[Short title + Author Name - P&H title] 17 (2024) 105703

Contents lists available at ScienceDirect

Arabian Journal of Chemistry

journal homepage: www.ksu.edu.sa

Original article

HPLC profiling for the simultaneous estimation of antidiabetic compounds

from Tradescantia pallida
Fariha Imtiaz e, *, Muhammad Islam a, Hamid Saeed a, Muhammad Ishaq a, Usman Shareef b,
Muhammad Naeem Qaisar c, Kalim Ullah d, Sibghat Mansoor Rana a, Anam Yasmeen a, f,
Aneeqa Saleem d, Romia Javaid Saddiqui a
a
Punjab University College of Pharmacy, University of the Punjab, Lahore 54000, Pakistan
b
Shifa Tameer-e-Millat University, Islamabad, Pakistan
c
Department of Pharmacy, University of Sargodha, Pakistan
d
Department of Pharmacy, Minhaj University, Lahore 54000, Pakistan
e
School of Pharmacy, University of Management and Technology, Lahore, Pakistan
f
Department of Pharmacy, University of South Asia, Lahore 54000, Pakistan

A R T I C L E I N F O A B S T R A C T

Keywords: Diabetes is a long-term metabolic disease epitomized by postprandial hyperglycemia. The prolonged use of
Diabetes synthetic drugs renders distinct side effects, necessitating the development of safe and cost-effective substitutes.
Phenolic compounds The aim of the current study is to isolate, evaluate the antidiabetic potential and HPLC method development for
Phenols
simultaneous estimation of antidiabetic compounds from the leaves of Tradescantia pallida. The leaves were
Tradescantia pallida
HPLC
extracted, fractionated and subjected to column chromatography. The isolated compounds’ antidiabetic potential
Method development was evaluated using α-amylase and glycosylation of hemoglobin assays. The study employed molecular docking
to scrutinize interactions between antidiabetic compounds and human α-amylase and hemoglobin protein. Prime
MM-GBSA calculations determined binding energies of ligand–protein complexes. Further analysis of morin and
catechin involved exploring dynamic and thermodynamic constraints through molecular dynamics simulations
under specific biological conditions. A rapid HPLC method was developed and validated for the simultaneous
estimation of isolated compounds. The column chromatography culminated in the isolation of four antidiabetic
compounds (syringic acid, catechin, p-coumaric acid and morin). The in vitro analyses revealed that morin and
catechin exhibited 72.67 % and 78 % α-amylase inhibition and 67 % and 71.66 % inhibition of hemoglobin
glycosylation, respectively. In silico studies substantiated the in vitro assay, confirming the stability of catechin
and morin complexes via root mean square deviation analysis. Interactions, encompassing hydrophilic, hydro­
phobic, water bridges, and ionic interactions, identified key residues involved in these processes. The validated
HPLC method exhibited excellent correlation coefficients ranged from 0.9909 to 0.9997. The antidiabetic
compounds were quantified from the extract in the range of 0.072 – 0.160 µg/mL. The study concluded that the
isolated compounds from Tradescantia pallida have remarkable antidiabetic activity, and the developed method
can be successfully used for the identification and quantification of phenolic compounds in Tradescantia pallida
and other plant-derived matrix.

1. Introduction 537 million individuals globally, and the worldwide prevalence has
surpassed 10 %, as reported by the 10th Edition of IDF Diabetes Atlas.
Diabetes is a multifaceted chronic disease that necessitates glycemic Oral antidiabetic medications like biguanides, sulfonylureas, thiazoli­
control and ongoing medical attention in order to implement risk- dinediones, and digestive enzyme inhibitors are the first-line treatment
reduction strategies (Association, 2022). In 2021, diabetes impacted for managing diabetes, with insulin acting as a last desperate measure

* Corresponding author.
E-mail addresses: [email protected], [email protected] (F. Imtiaz), [email protected] (M. Islam), [email protected]
(H. Saeed), [email protected] (U. Shareef), [email protected] (M.N. Qaisar), [email protected] (K. Ullah), aneeqa.pharmacy@mul.
edu.pk (A. Saleem), [email protected] (R. Javaid Saddiqui).

https://doi.org/10.1016/j.arabjc.2024.105703
Received 29 November 2023; Accepted 28 February 2024
Available online 1 March 2024
1878-5352/© 2024 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

