INTRODUCTION
Plants of medicinal values have been used to
cure many diseases since ancient times. Several studies indicate that many
medicinal plants exhibit antioxidant properties (Saeed et
al. 2012). Antioxidants are crucial in preventing the formation of reactive
oxygen species (ROS), neutralizing existing ROS, and repairing damage caused by
ROS (Ighodaro and Akinloye 2018). Putranjiva roxburghii Wall is an
underutilized but valuable plant (Emasushan and John
Britto 2018). It has effective medicinal value and plays a significant role
in the traditional Ayurvedic and Unani systems (Gupta
2016; Pandey and Flulara 2022). Various parts of P. roxburghii are used
for the treatment of different diseases (Unnikrishnan et al.
2015; Mradu 2016). It has an anti-inflammatory, antipyretic, analgesic, and
anti-rheumatic herb, that is also useful to treat gynaecological and fertility
disorders (Naik et al. 2023; Pandey and Flulara 2022). It is used to
treat many diseases such as treatment of mouth and stomach ulcers, hot
swellings, smallpox, burning sensation, ophthalmopathy and liver diseases (Mishra et al. 2021; Pandey and Flulara 2022).
Among the
attributes of P. roxburghii, the most important ones are anthelmintic,
anticancer, anti-inflammatory, antioxidant, aphrodisiac, diuretic and laxative.
Leaves and seed paste are used to treat burning sensation, filarial,
inflammatory and eye diseases (Samal and Dehury 2016). Seed paste had been used
in the treatment of various diseases like elephantiasis, constipation,
ophthalmic, semen disorders, infertility and diseases of the female genital.
The bark and seeds are used as an antidote in the treatment of snake bites. The
leaves are used in treating illness, phlegm, skin ailment, aridity, and are
also helpful in curing rheumatism. P. roxburghii possess
antioxidant, antipyretic, and anti-inflammatory activities (Pandey and Flulara 2022). Almost all parts of the
plant such as bark, stem, leaves, root, fruits, and seeds contain numerous
secondary metabolites such as flavonoids, phenolics, triterpenes, saponins,
glycosides, alkaloids, saponins, and glucosinolates. The presence of these phytochemicals
imparts efficient protection roles against various diseases. Various plant
parts and their extracts can be used as a cure for different diseases,
including cancer (Gupta 2016; Kumar et al. 2019; Naik et al.
2023).
Phytochemical information
of plants is required to fully explore the different parts of plants and
correlate its activity. P. roxburghii has remarkable ethnomedical
significance. However, its phytochemical profile in different parts has not
been fully explored. In this study, various extracts, including aqueous,
methanol and hydro-ethanol were prepared from the seeds of P. roxburghii
and subjected to phytochemical assessment. The qualitative analysis revealed
the presence of all representative groups, except tannins and glycoside, in the
samples. The primary objective of this study was to carry out an in-depth
screening of the plant’s phytochemical composition qualitatively and
quantitatively.
MATERIALS AND METHODS
Collection of plant samples and
identification
The seeds of plant were collected from
University of Agriculture, Faisalabad (UAF) and authentication of plant was
done by a taxonomist at the department of Botany at UAF. The photographic
documentation of plant and a voucher sample was provided under reference number
(255-1-2023) and identified as P. roxburghii (Fig. 1).
Preparation of seed extract
The seeds of P. roxburghii were washed and subjected to drying under shade. The
dried seeds were subsequently ground using a high-speed blender and preserved
in an airtight jar. P. roxburghii seed powder was used for preparing various extracts. For
each extraction, 10 g of the powder was placed in 250 mL conical flasks, and
100 mL of methanol, hydro-ethanol (a mixture of water and ethanol in a 1:1
ratio) and distilled water were added separately. The extraction process was
done by using an orbital shaker (IRMECO) set at 220 rpm for 24 h. Following the
extraction period, the resultant mixture was filtered using Whatman No. 1
filter paper. The filtrates obtained were subjected to evaporation at 45°C
using a vacuum drying oven (Memmert, GmbH, Dusseldorf, Germany) to obtain the
dry extract. Subsequently, these dry extracts were transferred to Eppendorf’s
and preserved at 4°C for subsequent use. For analysis, evaporated extracts
weighing 50 mg were dissolved in 5 mL of DMSO, stored at 4°C and subjected to
phytochemical screening and analysis including total phenolic contents (TPC),
total flavonoid contents (TFC), and DDPH radical scavenging assay.
