the pharmaceutical industry to create cutting-edge herbal treatments for a range of illnesses that have fewer adverse effects². The complex blends of organic compounds found in herbs can have varying concentrations based on several aspects of the plant's development, manufacturing, and processing³. Characterising novel lead structures from medicinal plants is made possible by their quantity and variety.

The exploration and utilization of phytoconstituents derived from plants have gained significant momentum in the field of natural product research due to their potential therapeutic benefits⁴. Quantitative analysis serves as the foundational step in phytochemical research, providing precise measurements of the concentration of specific constituents within a plant matrix. GC-MS allows for the comprehensive profiling of volatile and semi-volatile phytoconstituents, offering high sensitivity, specificity, and the ability to identify compounds even at trace levels. The isolation of phytoconstituents involves a series of extraction, purification, and characterization processes designed to obtain pure bioactive compounds from complex plant matrices⁵. The isolation of natural products from medicinal plants is of great importance in the search for new drugs⁶. These methodologies provide a robust framework for the systematic study of phytoconstituents, enabling the development of novel medications, the development of natural therapies, and the enhancement of our understanding of plant chemistry.

Sansevieria trifasciata (S.trifasciata), or viper's bowstring hemp, is an evergreen plant that thrives in tropical parts of Africa, ranging from east of Nigeria to the Congo and Southeast Asia. Bowstring hemp, produced by Sansevieria, is a durable plant fibre that was formerly used to make bowstrings. Like numerous other plants in its genus, it is now mostly utilised as a decorative plant, either indoors as a houseplant in colder climates or outside in warmer climates⁷. Historically, these species have been utilized to cure a variety of illness, including colds, diarrhoea, coughs, inflammation of the respiratory tract, swelling, bumps, bruise, ulcers, poisonous snake bites, hair fertilizers, ear-ache, jaundice, pharyngitis, skin itches, urinary diseases, analgesic and antipyretic⁸. The present work's main goal was to identify, quantify, and isolate the chemical components from S.trifasciata plant leaf extracts and then use GC-MS data to characterise them.

MATERIAL AND METHODS:

Plant collection:

Plants of S. trifasciata were collected (5kg) from the Shivamogga district, Karnataka, during July of 2022. The botanist Dr. Smitha Hegde Professor, NUCSER, Nitte (DU), verified the authenticity of the plant. The plant specimen is preserved by submitting it to the herbarium placed at the NGSM Institute of Pharmaceutical Sciences at Paneer, Deralakatte, Mangalore and it was assigned the herbarium ID 21PC008R.

Extraction:

The collected plant leaves were washed clean, dried under shade. Once the leaves were sufficiently desiccated, they were coarsely powered using a pulverizing grinder. This coarse powder was further taken for extraction. Extraction method chosen for the study was cold maceration method. 25g of plant material was taken in 6 different glass chambers and submerged in six distinct solvents, namely n-hexane, petroleum ether, methanol, ethanol, and aqueous. The glass chamber was then covered and kept in dark for 7 consecutive days with occasional stirring. Crude extract was obtained by vacuum filtering the extract using muslin cloth after seven days, and then drying the filtrate. These crude samples were semi-solid in nature and was soluble in their respective solvents. The extracted samples were further employed for phytochemical (qualitative) screening, quantitative analysis, characterization using GC-MS analysis and isolation.

Preliminary Phytochemical screening (Qualitative Analysis):

Six solvent extracts of S. trifasciata were subjected to a first phytochemical screening in accordance with established protocol to discover the phytoconstituents that are found in leaves by observing the color changes in reaction mixture^9,10,24.

Quantitative analysis:

Total phenolic content estimation:^11,12

The total phenolic content in various S. trifasciata leaf extracts was evaluated using the Folin-Ciocalteu colorimetric technique. Six distinct solvent extracts were weighed, and 1mg (1000μg/ml) of each extract was diluted in the appropriate solvent. Following the separation of 10, 20, 30, 40, and 50μg/ml from this solution, after adding 2ml of 7.5% Na₂CO₃, the mixture was then allowed to ferment at room temperature for roughly 20 minutes. After that, 200μl of FC reagent was added, well combined, and allowed to stand for 30 minutes at room temperature in the dark. At 650nm, the absorbance was measured. The standard was gallic acid, which was treated in the same way as the other substances. The total phenolic content was expressed as the extract's gallic acid equivalent (GAE), expressed in mg/g.