(Mohamed, 2021). The development of a safer, more affordable, and less USA. Sigma-Aldrich Chemire GmbH, Germany, supplied the dichloro­
toxic medication is required due to the common side effects of synthetic methane, acetonitrile, 3,5- Dinitrosalicylic acid, formic acid, porcine
antidiabetics, which include hepatic dysfunction, fluctuation in body pancreas’s α-amylase, hemoglobin and Acarbose. Merck Millipore,
weight, vision problems, peripheral arterial disease, and gastrointestinal Germany made available the silica gel, anhydrous glucose, TLC sheets
distress (Asghar, 2018; Marín-Peñalver, 2016). Due to their efficient, F254 and potato starch. SIGMA-ALDRICH, USA delivered the meth­
secure, and sustained mode of operation, medicinal plants have attrac­ anol‑d3 and Sephadex LH-20. Amros Pharmaceuticals, Pakistan pro­
ted the interest of researchers worldwide in the pursuit of finding a vided the Gentamicin 80 mg/2 mL. Assembly of the Merit water still
remedy for diabetes (Rahman, 2022). These medicinal plants contain from Cole-Parmer, based in the UK was used to prepare distilled water.
biologically active compounds possessing valuable therapeutic value PTFE filters in 0.45μ size were purchased from Sartorius, Germany.
(Mohamed, 2021; Abid et al., 2022).
Tradescantia pallida, also known as Purple Heart, is a member of the 2.2. Plant material
Commelinaceae family. This enduring herb has a history of application
within the food sector, where it has been conventionally utilized for its The leaves of Tradescantia pallida were gathered from the Botanic
preservative, coloring, and additive properties (Tan and Kwan, 2020). Garden of GCU (Government College University) (31o33′23.7″N
The plant has been employed medicinally to treat inflammation (Li and 74o19′41.6″). The plant was identified and authenticated by Prof. Dr.
Taiwanese native medicinal plants: phytopharmacology and therapeutic Zaheer-ud-Din Babar, (GCU), Lahore, Pakistan. A voucher specimen
values., 2006), bacterial infections (Tan, 2014) and to treat sore eyes bearing the reference number GC. Herb. Bot. 3627 was submitted to the
(Ragragio et al., 2013). Several species of the Tradescantia genus have Herbarium. The collection of leaves was performed with precision, and
manifested antidiabetic activity. Mexicans and Puerto Ricans have used our research posed no discernible threat or jeopardy to the biological
infusions made from Tradescantia zebrina and Tradescantia spathacea to integrity of the studied species.
treat diabetes (Andrade-Cetto and Heinrich, 2005; Gavillán-Suárez,
2015). In our previous studies, the extract of Tradescantia pallida has 2.3. Extraction and fractionation
been investigated for its antidiabetic potential in vitro as well as in vivo
(Imtiaz, 2023; Imtiaz, 2023). An analysis of Tradescantia pallida’s The leaves underwent a thorough washing and were dried in the
composition demonstrated a richness in polyphenols, tannins, flavo­ shade until about 90 % of their water content had dissipated. Following
noids, steroids, and alkaloids (Huq, 2016). However, as of now, there is a this, the dried leaves were finely grounded and kept for future use in an
lack of published quantitative data on these components and the airtight container. Soxhlet extraction assembly was used for solid–liquid
bioactive compounds responsible for antidiabetic activity. hot extraction (Imtiaz, 2017). In order to prevent the degradation of
High-Performance Liquid Chromatography (HPLC) stands out as a thermolabile compounds, 250 g of powdered leaves underwent
powerful tool, recognized for its precision, sensitivity, and capacity to sequential extraction with petroleum ether, chloroform, and methanol
dissect and quantify complex mixtures of compounds within a sample. at mild temperatures (45–50 ◦ C). After the extraction process, rotary
The primary objectives of method development are to identify and pu­ evaporation (Heidolph, Laborota 4002, Merck, Germany) was employed
rify drugs. Additionally, it provides crucial information regarding the to concentrate all the extracts. The antidiabetic potential of each extract
drug’s stability and bioavailability (Tartaglia, 2022). The simultaneous was then evaluated using in vitro models as described in section 2.5.
estimation is a rapid and cost-effective method which increases phar­ Among the examined extracts, the chloroform extract exhibited the most
maceutical sector productivity by performing a single procedure to notable antidiabetic efficacy and was consequently selected for subse­
analyze the mixture of two or more compounds in an extract or a quent assessment.
pharmaceutical dosage form (Habib, 2020). The adoption of HPLC In a separatory funnel, an enriched extract weighing 90 g underwent
profiling in our study stems from the technique’s well-established liquid–liquid extraction using Kupchan’s method, with minor adjust­
reputation for its analytical versatility. ments (Kupchan et al., 1973). The distribution involved a balanced
Natural products have more drug-like properties concerning struc­ partitioning between water and chloroform at a 1:1 volumetric ratio.
tural intricacy and functional groups, surpassing those derived from Concentration of the chloroform portion resulted Fraction α (80 g),
combinatorial chemistry in these aspects (Karageorgis, 2020). The while freeze-drying the aqueous fraction led to the creation of Fraction β
critical need to extract and isolate bioactive compounds from natural (5 g). Further fractionation of Fraction α involved the employment of an
sources for the advancement of human health underscores the signifi­ aqueous methanol solution with a volumetric ratio of 1:9 and n-hexane
cance of ongoing research. Despite the documented antidiabetic activity in 1:1 proportion, resulted in the generation of Fractions γ (60 g) and
of various Tradescantia species, there remains a notable gap in the un­ Fraction δ (15 g), respectively. Fraction γ was subjected to further sep­
derstanding of the antidiabetic evaluation of Tradescantia pallida leaves. aration, employing ethyl acetate (in a 1:1 ratio) and n-butanol (also in a
This gap in knowledge prompted our investigation into the compounds 1:1 ratio), leading to the isolation of Fraction ε (35 g) and Fraction θ (20
responsible for the antidiabetic activity of Tradescantia pallida, particu­ g).
larly considering its native presence in diverse regions, including
Pakistan, India, West Indies, Africa, and the United States of America. In 2.4. Isolation of bioactive compounds
this context, the principal objective of this investigation involves the
isolation of targeted bioactive compounds from Tradescantia pallida for Fraction ε underwent normal phase silica column chromatography,
comprehensive analysis and characterization and evaluate their antidi­ involving a silica gel column (200 g) and elution with methanol:
abetic potential through in vitro and in silico studies. Furthermore, this dichloromethane mixtures in varying ratios, ranging from 100:1 to
study aims to establish and validate an efficient and expeditious method 1:100, yielding 16 sub-fractions ranging from ε1 to ε16. While fraction θ
for the simultaneous estimation of these isolated antidiabetic provided six sub-fractions (θ1 to θ6). TLC was used to examine the
compounds. fractions.
Five sub-fractions, ε13a to ε13e, were obtained after further frac­
2. Materials and methods tionating sub-fraction ε13, employing a silica gel column (50 g), the sub-
fraction was subjected to elution using a mixture ranging from 90:10 to
2.1. Materials 50:50 methanol and dichloromethane. Bioactive compound SACL4 was
isolated from fraction ε13c (262.5 mg). Bioactive compound PACL7 was
All analytical grade solvents (n-hexane, methanol, chloroform, n- isolated from fraction ε13e (385 mg). Sephadex LH-20 was used to pu­
butanol and ethyl acetate) were procured from Emsure, Merck Millipore, rify the subfraction ε15 using a water:methanol ratio of 10:90 to 0:100,

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F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Fig. 1. Structures of antidiabetic compounds isolated from Tradescantia pallida leaves. where SACL4-syringic acid, PACL7-p-coumaric acid, MACL6-morin and
P42-catechin.