Phytochemical screening
The various extracts of P. roxburghii seeds were used for qualitative analysis of
various groups of compounds, such as flavonoids, carbohydrates, phenols,
saponins, alkaloids, glycosides and tannins by employing standard methodologies
as presented (Sharma1995; Treare and Evans 1985; Peach
and Tracy 1956; Varma et al. 2010).
Test for phenols: Potassium dichromate test: two mL of extract
was treated with 5% potassium dichromate solution. Positive result was
confirmed by a formation of brown precipitate (for phenol). Ferric chloride
test: 2 mL of extract was treated 2–3 drops of 5% ferric chloride solution.
Formation of bluish-black color showed presence of phenols and black color
showed tannins.
Test for flavonoids: Lead acetate test: one mL extract was treated
with 1 mL 10% lead acetate (Pb(OAc)4) solution. Formation of yellow
color precipitate indicated the presence of flavonoids.
Test for tannins: Braymer’s test: 2 mL of extract was treated
with 2 mL H2O and followed with 2–3 drops of FeCl3 (5%).
Green precipitate proved the presence of tannins.
Test for saponins: Foam test: two mL extract was diluted with 10
mL of distilled water and warmed gently. It was shaken for 5 min. Persistent
froth indicated the presence of saponins. The same extract was added with a few
drops of olive oil. Formation of a soluble emulsion confirmed the presence of
saponins (Treare and Evans 1985).
Test for glycosides: For Keller Kiliani test of glycosides,
2 mL extract was treated with 1 mL glacial acetic acid, one drop of 5% FeCl3
and 1 mL of conc. H2SO4. The brown ring of the interface
indicated the presence of cardiac glycosides (Treare and Evans 1985).
Test for alkaloids: Wagner’s test: Two mL of extract was treated
with few drops Wager’s reagent. Formation of reddish-brown precipitate
indicated the presence of alkaloids (Treare and Evans 1985).
Test for sterols: Salkowski’s test: two mL of extract was
treated with 2 mL chloroform and 2 mL of conc. H2SO4.
Chloroform layer appeared red, and the acid layer showed greenish yellow
fluorescence, which indicated the presence of sterols.
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+24010103+GP+Putranjiva+Dr.+Tahira_files/image004.png)
Test for terpenoids: Salkowski’s test: Two mL of chloroform and 1 mL of
conc.H2SO4 was added to 1 mL of extract and observed for
reddish brown color that indicated the presence of terpenoids (Sharma 1995).
Test for coumarins: Two mL of extract was treated with 3 mL of
10% NaOH solution. Yellow coloration indicated the presence of coumarins.
Analysis of Total phenolic and flavonoids
contents: A quantitative assessment was conducted to
determine Total Phenolic Contents (TPC) and Total Flavonoid Contents (TFC) in
the raw extracts was carried out by following standard protocols.
Total phenolic content: The total phenolic contents in the extracts
were determined by using Folin-Ciocalteu
procedure with Folin-Ciocalteu reagent (MERCK). Standard concentrations of 5,
10, 20, 30, 40, and 50 µg/mL of Gallic acid were prepared from a stock solution
of Gallic acid (1 mg/mL) in methanol. For each standard, 100 µL was combined
with Folin-Ciocalteu reagent, vortexed, and then 800 µL of sodium carbonate was
added and vortexed. After 1 h incubation, the absorbance was recorded at 765 nm with a spectrophotometer using a blank
to set the instrument at zero. The blank was composed of 100 µL of methanol instead of the standard.
The determination of total phenolic contents (TPC) was conducted for crude
extracts obtained from methanol, hydro-ethanol, and water were
determined following the same procedure as the standards (Jagadish et al.
2009). The findings are presented
in equivalence to Gallic acid (mg GAE/g). The standard curve of gallic
acid with the equation are given in Fig. 2a.
Total flavonoid contents
The determination of Total flavonoid content was conducted using
the aluminium chloride method. A range of standard concentrations (50, 100,
200, 300, 500, and 1000 µg/mL) of Catechin was prepared from a stock solution
of Catechin (1 mg/mL) in methanol. To 1 mL of each standard, 1 mL of 2% AlCl3
was added, vortexed, and absorbance was measured by using
spectrophotometer at 417 nm after a 15 min incubation. The spectrophotometer was set at zero by using blank and it was
prepared by substituting 100 µL of methanol in place of standard. The total
flavonoid contents (TFC) of crude extracts from methanol, hydro-ethanol, and
water were determined following the same procedure as the standards (Riaz et al. 2019). The results are expressed in equivalence to catechin (mg Catechin
E/10 g). The standard curve of catechin with the equation are presented in Fig.