Total flavonoid content estimation:^13,14

The total flavonoid concentration was ascertained using the colorimetric assay with aluminium chloride. Plant extract (1mg) was combined with 4 millilitres of distilled water. To this, 0.3 millilitre of 5% sodium nitrite was added. Two millilitres of 1M NaOH were added after six minutes, followed by 0.3 millilitres of 10% AlCl₃. Immediately after, distilled water was added to the mixture and thoroughly mixed. At 510nm, absorbance was measured. The amount of total flavonoid content per gramme of dry extract was represented as mg of catechin equivalents.

Total alkaloid content estimation:¹⁵

Preparation of reagent:

Bromocresol green solution:

Bromocresol green (69.8mg) was mixed with 3ml (2N NaOH) and 5ml of distilled water. The solution was heated to dissolve the components, and then distilled water was added to dilute it to 1000ml.

Phosphate buffer (4.7): Prepared by adding 0.2M citric acid (42.02g of citric acid in 1 litre of distilled water) to 2M sodium phosphate (71.6g Na₂HPO₄ in 1litre of distilled water) to bring its pH down to 4.7.

Atropine:

Dissolved 1mg of atropine in 10ml of purified water.

A basic spectrophotometric technique was employed to identify the total alkaloid content in the plant extracts with the principle that when bromocresol green reacts with the sample, gives yellow coloured complex, which was extracted using CHCl₃ at pH 4.7. The absorbance was measured and compared with standard atropine at the concentration of 10, 20, 30, 40 and 50μg/ml.

1mg of plant extract was dissolved in 1ml of 2N HCl and filtered. This solution was poured into a separatory funnel, and was rinsed three times with 10ml of CHCl₃. This solution's pH was brought to neutral using 0.1 N NaOH. Next, this solution was mixed with 5ml of BCG solution and 5ml of phosphate buffer. After vigorously shaking the mixture, the complex that had formed was extracted using 1, 2, 3, and 4 millilitres of CHCl₃. Chloroform was used to dilute the extracts to volume after they were collected in a 10ml volumetric flask. At 470nm, the complex's absorbance in CHCl₃was measured. Standard solution of atropine was also prepared in the similar manner as that of test solution.

GC-MS Analysis:¹⁶

A GC-MS analysis was performed on six extracts of leaves from S. trifasciata. The analytical tool utilised was the Clarus 680 GC. A fused silica column loaded with Elite-5MS, a mixture of 5% biphenyl and 95% dimethylpolysiloxane, was employed in the experiment. The column's dimensions were 30m × 0.25mm ID × 250μm df. The temperature was raised to 300°C at a rate of 10°C per minute after being held steady at 60°C for two minutes. After six minutes, the final temperature was kept constant. To create the component separation, helium was employed as the carrier gas at a constant flow rate of one millilitre per minute. Throughout the chromatographic run, the injector temperature was kept at 260°C. Both the ion source and the transfer line were

maintained at 240°C. Mass spectra were obtained at a rate of one scan per 0.1 second and measured at 70 eV. The run lasted for thirty-two minutes in total. By comparing the constituents' mass spectra with the accurate standards and those in the NIST 2008 database, the components were identified. Several compounds' identities were validated using relative retention indices that were based on established standards. A comparison of mass spectra from published works was also included.

Isolation of phytoconstituents:^17-19,25,26

200g of the leaf powder was added to a one-litre conical flask and linked to the Clevenger equipment in order to extract the essential oils. After adding 500mL of distilled water and heating it to boiling, the flask was sealed. After distilling the essential oils for 5hours in a graduated cylinder, the oil and aqueous layer were separated. Until further study, the oil was stored in the refrigerator. The oil obtained from the distillation was facilitated to column chromatography (10 X 300MM) for further purification. Thin-layer chromatography (TLC) methods were employed using various solvents ratio to choose the ideal mobile phase for the isolation of oils through column chromatography.

Preliminary TLC was performed using various ratios of n-hexane and ethyl acetate (n-hexane: ethyl acetate: 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1) with the oil obtained from steam distillation prior to the start of isolation by column chromatography. The dots were visible in UV light with a wavelength of 254nm. The iodine chamber was also used for better visualization of spots. n-hexane: ethyl acetate 4:6 and 5:5 was selected as the best solvent ratio for the isolation of the compounds which gave a significant two different spots with an Rf value of 0.94 and 0.92 respectively.