cm− 1, 1625 cm− 1, 1600 cm− 1, 1507 cm− 1, 1446 cm− 1, 1420 cm− 1, 1310
Table 1 cm− 1, 1280 cm− 1, 1241 cm− 1, 1211 cm− 1, 1172 cm− 1, 1103 cm− 1, 976
Gradient program of HPLC analysis of antidiabetic compounds from Tradescantia
cm− 1, 937 cm− 1, 911 cm− 1, 827 cm− 1, 797 cm− 1, 689 cm− 1 and 659
pallida.
cm− 1. ESI-MS [M]- 119.00 m/z and [M - H]-163.00 m/z corresponded to
Time Mobile Phase A Mobile Phase B the molecular formula C9H8O3. 1H NMR (CD3OD) (d, 1H − 8H, J =
(min) (percent v/v) (percent v/v)
15.89 Hz) 6.28 δppm, (d, 2H − 3/5H, J = 8.65 Hz) 6.80 δppm, (d, 2/6H
0 – 0.1 95 5 − 2H, J = 8.54 Hz) 7.43 δppm, (d, 7H, 1H, J = 15.92 Hz) 7.61 δppm.13C
0.1 – 4 95 → 65 5 → 35
and DEPT-135 (CD3OD) (8-C) 115⋅52 δppm, (3/5C) 116⋅77 δppm, (1C)
4–7 65 → 35 35 → 65
7 – 15 35 → 20 65 → 80 127⋅16 δppm, (2/6C) 131⋅09 δppm, (7C) 146⋅67 δppm, (4C) 161⋅14
15 – 20 20 → 5 80 → 95 δppm and, (9C) 171⋅07 δppm.
20.1 5 → 95 95 → 5 MACL6 (Morin) (227.5 mg) light yellow solid, melting point range
302–304 ◦ C. λmax 207 nm (4.31). ATR-FTIR; 3499 cm− 1, 1647 cm− 1,
1602 cm− 1, 1170 cm− 1, 1138 cm− 1, 1101 cm− 1, 1086 cm− 1, 788 cm− 1,
yielding a total of 227.5 mg of the MACL6.
and 683 cm− 1. ESI-MS [M]- 124.90 m/z and [M - H]- 301.06 m/z rep­
Sub-fraction θ3 was purified from fraction θ using Sephadex LH-20.
resented the molecular formula C15H10O7. 1H NMR (CD3OD) (d, 6-H, 8-
Water: methanol was used to elute the swelled beads (10:90 to 0:100). A
H, J = 1.81, 1.80 Hz) 6.16 and 6.32 δppm, (d, 1H − 3′-H, J = 2.14 Hz)
220 mg of P42 was isolated from the subfractions θ31 to θ33.
6.42 δppm, (dd, 1H − 5′-H, J = 2.18, 8.59 Hz) 6.47 δppm, and (d, 1H −
The determination of the compounds’ structure (Fig. 1) was achieved
6′-H, J = 8.58 Hz) 7.41 δppm.13C and DEPT-135 (CD3OD) Ring A; (6-C)
through NMR spectroscopy (Bruker AVANCE NEO spectrometer)
99.27 δppm, (8-C) 94.56 δppm, (5-C) 157.53 δppm, (7-C) 162.78 δppm,
comprising 1H NMR (600 MHz), 13C NMR (150 MHz) and DEPT-135
(9-C) 165.56 δppm. While for ring B; (C-3′) 105.09 δppm, (C-5′) 111.38
with a triple resonance probe in addition to several other spectro­
δppm, (C-6′) 132.27 δppm, (C-1′) 136.39 δppm, (2′-C) 149.93 δppm and,
scopic analysis mainly ATR-FTIR (Cary 630, Agilent, USA), UV–Visible
(4′-C) 158.97 δppm.
spectroscopy (2550 UV–Vis, Shimadzu, Japan), and LC-MS (1260 In­
P42 (Catechin) (220 mg) cream white solid, melting point ~ 211 ◦ C.
finity II and 6470 LC/TQ, Agilent, USA) (Imtiaz, 2023).
[α]25 − 1
D + 19.25. λmax 280 nm (4.44). ATR-FTIR; 3233 cm , 1608 cm ,
− 1
SACL4 (Syringic acid) (262.5 mg) A white, amorphous solid with a
1518 cm− 1, 1459 cm− 1, 1364 cm− 1,1282 cm− 1, 1239 cm− 1, 1142 cm− 1,
pale hue, melting point ~ 207 ◦ C. λmax 216 nm (4.33). ATR-FTIR; 3373
1077 cm− 1 and, 711 cm− 1. ESI-MS [M]- 245.09 m/z and [M - H]- 289.06
cm− 1, 2843 cm− 1, 1694 cm− 1, 1455 cm− 1, 1316 cm− 1, 1194 cm− 1, 1174
m/z correlated to the molecular formula C15H14O6. 1H NMR (CD3OD);
cm− 1, 1101 cm− 1, 1038 cm− 1, 907 cm− 1, 862 cm− 1, 767 cm− 1, 687
(dd, 1H − 10-H, J = 8.21 Hz) 2.50 δppm, (dd, 1H − 4-H, J = 5.41 Hz)
cm− 1 and 669 cm− 1. ESI-MS [M]- 182.00 m/z and [M - H]- 197.03 m/z
2.83 δppm, (m, 1H − 3-H, J = 5.49, 5.23, 5.44 Hz) 3.96 δppm, (d, 1H −
denoted to the molecular formula C9H10O5. 1H NMR (CD3OD) (s, 6H and
7-H, J = 7.55 Hz) 4.55 δppm, (d, 1H − 8-H, J = 2.29 Hz) 5.84 δppm, (d,
2H) 7.31 δppm and (s, 5OCH3 and 3OCH3 − 6H) 3.82 δppm.13C and
1H − 6-H, J = 2.30 Hz) 5.92 δppm, (dd, 1H − 6′-H, 1.95) 6.71 δppm, (d,
DEPT-135 (CD3OD) (6OCH3) 56.69 δppm, (6C and 2C) 108.15 δppm,
1H − 5′-H, J = 1.95 Hz) 6.72 δppm, (d, 1H − 2′-H, J = 1.97 Hz) 6.83
(1-C) 121.83 δppm, (5C and 3C) 141.64 δppm, (4C) 148.79 δppm,
δppm.13C and DEPT-135 (CD3OD); (4-C) 28.51 δppm, (3-C) 68.77 δppm,
(–COOH) 169.93 δppm.
(2-C) 82.82 δppm, (6-C) 95.39 δppm, (8-C) 96.16 δppm, (10-C) 100.71
PACL7 (p-coumaric acid) (385 mg) pale yellow solid, melting point
δppm, (2′-C) 115.17 δppm, (5′-C) 119.99 δppm, (1′-C) 132.13 δppm, (3′-
~ 211 ◦ C. λmax 310 nm (3.49). ATR-FTIR; 3345 cm− 1, 2815 cm− 1, 1666
C) 146.19 δppm, (4′-C) 146.22 δppm, (9-C) 156.88 δppm, (5-C) 157.56

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F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Fig. 2. Percentage inhibition of α-amylase (a) standard and isolated compounds (b) SACL4 (c) PACL7 (d) MACL6 and (e) P42 from the leaves of Tradescantia pallida.
where standard-acarbose, P42-catechin, SACL4-syringic acid, PACL7-p-coumaric acid and MACL6-morin.