2b.
DPPH radical
scavenging assay
The antioxidant capacity of the
extracts was determined by their capability to neutralize DPPH assay (Riaz et
al. 2019). In a 250 μL plant extract, 1 mL of the DPPH solution was added,
thoroughly mixed, and the mixture was left for 30 min for incubation in
darkness. Subsequently, the spectrophotometer was used to measure the
absorbance of both DPPH and extracts at 515 nm. The spectrophotometer was set at
zero with blank (methanol) (Gulcin and Alwasel 2023). The percentage
DPPH scavenging capability was calculated using the formula given below:
% inhibition = (A control – A sample
/A control) × 100
Where A control
is absorbance by the DPPH and A sample is absorbance
by the test samples
Statistical analysis
Triplicate analysis was done for each parameter
and the data have been presented as mean ± standard deviation of triplicate
analysis. Correlation coefficient (r) of phytochemical activities was
calculated by employing the correlation and regression function of Microsoft
Excel program (Microsoft, Redmond, WA, USA).
RESULTS AND
DISCUSSION
Importance of phytochemical constituents
Phytoconstituents are generated by the plants as a defense system
against pathogens and predators. They are helpful for the treatment of diseases
including antimicrobial, antioxidant, and stimulation as well as inhibition
enzymes (Ahmed et al. 2017). Plant-based therapeutic agents possess
fewer side effects as compared to synthetic agents and are also cost-effective
(Mustafa et al. 2017). Plants contain a wide range of constituents such
as alkaloids, polyphenolics, tannins terpenoids, etc. which are attributable to
therapeutic potential (Verma and Singh 2008). Phytochemicals, either as crude
extracts or isolated compounds, provide opportunities for drug discovery (Sasidharan et al. 2011). The various
extracts of seeds were used for qualitative analysis of various groups of
compounds, such as flavonoids, carbohydrates, phenols, saponins, alkaloids,
glycosides and tannins by employing standard methodologies as presented in
Table 1 (Auwal et al. 2014; Gul et al.
2017; Amine et al. 2019; Hussain et al. 2023). Furthermore,
it aimed to determine the qualitative phytochemical composition and antioxidant
potential as TPC, TFC and DPPH % qualitatively of seed extracts of P. roxburghii.
Qualitative phytochemical screening
A comprehensive estimation
of phytochemicals within their respective categories was carried out in P. roxburghii. Positive and
negative results were obtained for all tests conducted. According to the identification of noteworthy
chemicals entitles the various extracts of P. roxburghii seeds, including methanol (M), Hydro ethanol (HE) and aqueous (A)
extract. The results of qualitative screening of phytochemicals of P.
roxburghii seeds showed the presence of Phenols, Flavonoids, Saponins,
Alkaloids, Carbohydrates, Sterols and Terpenoids in all examined samples (Table 1). Our results showed
absence of tannins and glycosides in all extracts which are in line with Siwach
et al. (2024) but contrary to Sarath and Sudha (2019) that shows
presence of tannins in methanolic extract. Siwach et al. (2024) reported
absence of cardiac glycosides, tannins and saponins in methanolic extracts of
seeds of P. roxburghii.
Different parts
like leaves, fruits, seeds, root and stem bark of P. roxburghii showed the presence of many phenols,
alkaloids, saponins, steroids, flavonoids and glycosides and triterpenes
(Raghavendra et al. 2010; Kumar 2020; Balkrishna et al.
2021; Mishra et al. 2023). Qualitative phytochemical tests help to
understand the role of chemical compounds and their usefulness as
pharmaceuticals (Gupta 2016; Emasushan and Jhon Britto; 2018; Pandey and Fulara
2022).