The column was initially saturated with the solvent system – n-hexane: ethyl acetate:: 4:6 by pouring the solvent into a pre-packed column. The oil (15ml) was dissolved in the mobile phase and was loaded into the column. As the compound separation process developed via a number of steps, the various eluents were gathered in varied fractions, and these separated eluents were further subjected to TLC until the fraction showed the desired spot with the expected Rf value. The fraction showing spot were collected, concentrated and recrystallized using ethyl acetate.

Table 1: Phytochemical screening of S. trifasciata extracts from the leaves

Sl. NO.	Phytoconstituents	Presence in solvents
Sl. NO.	Phytoconstituents	H₂O (Aqueous extract)	EtOH (Ethanolic extract)	MeOH (Methanolic extract)	CHCl₃ (Chloroform extract)	PE (Petroleum ether extract)	n-H (n-Hexane extract)
1	Carbohydrates	+	+	+	+	+	+
2	Proteins	+	-	+	-	-	+
3	Fats / oils	-	+	+	+	+	+
4	Alkaloids	+	+	+	+	+	+
5	Flavonoids	+	+	-	-	-	+
6	Phenolic compound	+	+	+	-	-	+
7	Saponins	+	-	-	+	-	-
8	Glycosides	+	+	+	+	+	+
9	Steroids and terpenoids	-	-	-	-	-	+

(+): presence of compound; (-): absence of compound.

The column was then washed and saturated with the solvent system – n-hexane: ethyl acetate : 5:5 by pouring the solvent into a pre-packed column. The various eluents were gathered in varied fractions, and these separated eluents were further subjected to TLC until the fraction showed the desired spot with the expected Rf value. The fraction showing spot were collected, concentrated and recrystallized using ethyl acetate.

RESULTS AND DISCUSSION:

Preliminary Phytochemical screening (Qualitative Analysis):

A preliminary phytochemical investigation revealed that the leaf extract included alkaloids, flavonoids, terpenoids, glycosides, phenolic compounds, and carbohydrates. (Table-1).

Quantitative analysis:

Qualitative analysis revealed that all six solvent extracts contained consistently identified amounts of the three primary phytoconstituents: phenolic chemicals, flavonoids, and alkaloids. Thus, these components were selected for additional evaluation using quantitative methods.

The six extracts total flavonoid content was ascertained by applying a standardised process with the aluminium chloride colorimetric method. Milligrams of quercetin equivalent (QE) per gramme of dry extract were used to express the results. A curve was created by graphing the absorbance of quercetin standards, and an equation y=0.008x-0.014 was deduced, with an R² value of 0.9939. The extract's concentration (x) and absorbance (y) are represented in this equation, respectively. The extracts with the highest content of flavonoids were methanol, pet, ethanol, and aqueous. In descending order, flavonoids were discovered in the extracts of ether, chloroform, and n-hexane.

A folin-ciocalteu colorimetric test was performed in accordance with known methods to ascertain the total phenolic content of the six extracts. In milligrams of gallic acid equivalent (GAE) per gramme of dry extract, the results were reported. The equation y=0.0046x + 0.0999 with an R² value of 0.9832 was obtained by graphing the absorbance of gallic acid standards to create a calibration curve. The extracts containing phenolic chemicals included methanol, n-hexane, pet ether, aqueous, and ethanol extracts at different concentrations; the extract including chloroform had the greatest concentration.

A total alkaloid content measurement was made, and the results were reported in mg of atropine equivalent (AE) per gram of extract. The equation y = 0.0033x + 0.0069, which has an associated R² value of 0.9902, was obtained by graphing the absorbance of atropine standards to create a calibration curve. The extract with the greatest alkaloid concentration among the others was found to be the n-hexane extract. Alkaloids were also found in descending order of concentration in the ethanol, chloroform, methanol, aqueous, and ethanolic extracts.

Table 2: Total flavonoid, total phenolic and total alkaloid contents of S. trifasciata extracts

Solvent	TFC mg of QE/gm	TPC mg of GAE/gm	TAC mg of AE/gm
Aqueous	6.82±0.084	4.70±0.155	28.81±0.212
Ethanol	5.56±0.169	4.56±0.311	20.93±0.431
Methanol	10.85±0.084	12.38±0.304	30.53±0.424
Chloroform	3.53±0.084	17.60±0.148	36.97±1.889
Pet. ether	3.9±0.084	5.64±0.304	38.20±0.212
n-hexane	3.53±0.084	11.37±0.304	45.48±0.212

Each column represented as mean ± SEM (n=3); level of significance <0.05; QE -quercetin equivalent; GAE – gallic acid equivalent; AE – atropine equivalent.

Total phenolic content:

Figure 1: Total phenolic content calibration curve using gallic acid as the standard.