Fig. 3. α-amylase median inhibitory concentration of (a) standard and isolated compounds (b) SACL4 (c) PACL7 (d) MACL6 and (e) P42 from the leaves of
Tradescantia pallida. where standard-acarbose, P42-catechin, SACL4-syringic acid, PACL7-p-coumaric acid and MACL6-morin.

4
F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Fig. 3. (continued).

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F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Fig. 3. (continued).

Fig. 4. Percentage inhibition of non-enzymatic glycosylation of hemoglobin assay of (a) standard and compounds (b) SACL4 (c) PACL7 (d) MACL6 and (e) P42 from
the leaves of Tradescantia pallida. where standard-acarbose, P42-catechin, SACL4-syringic acid, PACL7-p-coumaric acid and MACL6-morin.

δppm and (7-C) 157.81 δppm. Subsequently, the samples were positioned in a water bath adjusted to
85 ◦ C after the addition of coloring reagent (3,5-dinitro salicylic acid) for
2.5. In vitro antidiabetic assays 5 to 10 min. The samples were subsequently given time to cool to room
temperature. The same procedures were used to prepare the standard
2.5.1. α-amylase inhibition assay acarbose solution (1 % w/v), and the absorbance of each test solution
The inhibitory α-amylase activity measured using the method and standard was measured at 540 nm. Equation (1) provides the
described by Saleem, Islam (Saleem, 2018). The sample solutions following formula for calculating percentage inhibition activity;
(SACL4, PACL7, MACL6 and P42) were prepared in 1 mg/mL concen­ (Absorbance of Control − Absorbance of Sample)
tration. Briefly, 1 mL of 1 % w/v α-amylase was added in 1 mL from Percentage Inhibition =
Absorbance of Control
sample solutions and the mixtures were incubated at 37 ◦ C for 15 min. × 100
The solutions underwent further incubation at 37 ◦ C for an additional
(1)
15 min following the addition of a 1 % w/v starch solution (1 mL).

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Fig. 5. Median inhibitory concentration of non-enzymatic glycosylation of hemoglobin assay of (a) standard and compounds (b) SACL4 (c) PACL7 (d) MACL6 and (e)
P42 from the leaves of Tradescantia pallida. where standard-acarbose, P42-catechin, SACL4-syringic acid, PACL7-p-coumaric acid and MACL6-morin.

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F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Fig. 5. (continued).

The antienzyme potential of the isolated compounds was screened and 2.5.2. Glycosylation of haemoglobin
those demonstrating highest enzyme inhibition were chosen to deter­ The determination of glycosylation of hemoglobin protein was con­
mine the median inhibitory concentration. ducted using slight modifications in Parke’s method (Parker, 1981). The

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F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Fig. 5. (continued).

samples (SACL4, PACL7, MACL6 and P42) were prepared in 1 mg/mL the molecular docking procedure for improved reliability and accuracy.
concentration using methanol whereas, phosphate buffer (o.o1 M; pH
7.4) was used to prepare the reagent solutions. Precisely, 0.06 % w/v 2.6.2. Prime molecular Mechanics-Generalized Born surface Area (MM-
hemoglobin (1 mL), 2 % w/v anhydrous glucose solution (1 mL), and GBSA)
0.02 % v/v gentamicin (5 µL) were combined with 1 mL of each sample The PRIME module within the Schrödinger Suite was employed for
solution. Subsequently, the mixtures underwent incubation at 37 ◦ C Molecular Mechanics Generalized Born Surface Area (MM-GBSA) anal­
(MIR-153 Sanyo, Japan) for a period of 3 days. The absorbance mea­ ysis on the Extra Precision (XP)-docked complexes. The estimation of
surements were taken at 443 nm after 72 h. The standard used was binding energies was facilitated through Pose viewer files, and subse­
Acarbose and underwent the same treatment as the test solutions.. The quent to docking, the poses underwent minimization using the PRIME
following equation was used to calculate the percentage of inhibition; local optimization tool. For the calculation of binding free energies, a
comprehensive model was employed, incorporating the OPLS4 force
(Absorbance of Control − Absorbance of Sample)
Percentage Inhibition = field, rotamer search algorithms, and the Variable dielectric Generalized
Absorbance of Control
Born (VSGB) solvent model.
× 100
(2) 2.6.3. Molecular dynamic simulations
Molecular Dynamics (MD) simulation was employed to scrutinize the
The inhibitory potential of the isolated compounds was screened and
ligand–protein complex that showed the lowest MM-GBSA binding free
those demonstrating highest inhibition were chosen to determine the
energy. Utilizing DESMOND, MD simulations were conducted over a
median inhibitory concentration.
100 ns timeframe to gain deeper insights into the ligand–protein com­
plex. The DESMOND Schrödinger interface’s system builder panel
2.6. In silico studies facilitated the selection of an orthorhombic simulation box, and an
explicit TIP3P water model was constructed. Maintaining a constant
2.6.1. Molecular docking distance of 10 Å between the protein surface and the simulation box,
Human pancreatic α-amylase (PDB: 5U3A, 0.95 Å) and human he­ potential acidic or basic disruptions were mitigated by introducing 150
moglobin protein (PDB ID: 2DN1, 1.25 Å and) were retrieved from the millimolar (mM) sodium chloride for neutralization and establishment
PDB databank. The Protein Preparation Wizard integrated into the of an isosmotic salt environment. The system underwent 2000 iterations
Schrödinger interface was utilized for the preparation of both proteins, to attain its optimal configuration. Subsequently, a 100 ns MD simula­
ensuring their suitability for subsequent docking analysis. During the tion was initiated under the NPT ensemble, employing default relaxation
preparatory phase, a receptor grid was established with specific co­ parameters at 300 Kelvin and 1.01 bars. Temperature and pressure
ordinates for each protein. For 5U3A, the coordinates were set at 3.99 (x- control during the simulation were managed by the Nose-Hoover Chain
axis), 79.23 (y-axis), and 145.0 (z-axis), while for 2DN1, the coordinates thermostat and the Martyna-Tobias-Klein barostat, respectively. Tra­
were set at 39.0 (x-axis), 35.0 (y-axis), and 10.0 (z-axis). Additionally, jectory files recorded both energy and structural data at 10-picosecond
the scaling factor was adjusted to 1.0 \AA to optimize the grid param­ (ps) intervals. The simulation operated with a time step of 2 fs (fs),
eters. The molecular docking investigation was carried out using Glide. and trajectory analysis, as well as three-dimensional structure exami­
Additionally, the extra precision (XP) docking method was employed in nation, were performed using MAESTRO.