Total phenolic content
The results of quantification of total
contents of phenols and flavonoid in the various extracts of the P. roxburghii seeds are presented in
Table 2. The determination
of TPC is expressed as milligrams of Gallic acid equivalent per grams of the
extract, determined using a reference standard curve (Fig. 2). The contents
exhibited variation among fractions. Total phenolic content ranged from 48.8 ±
3.29 to 83.0 ± 2.88 mg GAE/g of extract. The hydro-ethanol extract exhibited
the highest phenolic contents (83.0 ± 2.88 mg/g of GAE), followed by the
methanol extract with phenolic quantities of 65.0 ± 9.5 mg/g of GAE. In
comparison the aqueous extract displayed a lower phenolic concentration (48.8 ±
3.29 mg GAE/ g). These results align with findings reported in leaf by Keshav et
al. (2021) reported TPC in hydro ethanol (30:70) leaf extract of P. roxburghii as 46.58 ± 2.52 mg
GAE/g which is lower than from present study. Shahwar et al. 2012
reported total phenols as 176.0 ± 1.3 and 36.9 ± 3.0 mg GAE/g of stem extract
of methanol and distilled water, respectively. These values are higher for
methanol extract and lower for water extract. TPC notified in our research was
somewhat higher than a recently published study which revealed that P.
roxburghii leaf (hydroethanol, 30:70) extract contains 46.58 ± 2.52
mg/g GAE polyphenolic content (Keshav et al. 2021). Alterations in
agro-climatic conditions accompanied with temperature and rainfall impart a
significant impact on the amount of phytoconstituents within similar species of
plants growing in different regions (Kumar et al. 2017) and different
parts of the same plant also show variation in TPC and TFC contents (Shahwar et
al. 2012; Sarath and Sudha 2019; Nazli et al. 2022). Hence, differences in solvent composition,
plant collection sites and parts might be responsible for the variations of
estimated TPC.
Total flavonoid content (TFC)
The results of
total flavonoid contents as in the extracts are presented as milligrams of
catechin equivalent per 10 g of the extract are presented in Table 2. The flavonoid quantities ranged from 565 ± 121
to 915 ± 185 mg CE/10 g. Hydro-ethanol extract exhibited the highest flavonoid
content (915 ± 185 mg CE/100 g), followed by the aqueous extract (728 ± 130 mg
CE/10 g) while the methanol extract displayed the least quantity (565 ± 121 mg
CE/10 g). The observed flavonoid contents in P. roxburghii seeds extracts were higher than previous reported
(Nazli et al. 2022; Keshav et al. 2021).
+24010103+GP+Putranjiva+Dr.+Tahira_files/image005.png)
DPPH radical
scavenging assay
Metabolic processes within the body and
environmental factors produce free radicals mainly reactive oxygen species
(ROS) which cause various ailments including ageing, carcinogenesis,
mutagenesis, and cardiovascular abnormalities. Antioxidants are the agents
which counteract the effects of free radicals and limit oxidative stress
(Kedare and Singh 2011). DPPH assay is a standard method to investigate the
free radical scavenging ability of test samples (Mishra
et al. 2012). The
ability of various solvent extracts to donate hydrogen atoms or electrons was
assessed by reducing a purple DPPH into 1,1-diphenyl-2-picryl hydrazine
(Raclariu-Manolică and Socaciu 2023). The ability of each extract to
scavenge DPPH radicals was then measured and presented in Table 2. The
methanol extract demonstrated DPPH radical scavenging activity 69.2 ± 8.0%
whereas hydro ethanol and aqueous extract show almost similar activity as 32.8
± 1.86 and 35.6 ± 9.60, respectively.
The plants rich in phenols and flavonoids possess substantial antioxidant potential and are believed to be accountable for the
observed antioxidant capacity in the DPPH experiment. Nazli et al.
(2022) reported maximum percent free radical scavenging activity by methanol
stem (MeOH-S) and distilled water leaf (DW-L)-L extracts as 86 ± 0.56% which is
higher than our study.
CONCLUSIONS
Based on the
results, it has been established that hydro ethanolic extract of P. roxburghii seeds contain high
contents of both TPC and TFC while methanolic extract showed highest percentage
of DPPH inhibition. P. roxburghii seeds can be used for free radical scavenging and attributed to its
flavonoids and phenolic acids. However, further testing is necessary to
demonstrate specific chemical composition.
AUTHOR CONTRIBUTIONS
MJ did analytical work; TI conceptualized and supervised the work; SZ and
RR wrote and reviewed the first draft; MS performed formal data analysis, partially
supervised lab work. All authors read and approved the final draft.
CONFLICTS OF INTEREST
The authors affirm that they possess no conflicts of
interest.
DATA AVAILABILITY
The data will be made available on a fair request to
the corresponding author
ETHICS APPROVAL
Not applicable
FUNDING SOURCE
This project is not funded by any agency.
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