Total flavonoid content estimation:

Figure 2: Total flavonoid content calibration curve using quercetin as the standard.

GC-MS Analysis:

In the GC-MS analysis of the six different extracts derived from S. trifasciata leaves, numerous phytoconstituents were identified, each possessing unique pharmacological properties. The following are some of the compounds found in the extracts along with their medicinal applications:

Tridecane- antibacterial, antiproliferative²⁰, isophytol- anti-inflammatory, antiarthritic, anticoronary, and antidiabetic agents²¹, Nitric acid- hypotensive²², sulfurous acid- antioxidant ²³.

Total alkaloid content estimation:

Figure 3: Total alkaloid content calibration curve using atropine as the standard.

Table 3: The GC-MS analysis of 6 different extracts of leaves of S. trifasciata.

S. No	IUPAC name of the constituents	Molecular formula	Molecular weight
1	Isophytol	C₂₀H₄₀O	296
2	Dichloroacetaldehyde	C₂H₂OCl₂	112
3	1-butene-3yne-1-chloro	C₄H₃Cl	86
4	3,9-diazabicyclo[4,2,1]nonan-4-one,3,9-dimethyl	C₉H₁₆ON₂	168
5	Sulfurous acid	C₉H₂₀O₃S	208
6	Nitric acid	C₃H₇O₃N	105
7	1-propanesulfonyl chloride	C₃H₆O₂Cl₂S	176
8	Propane	C₃H₆ClBr	156
9	2-hydroxy-Propenamide	C₃H₇O₂N	89
10	Tridecane	C₁₄H₂₈	196
11	2,2-dimethyl-propyl 2,2 dimethyl-propane sulfinyl sulfone	C₁₀H₂₂O₃S₂	254
12	3-[N-aziridyl] propionyl hydrazide	C₅H₁₁ON₃	129
13	1,6-heptadiene,3-methyl	C₈H₁₄	110
14	Dichloro acetic acid	C₂H₂O₂Cl₂	128
15	But-1-en-3ynyl methyl sulfide	C₅H₆S	98
16	4-chlorobuten-3-yne	C₄H₃Cl	86
17	Sulfurous acid, dipentyl ester	C₁₀H₂₂O₃S	222
18	Methyl phosphine	CH₂P	48
19	Cyclopropane, ethynyl	C₅H₆	66
20	2,2,3,3,3-pentafluro-1 propanol	C₃H₃OF₅	150
21	Acetamide, N-(2,5 dihydro-5-oxo-2-furanyl)	C₁₀H₁₆O₃	184
22	2-hexanone,3,4-dimethyl	C₈H₁₆O	128
23	1-pentene,3,4-dimethyl	C₇H₁₄	98
24	Methylphosphinic acid, ethyl ester	C₃H₉O₂P	108
25	2-butyne,1,1-dimethoxy	C₆H₁₀O₂	114

The GC-MS spectra of different solvent extracts:

Figure 4: Phytoconstituents found in the GC-MS spectrum of aqueous extract

Figure 5: Phytoconstituents found in the GC-MS spectrum of chloroform extract

Figure 6: Phytoconstituents found in the GC-MS spectrum of ethanolic extract

Figure 7: Phytoconstituents found in the GC-MS spectrum of methanol extract

Figure 8: Phytoconstituents found in the GC-MS spectrum of n-hexane extract

Figure 9: Phytoconstituents found in the GC-MS spectrum of pet.ether extract

Isolation of phytoconstituents:

Using column chromatography, two compounds were generated with the yield of 26 and 28ml. For improved identification, both compounds were submitted to characterization using three different techniques: GC-MS, FT-IR, and NMR.

Isolation of Tridecaine, 3-methylene

Physical parameters:

Colour – colourless liquid;

Consistency – thick liquid/semi solid

Molecular formula – C₁₄H₂₈;

Molecular weight – 196

Yield – 26ml;

Rf value –0.94 (n-hexane: ethyl acetate :: 4:6)

Boiling point-248°C

Table 4: Spectral data of Tridecaine

Spectra	Spectral values
IR spectrum (ATR-IR-cm¹)	2932.38 (-CH alkane); 1462.62 (-CH₂); 1376.31(CH₃)
¹H NMR (CDCl₃- ppm)	δ 1.253 (-H alkane positions: 2,3,4,5,6,7,8,9,10,11,12,); 0.889 (-Hpositions 1 and 13)
¹³C NMR (CDCl₃-ppm)	δ 29.690 (CH₂position 2 and 12) and 22.680 (-CH₂position 4,6,7,8,9,10)
GC-MS(m/z)	M⁺ peak-196,1221

*Position of the functional group or atoms are specified from the structure mentioned in spectrum.