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F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Fig. 6. Molecular docking of isolated compounds from Tradescantia pallida with human pancreatic α-amylase protein 5U3A (a) MACL6 (b) P42 (c) PACL7 (d) SACL4.
where MACL6-morin, P42-catechin, PACL7-p-coumaric acid and SACL4-syringic acid.

2.7. RP-HPLC method development before being diluted to 50 mL with diluent. PTFE 0.45 µ filters were used
to filter the working standards and samples into HPLC vials.
2.7.1. Working standards and samples
As working standards, syringic acid, morin, p-coumaric acid, and 2.7.2. HPLC conditions
catechin were used. The chloroform extract of leaves of Tradescantia A HPLC (Shimadzu LC20 HPLC, with a degassing unit DGU-20A 5R,
pallida was analyzed to quantify the isolated compounds. A 50 mL auto-sampler SIL-20AC HT, LC-20 AT pump, diode array detector (DAD)
volumetric flask was filled with diluent and 40 mg of each standard. The SPD-M20A, column oven CTO 20AC, and compliant software CFR 21
ultrasonic mixer was used to sonicate the solutions. The ultimate volume Lab Solutions (version 6.83) with the thermo ODS column with sta­
was adjusted to 50 mL. Subsequently, 5 mL of each standard was tionary phase C18, a length of 150 mm, a diameter of 4.6 mm, and a
transferred to a 50 mL volumetric flask, and additional diluent was particle size of 5 µm was used. The flow Rate of 1.5 mL/min, 30 ◦ C
introduced to achieve a total volume of 50 mL. For each of the four column oven temperature, injection volume of 10 µL and 254 nm
standards, the standard solutions underwent another sonication to wavelength were optimized. The HPLC analysis was carried out in
achieve a final concentration of 0.08 mg/mL. Six individual samples of gradient mode. The formulation of mobile phase A involved dissolving
extract were prepared. In a 50 mL volumetric flask, the transfer involved 100 mL of formic acid in 1000 mL of water, while mobile phase B
5 mg of the extract along with 40 mg of compounds, and the subsequent consisted of HPLC grade acetonitrile. Table 1 describes the gradient
addition of 25 mL of diluent. The samples were sonicated for 30 min program used to perform the analysis. The compounds were identified

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F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Fig. 6. (continued).

according to the retention time and spiking with working standards. The linearity test.
quantification of the compounds in the sample were calculated ac­
cording to the percent peak area, reported in the calibration curve of the 2.7.5. LOD / LOQ
working standards. The LOD and LOQ values were determined through the analysis of a
linearity curve utilizing the slope and standard deviation of the
2.7.3. Validation response. The formulas for computing LOD and LOQ are as follow:
The linearity, Limit of Detection (LOD), Limit of Quantitation (LOQ), LOD = 3.3σ/S.
precision, accuracy, robustness, stability, range, and specificity of the Where σ is the standard deviation of the response.
developed analytical method were validated in accordance with ICH S is the slope of curve drawn between concentration and response.
guideline Q2 (R1) (ICH, Ich, 2017). LOQ = 10σ/S.
Where σ is the standard deviation of the response.
2.7.4. Linearity S is the slope of curve drawn between concentration and response.
At a concentration of 1 mg/mL, syringic acid, morin, p-coumaric
acid, and catechin were solubilized in a consolidated stock solution. It 2.7.6. Precision
was used to make the following dilutions: 0.06 mg/mL (60 ppm); 0.08 The precision evaluation of each sample involved conducting mul­
mg/mL (80 ppm); 0.1 mg/mL (100 ppm); 0.12 mg/mL (120 ppm); and tiple series of measurements. The precision within a day (intra-day) and
0.14 mg/mL (140 ppm). In the HPLC, a 10 µL injection volume was used across consecutive days (inter-day) was assessed by analyzing six rep­
for each of these dilutions. T-test was applied to confirm the reliability of licates at various concentration levels (0.06 mg/mL; 0.08 mg/mL; 0.1

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F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Fig. 7. 2D LID of (a) MACL6 (b) P42 (c) PACL7 (d) SACL4 with 5U3A. where MACL6-morin, P42-catechin, PACL7-p-coumaric acid and SACL4-syringic acid.

Table 2
Molecular docking and Prime MM-GBSA analysis of isolated compounds from Tradescantia pallida.
PDB Ligands Interacting Residues Distances Å Types of interactions XP Docking Glide Energy Scores MM-GBSA ΔG
ID scores kcal/mol Bind

2DN1 MACL6 HIS87, TYR42, HIE58 4.19, 5.04, 5.42, 1H-Bond, 3 hydrophobic interactions − 9.779 − 44.113 − 41.08
1.96
P42 TYR42 5.17 1 hydrophobic interaction − 8.906 − 44.220 − 20.14
PACL7 ASN97 2.00 1H-Bond − 8.992 − 49.118 5.62
SACL4 No interacting —— − 6.572 − 31.021 − 25.76
residues
5U3A MACL6 HIS201, GLH233, 5.14, 1.58, 2.37, 2.2, 4H-Bond and 1 hydrophobic interaction − 8.805 − 48.497 − 4.08
ASP197 1.97
P42 ASP197, ASP300, 1.65, 1.70, 1.67 3H-Bonds − 5.612 − 46.133 − 25.82
THR163
PACL7 GLH233, LYS200, 1.79, 4.22, 2.02, 2H-bonds, 1 salt bridge and 1 − 3.664 − 29.515 − 2.35
TYR151 2.17 hydrophobic interaction
SACL4 LYS200, TYR151, 1.78, 2.91, 2.13 2H-Bonds and 1 salt bridge − 3.172 − 19.315 − 2.29
HIP305

where P42-catechin, SACL4-syringic acid, PACL7-p-coumaric acid and MACL6-morin

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F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Fig. 8. Molecular docking of isolated compounds from Tradescantia pallida with human pancreatic hemoglobin protein 2DN1 (a) MACL6 (b) P42 (c) PACL7 (d)
SACL4. where MACL6-morin, P42-catechin, PACL7-p-coumaric acid and SACL4-syringic acid.

