ORIGINAL ARTICLE
Comparative phytochemical analysis of Senna alata (L.) Roxb.
and Gliricidia sepium (Jacq.) Walp. leaf extracts
O. Veena, C.V. Lekshmipriya, V. Soorya, Greeshma, Tschidiso Herman Pheko,
T.S. Swapna
Department of Biotechnology, University of Kerala, Thiruvananthapuram, Kerala, India
Corresponding author: O. Veena, Email: dr.veena@keralauniversity.ac.in
Journal of Experimental Biology and Zoological Studies. 1(2): p 113-200, Jul-Dec 2025.
Received: 14/06/2025; Revised: 19/06/2025; Accepted: 24/06/2025; Published: 01/07/2025
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Abstract
Phytochemicals remain a cornerstone of drug development due to their natural origin, cost-
effectiveness, and lower toxicity. Senna alata (S. alata) and Gliricidia sepium (G. sepium), both
belonging to the family Fabaceae, are recognized for their diverse bioactivities, including
pharmacological and insecticidal properties. However, the limited and seasonal availability of medicinal
plants like S. alata, a perennial shrub with an uneven distribution in Kerala, poses challenges for large-
scale extraction of compounds with therapeutic and insecticidal value. In contrast, G. sepium is a fast-
growing, widely distributed tree that can be a promising source of bioactive compounds, owing to its
abundant biomass and year-round availability. The present study compares the phytochemical profiles
of S. alata and G. sepium to evaluate their potential as sources of valuable bioactive compounds. In this
study, sequential solvent extraction using hexane, chloroform, and ethanol was performed on dried
leaves, followed by both qualitative and quantitative analyses through biochemical assays and thin-
layer chromatography (TLC). The findings of the present study demonstrate substantial overlap in the
qualitative and quantitative profiles of major bioactive compounds. This study further signifies the need
for exploration of underutilized plant species to overcome supply constraints in phytopharmaceuticals
and for facilitating drug discovery and the development of related products. Further research involving
advanced analytical techniques and bioactivity assays is recommended to confirm functional
equivalence.
Keywords: Senna alata, Gliricidia sepium, medicinal plant, phytochemicals, biopesticide, bioactivity,
TLC
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Introduction
Traditional as well as modern medicine often rely on the phytochemical compounds from the medicinal
plant that show various bioactivities, offering a wide range of benefits. Phytochemicals have long been
the choice of drug development as they are easily available, natural, and have less nontarget action and
toxicity. Low cost and easy availability make these plant-based products attractive. Senna alata (S.
alata) and Gliricidia sepium (G. sepium) are two economically important plants known for their
pharmacological properties, like antimicrobial, anti-inflammatory, and antioxidant activities[1,2] and
antifeedant properties.[3] The phytochemicals, such as alkaloids, flavonoids, tannins, and phenolic
compounds found in these plants have been shown to exhibit significant bioactivity and are often
targeted in natural product research.[4] G. sepium is a fast-growing, medium-sized tree available
throughout the season and has a wide distribution, whereas S. alata is a perennial shrub typically found
along riversides, near water bodies, and in cultivated areas. Both plants belong to the family Fabaceae.
This study aims to evaluate and compare the phytochemical compositions of G. sepium and S. alata to
assess the degree of similarity that may justify their analogous uses, an approach commonly employed
in medicinal plant research. Compounds with potential bioactivity have been serially extracted from
the dried leaves of S. alata and G. sepium using hexane, chloroform, and ethanol as solvents.
Qualitative and quantitative analyses were subsequently conducted using biochemical assays, thin layer
chromatography (TLC), and spectrophotometric techniques[5,6] This is a comprehensive and
comparative account of the phytochemical profiles of S. alata and G. sepium.
Materials and Methods
The leaves of S. alata (Figure 1) and G. sepium (Figure 2), collected from Thiruvananthapuram and
surrounding areas, were shade-dried and subjected to serial hot extraction using hexane, chloroform,
and ethanol. The extracts were dried in a vacuum concentrator.