Figure 10: FTIR spectrum of Tridecaine,3-methylene

Figure 11: ¹H NMR spectrum of Tridecaine,3-methylene

Figure 12: ¹³ C NMR spectrum of Tridecaine,3-methylene

Figure 13: Mass spectrum of Tridecaine,3-methylene

Characterization of Tridecaine:

IR spectrum showed prominent peaks at 2932.38 cm^-1 indicating the -CH stretching of alkane; 1462.62 cm^-1 indicating the -CH₂ bending of alkane; 1376.31 cm^-1 indicating the -CH₃ bending of alkane. Both ¹H NMR and ¹³C NMR were performed using CDCl₃ as the solvent. ¹H NMR showed signals at the chemical shift values (ppm) of δ 1.253 (-CH group); 0.889 (-CH₃ group). ¹³C NMR showed signals at the chemical shift values (ppm) of δ 29.690, 22.680 (-CH₂).

Figure 14: Structure of Tridecane

Isolation of Isophytol :

Physical parameters:

Colour–Colourless; Consistency – viscous liquid/semi-solid

Molecular formula-C₂₀H₄₀O;

Molecular weight - 296

Yield -28ml;

Rf value – 0.92 (n-hexane: ethyl acetate: 5:5)

Melting point: 4.08 °C

Boiling Point:309°C

Table 5: Spectral data of Isophytol

Spectra	Spectral values
IR spectrum (ATR-IR-cm^-1)	3428.24 (-OH of alcohol); 2920.28 and 2851.28 (-CH alkane); 1641.31(C=C alkene); 1463(-CH₂); 1377.35 (-CH₃ alkane)
¹H NMR (CDCl₃-ppm)	δ 7.260 (H); 1.685 (-CH); 1.30 (CH₂); 1.013 , 0.954(CH₃)
¹³C NMR (CDCl₃-ppm)	δ 76.67 (C); 31.91 (CH); 29.685(CH); 22.680(CH₃)
GC-MS(m/z)	M⁺ peak – 296.1946

*Position of the functional group or atoms are specified from the structure mentioned in spectrum.

Figure 15: FTIR spectrum of compound Isophytol

Figure 16: ¹H NMR spectrum of compound Isophytol

Figure 17 :¹³ C NMR spectrum of compound isophytol

Figure 18: Mass spectrum of compound Isophytol

Characterization of Isophytol:

IR spectrum showed prominent peaks at 3428.24 cm^-1 indicating the OH alcohol; 2920.28 and 2851.28 cm^-1 indicating -CH stretching of alkane; 1641.31 cm^-1 indicating -C=C alkene; 1463 cm^-1 indicating -CH₂ bending of alkane; 1377.36 indicating -CH₃ of alkane. Both ¹H NMR and ¹³C NMR were performed using CDCl₃ as the solvent. ¹H NMR showed signals at the chemical shift values (ppm) of δ 7.260 (H); 1.685 (-CH₂); 1.013, 0.954 (-CH₃).

Figure 19: Structure of Isophytol

CONCLUSION:

Alkaloids, flavonoids, terpenoids, phenolic compounds, and carbohydrates were among the several kinds of phytoconstituents that were discovered during the first screening of six distinct extracts, which were water, ethanol, methanol, pet.ether, n-hexane, and chloroform. Promising findings were obtained from a quantitative examination of the phenolic, flavonoid and alkaloids present in each extract. Among the extracts, methanol, n-hexane, and chloroform showed the highest levels of flavonoids, phenolic compounds, and alkaloids, respectively. Further analysis using GC-MS identified 25 major phytoconstituents, primarily consisting of pharmacologically significant alkanes, esters, terpenoids, and acids. Through the isolation and characterization of S. trifasciata leaf extracts, two specific chemicals, tridecane and isophytol, were identified.

ACKNOWLEDGEMENT:

The NGSM Institute of Pharmaceutical Sciences, Nitte (Deemed to be University), Mangalore, India is acknowledged with gratitude by the authors providing all the necessary support to carry out this work.

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Received on 04.10.2024 Revised on 07.02.2025

Accepted on 11.04.2025 Published on 12.06.2025

Available online from June 14, 2025

Research J. Pharmacy and Technology. 2025;18(6):2567-2574.

DOI: 10.52711/0974-360X.2025.00367

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