mg/mL; 0.12 mg/mL; and 0.14 mg/mL) on a single day and over three amount of placebo and were properly labeled. Three dilutions of 60 %,
consecutive days, respectively. The outcomes were presented in terms of 100 %, and 140 % were used to test the accuracy.
the relative standard deviation (RSD).
2.7.8. Robustness
2.7.7. Accuracy The method’s robustness was tested by adjusting the flow rate,
Sonication was applied to dissolve 40 mg of each standard in 30 mL wavelength, and column oven temperature. The flow rate was reduced
of diluent, and the volume was then increased to 50 mL, representing from 1.0 to 0.9 and then to 1.1 mL/min. The wavelength was changed
dilution 1. Subsequently, in a 100 mL volumetric flask, an aliquot of 10 from 254 to 252, and then to 256 nm. The oven temperature was
mL from the initial dilution was combined with additional diluent, reduced from 40 ◦ C to 28 ◦ C and 32 ◦ C.
resulting in dilution 2. The sample solutions were prepared in nine 100
mL volumetric flasks. Each 100 mL volumetric flask received a specific 2.7.9. Analytical solution stability
amount of placebo. One of the nine flasks received 7.5 mL of the Six carefully prepared samples for precision were reassessed using a
dilution-1 standard. The diluent was then used to make up the difference freshly prepared standard after a period of 24 h.
in volume. The procedure was repeated to prepare two more samples,
and the three flasks were labeled. This method produced three flasks of 2.7.10. Range
60 % dilutions. Similarly, three 140 % dilutions were made by adding Linearity was used to calculate range. This scientific method has
12.5 and 17.5 mL of the standard from dilution-1, respectively, to the been validated for a percentage range of 60 % to 140 %.
remaining six 100 mL volumetric flasks that contained the specified

13
F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Fig. 8. (continued).

2.7.11. Quantification of antidiabetic compounds in extract indicated compound MACL6 possesses the most significant antienzyme
The antidiabetic compounds were quantified from the extract of potential in comparison to the other isolated compounds (Fig. 2).
leaves of Tradescantia pallida by using calibration curve of the standards. After normalizing the data and calculating the log values of per­
centage inhibition, the median inhibitory concentration was determined
(Fig. 3a-e).
2.8. Statistical analysis
3.1.2. Glycosylation of hemoglobin
The data is presented in mean ± standard error mean. GraphPad
This assay revealed that compound P42 had the highest antidiabetic
prism (8.0.1 version, California, USA) was used to calculate the data. In
potential among the isolated compounds, inhibiting 71.66 % glycosyl­
vitro analysis utilized Two-way ANOVA with Dunnett’s post hoc test.
ation non-enzymatically, followed by MACL6, SACL4, and PACL7
Statistical significance was attributed to a p-value below 0.05.
(Fig. 4). At 750 µg/mL concentration, the results of P42 were compa­
rable with standard (p = 0.0016). Fig. 5a-e displays the IC50 values of
3. Results standard acarbose and isolated compounds. In silico studies.

3.1. In vitro analysis 3.1.3. Molecular docking and PRIME MM-GBSA analysis
Among the four ligands evaluated (Fig. 6a-d), catechin demonstrated
3.1.1. α-amylase inhibition a notable interaction with 5U3A, forming a total of three bonds in the
The outcome of compounds SACL4, PACL7, MACL6 and P42 ligand–protein complex, all of which were hydrogen bonds (Fig. 7a-d).
demonstrated that with the increase in the concentrations of these The first hydrogen bond interaction occurred between ASP197 and the
compounds, the enzymatic inhibition increased which eventually phenolic ring, with a bond distance of 1.65 Å. The second and third
reached a steady state. The assessment of the dose–response relationship

14
F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Fig. 9. 2D LID of (a) MACL6 (b) P42 (c) PACL7 (d) SACL4 with 2DN1. where MACL6-morin, P42-catechin, PACL7-p-coumaric acid and SACL4-syringic acid.

hydrogen bonds were observed between ASP300 and THR163, with interaction occurred between TYR42 and the phenolic ring of the ligand
bond distances of 1.70 and 1.67 Å, respectively. The XP docking score at a bond distance of 5.42 Å. The second and third hydrophobic in­
and MM-GBSA ΔG bind were determined to be − 5.612 kcal/mol and teractions were noted between HIS87 and the additional phenolic ring of
− 25.82 kcal/mol, respectively. Details of the docking and MM-GBSA morin at bond distances of 4.19 and 5.04 Å, respectively. These in­
scores for other ligands are provided in Table 2. teractions indicated the formation of stable connections, supported by
Fig. 8(a-d) depicts the docked compounds with 2DN1. Fig. 9a illus­ an XP docking score of − 9.779 kcal/mol and an MM-GBSA ΔG Bind of
trates the Local Interaction Density (LID) of morin and 2DN1, show­ − 41.08 kcal/mol. Notably, among the four ligands assessed, morin
casing the establishment of four noteworthy interactions as compared to exhibited the most substantial docking and MM-GBSA scores.
others (Fig. 9b-d). Among these, three were identified as hydrophobic As depicted in Table 2, Morin exhibited significant interactions and
interactions, and one was characterized as a hydrogen bond. Specif­ binding free energy with 2DN1, while catechin demonstrated note­
ically, a hydrogen bond was observed between HIE58 and the hydroxyl worthy interactions with 5U3A. Subsequently, both Morin-2DN1 and
residue of morin, with a distance of 1.96 Å. The first hydrophobic Catechin-5U3A complexes underwent Molecular Dynamics (MD)

15
F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Fig. 10. Root mean square deviations of (a) P42 simulated with human pancreatic protein 5U3A and (b) MACL6 simulated with human hemoglobin protein 2DN1.
where P42-catechin and MACL6-morin.