Figure 1: Senna alata
Figure 2: Gliricidia sepium
Qualitative phytochemical analysis
Various standard tests were employed for the qualitative phytochemical analysis of the extract by
mixing it with an equal volume of the appropriate reagent and observing the resulting colour change
indicative of specific phytochemicals. Alkaloids were detected using Hager’s[7] and Wagner’s
reagents;[8] flavonoids with ferric chloride test[9] and alkaline reagents;[10] and glycosides using Baljit’s
and foam tests.[11] Steroids were identified by Hesse’s response and Liebermann-Burchard’s test.[12]
Tannins and phenols were detected with Braymer’s[9] and Ferric chloride tests[13] respectively; proteins
with Biuret and Ninhydrin tests;[8,13] lipids with spot and filter paper tests[13,14] and carbohydrates with
Fehling’s and Benedict tests.[9,13]
Thin layer chromatography profiling and staining
Thin layer chromatography was used to separate and visualize phytochemicals in each of the fractions,
with silica gel as the stationary phase and a solvent system of petroleum ether, cyclohexane, ethyl
acetate, acetone, and methanol in the ratio 6: 1.6: 1: 1: 0.4 as the mobile phase. The bands separated
were observed under ultraviolet as well as visible light, and Rf values were recorded.
Quantitative phytochemical analysis
The phenol and tannins were quantitatively measured using Folin-Ciocalteu method at 725nm,700nm,
respectively, with gallic acid as standard.[14] Flavonoid content was estimated by the aluminium chloride
colorimetric assay at 415nm, using quercetin as the standard.[15] Protein content was determined using
Bradford assay at 595nm, with bovine serum albumin as the standard.[16] For all assays, stock solutions
of hexane, chloroform and ethanolic leaf extracts were prepared in methanol (1mg/mL stock
concentration), serially diluted, and analysed in Varioscan. Appropriate controls and enzyme blanks
were used, and absorbance were compared against calibration curves of respective standards to
determine the exact concentrations phytochemicals in the samples.
Yield of crude extracts
For calculating the yield of extracts, hexane, chloroform, and ethanol extracts of S. alata and G. sepium
were transferred into petri dishes and allowed to air dry. Upon complete solvent evaporation, the
remaining residues were weighed, and the percentage yield of each extract was determined. For
calculating the yield of TLC fractions, the bands from developed TLC plates were scraped off, extracted
using ethyl acetate, and separated from silica by centrifugation (at 10000 rpm for 10 mins). The yield
of each fraction was calculated by evaporating the solvent in a vacuum concentrator and subsequently
weighing the residue.
Results and Discussion
Qualitative analysis
The results of qualitative phytochemical analysis of S. alata and G. sepium leaf extracts are summarised
in Table 1. Analysis of leaf extracts of S. alata revealed that the hexane extract was rich in glycosides,
tannins, phenols, carbohydrates, and fats. The chloroform extract consisted of higher levels of alkaloids,
flavonoids, glycosides, phenols, tannins, carbohydrates, fats, and oils. This observation agrees with the
study by Akinmoladun et al., which also reported chloroform as an effective solvent for extracting
alkaloids and flavonoids from medicinal plants.[17] In the case of G. sepium, the hexane extract contained
Table 1: Qualitative analysis of phytochemicals identified in the thin layer chromatography fractions of
Senna alata and Gliricidia sepium leaves
Phyto-
chemicals
Tests
Senna alata
Gliricidia sepium
Hexane
extract
Chloroform
extract
Ethanol
extract
Hexane
extract
Ethanol
extract
Alkaloids
Hager’s Test
+
+
++
+
++
Wagner’s Test
+
++
++
++
++
Flavonoids
FeCl3 Test
+
++
++
+
++
Alkaline Reagent
Test
+
+
++
+
+
Glycosides
Baljet’s Test
+
+
++
+
++
Foam Test
++
++
++
++
+
Steroids
Salkowski Test
+
++
+
++
++
Hesse’s Response
+
++
+
++
++
Tannins
10% NaOH Test
++
+
+
+
+
Braymer’s Test
+
++
++
+
++
Phenols
Iodine Test
++
++
++
+
+
FeCl3 Test
+
++
++
++
+
Carbo-
hydrates
Fehling’s Test
++
++
++
++
++
Benedict’s Test
+
+
++
++
++
Proteins
Biuret’s Test
+
+
++
+
++
Ninhydrin Test
+
++
++
++
++
Fats and
Oil
Spot Test
++
++
+
++
+