simulations to further validate their interactions. simulated systems was confirmed by the MACL6 complex with 2DN1,
demonstrating a stable complex with minimal fluctuations. However, in
3.1.4. MD simulations the case of 5U3A, the P42 component stabilized within 24 ns, experi­
To assess the stability and intermolecular interactions within the enced destabilization at 65 ns, and then re-engaged with the protein
ligand–protein complex, Molecular Dynamics (MD) simulations were around 70 ns.
conducted on the top-docked compounds. Each compound and its Fig. 11(a-b) presents an overview of the intermolecular interactions
respective protein underwent MD simulations for a duration of 100 ns and key residues involved in hydrophilic and hydrophobic interactions,
(ns). The resulting trajectories were scrutinized using standard metrics, water bridges, and ionic interactions. In the simulation with 5U3A, P42
including Root Mean Square Deviation (RMSD) for α-carbons and the exhibited hydrophilic linkage with residues ASP197 and ASP356, ac­
examination of interactions between ligands and proteins. Fig. 10a–b counting for almost 100 % of the interactions. Similarly, in the simu­
depict the RMSD plots for the ligand-5U3A and ligand-2DN1 complexes. lation with 2DN1, MACL6 demonstrated approximately 100 % and 50 %
The RMSD graphs illustrate the atomic displacement over the 100 ns of hydrophilic contacts attributed to SER102, HIS87, and PHE108. These
simulation period for the 5U3A and 2DN1 systems. The RMSD trajec­ findings highlight the specific residues and nature of interactions
tories of the proteins revealed that, within the processing frame, atomic contributing to the stability and behavior of the ligand–protein
fluctuations peaked at a maximum of 2.4 Å. Equilibration of the complexes.

16
F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Fig. 11. Intermolecular interactionsof (a) P42 simulated with human pancreatic protein 5U3A and (b) MACL6 simulated with human hemoglobin protein 2DN1.
where P42-catechin and MACL6-morin.

3.2. HPLC method development each compound, a linear curve was obtained, as shown in Fig. 13. Each
curve was assigned a correlation coefficient. The correlation coefficient
3.2.1. Standard and sample preparation values for syringic acid, p-coumaric acid, morin, and catechin were
The standard was injected into HPLC in a 10 µL volume.The samples 0.9994, 0.9996, 0.9993, and 1.0000, respectively. The results were
were processed using the same method and injected with a volume of 10 confirmed by applying the statistical analysis (t-test) which presented
μL. Fig. 12 presents chromatograms of standard and samples. Table 3 good correlation and p value 0.0019.
entails the retention times for each component.
3.3.3. LOD/LOQ
The calibration equations’ determination coefficients (R2) were
3.3. Method validation
greater than 0.999 for all analytes. lists the calculated LOQ and LOD
values for the compounds (Table 5). LOD and LOQ were in the range of
3.3.1. System suitability parameters
3.37 – 4.98 ppm and 10.23 and 15.10 ppm, respectively.
This test was carried out to ensure that the chromatographic condi­
tions were effective and fit for use in HPLC analysis. The parameters
3.3.4. Precision
considered were the separation factor, capacity factor, resolution,
Analyses of standard compounds were replicated (n = 6) to deter­
tailing factor, and number of theoretical plates. The parameters were
mine instrumental precision. Table 5 shows the precision results for
calculated automatically by the HPLC system’s software. Table 4 dis­
individual compounds. The RSD values of the intra- and inter-day pre­
plays the results of the analysis.
cisions of each compound were < 2 %.

3.3.2. Linearity
3.3.5. Accuracy
A slope was drawn between the area of each dilution and the con­
The results showed that the recovered amount ranged between 98
centrations of 60, 80, 100, 120, and 140 ppm to test for linearity. For

17
F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Fig. 12. HPLC chromatogram of (a) standards and samples (b) isolated compounds (c) extract of Tradescantia pallida. where P42-catechin, SACL4-syringic acid,
PACL7-p-coumaric acid and MACL6-morin.

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F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Table 3 HPLC method for the simultaneous identification of these compounds.