Filter paper Test
++
++
+
++
+
+ indicates the presence of the compound; ++ indicates a relatively higher level. FeCl3 = Ferric chloride;
NaOH=Sodium hydroxide
alkaloids, flavonoids, steroids, glycosides, phenols, carbohydrates, proteins, fats, and oils. The
chloroform extract was rich in alkaloids, flavonoids, glycosides, tannins, phenols, carbohydrates, and
fats, as reported in an earlier study on G. sepium leaf extracts.[18] The ethanolic extracts of G. sepium
leaf, contained alkaloids, flavonoids, glycosides, steroids, carbohydrates, and proteins, which agreed
with Karthika et al., who reported the presence of bioactive constituents with antioxidant and anti-
inflammatory properties in ethanol extracts of G. sepium.[1] Extraction of phytochemicals from the plant
material mainly depends on the type of solvents used.[19] In the present study, the ethanolic extract was
found to contain alkaloids, flavonoids, glycosides, tannins, carbohydrates, and proteins, indicating that
ethanol is effective in the extraction of both polar and moderately polar compounds, consistent with the
findings of Harborne..[5]
Thin layer chromatography profiling and visualization
Thin layer chromatography analysis of S. alata and G. sepium leaf extracts in different solvents revealed
distinct differences in band patterns under UV-visible light. In S. alata, the hexane extract showed a
maximum of nine bands, with Rf values ranging from 0.98 to 0.31 (Figure 3). The highest Rf value
recorded was 0.98 in the hexane extract, while the lowest was 0.10 in the chloroform and ethanol
extracts. In G. sepium, the hexane extract revealed nine bands, and chloroform extracts had 10 bands
(Figure 4). The highest Rf value of 0.98, and the lowest Rf value of 0.08 were obtained with the ethanol
extract.
Figure 3: Thin layer chromatography profiles of extracts of Senna alata with hexane (SAH),
chloroform (SAC) and ethanol (SAE)
Figure 4: Thin layer chromatography profiles of extracts of Gliricidia sepium with hexane (GSH),
chloroform (GSC) and ethanol (GSE)
The TLC profiles of S. alata and G. sepium observed in this study may be compared to the patterns in
previous studies. Alvarez et al., reported three bands with Rf values of 0.123, 0.708, and 0.831 in
methanol extract of G. sepium leaf extract, suggesting the presence of flavonoid, phenolic, and alkaloid
compounds.[20] Likewise, Lahare et al., performed TLC on methanol, chloroform, and aqueous extracts
of S. alata and observed up to six bands in the methanol extract, comprising flavonoids, tannins,
alkaloids, and phenols, the Rf values of which corresponded to those obtained in the current study.[21]
These reports support our findings of multiple bands in both plant extracts. It is noteworthy that the
chloroform extracts of both plants exhibit identical Rf values viz., 0.98, 0.74, and 0.63 for alkaloids,
phenols, and tannins, respectively. This observation is further supported by staining with appropriate
reagents. The similarity in Rf values across species suggests the potential presence of identical or
structurally analogous bioactive compounds. Such cross-species similarity in phytochemical profiles
has also been reported in comparative studies of certain medicinal plants.[22,23] The present study
indicates that S. alata shares similarities in its phytochemical profile with G. sepium leaves, as revealed
by TLC analysis. Given that the availability of S. alata is limited to certain seasons, while G. sepium
leaves are available throughout the year, the latter could serve as a viable source for extracting several
of the beneficial bioactive components found in S. alata. This suggestion agrees with the ethnobotanical
practices that promote the use of alternative plant species during seasonal unavailability and to conserve
overexploited medicinal plants.[24-27] However, such a proposition necessitates further studies
elucidating the structural similarities, biological activities, and pharmacological properties of the
compounds in question.[28,29] Careful evaluation and optimization of extraction techniques are also
critical in this context.
Quantitative analysis
Quantitative analysis revealed that phenols were the most abundant compounds present in both plants.
The phenol content in S. alata ethanol extract was estimated to be 0.35 ± 0.010 mg/mL and in G. sepium
chloroform extract this was 0.18 ± 0.003 mg/mL (Figure 5). Flavonoid content was relatively lower,
revealing a maximum at 0.04 ± 0.004 mg/mL in S. alata ethanol extract (Figure 6). It was same for the
Figure 5: Total phenol in Senna alata and Gliricidia sepium leaf extracts
Figure 6: Total flavonoid in Senna alata and Gliricidia sepium leaf extracts
Figure 7: Total protein in Senna alata and Gliricidia sepium leaf
Figure 8: Total tannins in Senna alata and Gliricidia sepium leaf extracts
chloroform extract of G. sepium. Protein levels were apparent, particularly in S. alata, which measured
up to 0.30 ± 0.003 mg/mL in ethanol extract, while G. sepium chloroform extract contained 0.16 ± 0.003
mg/mL (Figure 7). Tannin levels were the lowest among the compounds tested, with 0.14 ± 0.005
mg/mL in S. alata ethanol extract and 0.13 ± 0.005 mg/mL in G. sepium chloroform extract (Figure 8).
The phenolic content of S. alata ethanol extract (0.35 ± 0.010 mg/mL) observed in the present study is
consistent with the findings of Rajendran et al., reporting a high phenolic composition of 69.72% in
hydroalcoholic leaf extracts.[30] Similarly, the recorded low flavonoid content (0.04 ± 0.004 mg/mL)
aligns with their findings (6.96%). Tannin levels in both plants were relatively low, in agreement with
previously reported values.[31,32]
Yield of different extracts of S. alata and G. sepium leaves
Following the extraction, the yield was calculated and compared. In S. alata, ethanol produced the
highest yield at 0.092 g, followed by hexane at 0.056 g and chloroform at 0.028 g. Similarly, in G.
sepium, the highest yield was also obtained with ethanol at 0.098 g, followed by chloroform at 0.088 g,
while hexane yielded the least at 0.048 g (Table 2).
Table 2: Percentage of yield in Senna alata and Gliricidia sepium crude leaf extracts.
Plant
Solvent
Colour of extract
Yield of extract*
(g)
Senna alata
Hexane
Brown
0.056
Chloroform
Green
0.028
Ethanol
Brown
0.092
Gliricidia sepium
Hexane
Brown
0.048
Chloroform
Green
0.088
Ethanol
Brown
0.098
* Represents the dry weight of the residue in the extract obtained from 100g of dry leaf powder
These results from S. alata agree with earlier findings that polar solvents like ethanol and methanol are
more efficient in extracting phytochemicals due to their ability to solubilize a broader range of polar
compounds. [33,34] Similarly, it is reported that Soxhlet extraction using methanol has produced a yield
of up to 25.14%, which is significantly higher than that of hexane (11.24%).[21] The present study also
showed that the yield was lowest when hexane was used for extraction. When TLC fractions were
analysed for yield it was found that the amount of residue obtained was highest in fraction 2 of ethanol
extracts in both plants (0.140 mg/mL in S. alata and 0.134 mg/mL in G. sepium), further supporting
ethanol's effectiveness in concentrating bioactive compounds. This agrees with previous studies
reporting high antioxidant and phytochemical content in ethanol and hydroethanolic extracts of
S. alata.[30,34]
Conclusion
The present study demonstrates that S. alata and G. sepium leaves are rich sources of bioactive
compounds, with ethanol and chloroform extracts showing particularly high concentrations of
phytochemicals. These findings are consistent with those of a recent study by Veena et al., which has
demonstrated the antifeedant properties of G. sepium leaves against the larvae of the coconut pest
Oryctes rhinoceros, thereby underscoring their potential in sustainable pest management.[35] Further
research is warranted to isolate and characterize the active compounds, evaluate their biological
activities through targeted bioassays, and explore their pharmacological potential for use in both pest
control and drug development.
To advance the present findings, future research should focus on the isolation, purification,
characterization, and identification of key active constituents using advanced analytical techniques such
as high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-
MS), and nuclear magnetic resonance spectroscopy (NMR). Bioactivity validation is recommended
through targeted bioassays to establish insecticidal, antimicrobial, or pharmacological properties,
thereby confirming efficacy. Another requirement is to elucidate the mechanisms of action of these
bioactive compounds to justify their use for a specific application. Studies should also explore the
development of formulations for agrochemical or therapeutic applications, with particular attention to
safety and scalability. By bridging phytochemical analysis with functional validation, these plants,
especially the more abundant Gliricidia sepium, could provide sustainable solutions to agricultural and
medical challenges, including the issue of herb scarcity.
Financial support and sponsorship
Nil
Conflicts of interest
There are no conflicts of interest.
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