Retention times of standard and samples. Thus, the present investigation involves the isolation of these com­
No. Name Retention time pounds, exploration of their antidiabetic potential, development of an
HPLC method, and the subsequent validation of the assay.
Standard Sample 1 Sample 2
Compound SACL4 was identified as syringic acid through spectro­
1 P42 11.577 11.522 11.547 scopic methods, including NMR (Maity, 2011; Mogana, 2014. 2014.;
2 SACL4 13.350 13.327 13.339
3 PACL7 14.312 14.300 14.311
Mogana, et al., 2020). Similarly, p-coumaric acid was confirmed as
4 MACL6 15.800 15.797 15.815 compound PACL7 (An, 2008; Karthikeyan et al., 2015. 2015.); morin as
compound MACL6 (Bunyapraphatsara, 2000; Hussain, 2014) and cate­
where P42-catechin, SACL4-syringic acid, PACL7-p-coumaric acid and MACL6-
chin as compound P42 (Annamalai et al., 2021; Zhou, 2020). The
morin
identification process aligns with established studies, providing consis­
tency and validation to our findings.
and 102 % of all compounds (Table 6). The statistical analysis was
In this current study, phenolic compounds exhibited significant
carried out using GraphPad Prism 8.0 and applying Residual Analysis.
inhibitory effects on the activity of the digestive enzyme α-amylase and
The t-test was applied and the values obtained was; t = 133.1, df = 5.000
effectively prevented the glycosylation of hemoglobin. P42 and MACL6
and p < 0.0001.
emerged as particularly potent in their antienzymatic and anti-non-
enzymatic (glycosylation of hemoglobin) activities. While literature
3.3.6. Robustness
has previously reported on the potential of catechin and its derivatives
Table 7 compared the resolution for normal parameters and modified
in combating diabetes through the inhibition of digestive enzymes
parameters. The RSD of retention time and area of standard were
(Zhou, 2020; Bhandari, 2008; Justino, 2018), there is a notable absence
calculated and displayed in Table 8. Each parameter change was thor­
of recognized research focused on their impact on glycated hemoglobin.
oughly monitored for resolution between drug peaks. The chromato­
Moreover, the antidiabetic efficacy of morin has been demonstrated in a
grams under normal conditions, at wavelength 252 nm, at wavelength
streptozotocin-induced rat model and through the monitoring of insulin
256 nm, at column oven 28 ◦ C, at column oven 32 ◦ C, at flow rate 0.9
signals in HepG2 cells (Jiang, 2020; Razavi et al., 2019). SACL4 dis­
mL/min, and at flow rate 1.1 mL/min are presented in Fig. 14a-f.
played a noteworthy 61 % inhibition of α-amylase and a 57 % glyco­
sylation inhibition of the hemoglobin protein in our study. Previous
3.3.7. Analytical solution stability
research has supported the potential of syringic acid in diabetes treat­
After 24 h, the precision assay samples were analyzed again with
ment by enhancing insulin production, revitalizing pancreatic β-cells,
freshly formulated standard solutions. Table 9 displays the results of
and impeding advanced glycation end products (AGEs) (Bhattacherjee
each compound. The results showed that the solutions were remarkably
and Datta,2015;Jan, 2022;Muthukumaran,2013). Our observations
stable after 24 h.
align with these findings, underscoring the potential therapeutic impact
of syringic acid. Similarly, PACL7 demonstrated a substantial 45 % in­
3.3.8. Range
hibition of α-amylase and a 52 % inhibition of non-enzymatic processes.
The linearity test determined the assay’s upper and lower limits. The
Consistent with previous research, the antidiabetic properties of p-cou­
40 to 160 % range chosen for this analysis validated the method and
maric acid were attributed to its ability to modulate glucose levels in the
confirmed that the compounds can be precisely and accurately analyzed
pancreas by stimulating the glucose transporter 2 (Amalan, 2016), and
using this method.
5′-adenosine monophosphate-activated protein (AMPK) release (Yoon,
2013). The results from our in vitro antidiabetic assessment are in
3.3.9. Quantification of antidiabetic compounds in extract
concordance with previously published data.
The quantity of the polyphenolic compounds isolated from the ex­
Molecular docking, a contemporary computer-based methodology
tracts of leaves of Tradescantia pallida are expressed in Table 10. The
(Dissanayake, 2022), offers insights into potential compounds and
findings indicate that morin possesses the highest concentration, while
streamlines the drug discovery process by revealing intricate receptor-
catechin has the lowest concentration.
ligand interactions, assessing energetic compatibilities, and eluci­
dating binding modes (Guedes et al., 2014). Our investigation un­
4. Discussion derscores that MACL6 and P42 exhibit robust binding affinities with
α-amylase and hemoglobin proteins. The α-amylase binding pockets are
Phenolic compounds derived from natural sources, predominantly categorized into − 1 to N subpockets, where − 1 represents the innermost
plants, exhibit a myriad of health benefits (El Gizawy, 2021; Pandey and subpocket, followed by + 1, +2, +3, and so forth. The glycosidic linkage
Rizvi, 2009). Diets rich in polyphenols have been associated with targeted for cleavage resides in the − 1 to + 1 subpockets (Proença,
significantly positive impacts on human health, offering protection 2019). The catalytic pocket in the α-amylase enzyme comprises triad
against various serious ailments such as diabetes (Imtiaz, 2023), car­ residues ASP197, ASP300, and GLU233, positioned in the − 1 subpocket
diovascular disease, and cancer (Arts and Hollman, 2005; Tung­ (Zhang, 2022). Our findings indicate that all isolated polyphenols
munnithum, 2018). Polyphenols play an important role in diabetes interacted within the same binding pocket, with key residues including
(Imtiaz, 2022) treatment by activating β-cells and regulating the diges­ HIS201, GLH233, ASP197, ASP300, THR163, LYS200, TYR151, LYS200,
tive enzymes (Imtiaz, 2023) responsible for carbohydrate metabolism and HIP305, suggesting a deep pocket within the − 1 subpocket. This
(Zhang, 2022; Zheng, 2020). This study is novel in isolation of phenolic interaction is likely facilitated by the formation of hydrogen bonds.
compounds from Tradescantia pallida leaves. Notably, there is a lack of a

Table 4
System suitability parameters for HPLC method development.
Compound Number of Theoretical Plates (N) Tailing Factor (T) Resolution (Rs) Capacity Factor (K) Separation Factor

P42 40,721 0.973 — — —

SACL4 91,243 1.164 8.728 0.163 —
PACL7 112,380 1.206 5.533 0.251 1.537
MACL6 118,558 1.221 8.402 0.384 1.534

where P42-catechin, SACL4-syringic acid, PACL7-p-coumaric acid and MACL6-morin

19
F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Fig. 13. Linearity curve of the isolated compounds (a) P42 (b) SACL4 (c) PACL7 (d) MACL6. where P42-catechin, SACL4-syringic acid, PACL7-p-coumaric acid and
MACL6-morin.

Similarly, in molecular docking with 2DN1, MACL6 exhibited in­ energies govern all processes at the molecular level (Rastelli, 2010). Our
teractions with HIS87, TYR42, and HIE58 as the primary amino acid data indicated that MACL6 and P42 exhibited the most negative energy,
residues, displaying an energy range of − 44.113 Kcal/mol. The hemo­ underscoring the stability of the optimized complexes. Our study is
globin protein encompasses ten binding pockets, with four larger than innovative in conducting Prime MM-GBSA calculations on MACL6 and
the others and positioned in each hemoglobin chain. Our investigation P42 against the 5U3A and 2DN1 targets.
unveiled that the binding pocket for the isolated polyphenols in the MD simulation studies provide insight into the dynamic and ther­
2DN1 protein is located in helix 6 and helix 7. This aligns with prior modynamic characteristics of biological systems under specific physio­
studies on molecular docking, supporting the potential of phenolic logical conditions (Azam, 2019; Azam, 2018). The study aimed to
compounds as potent agents against diabetes (Diker and Kutluay, 2021; validate and assess the stability of the P42 and MACL6 docked com­
Gancar, 2020; Nazir, et al., 2018). plexes with 5U3A and 2DN1. The root mean square deviation (RMSD)
The Prime MM-GBSA method is a rigorous and widely accepted trajectories of both ligand–protein complexes and the Apo proteins
approach for validating docked complexes by computing binding free initially followed similar patterns. Specifically, the RMSD of the P42-
energies (Genheden and Ryde, 2015). In biomolecular studies, the pre­ 5U3A complex and the 5U3A Apo form exhibited somewhat parallel
cise computation of binding free energies is a crucial goal, as these trajectories, with convergence points occurring around 40–50 ns.

20
F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Fig. 13. (continued).

Table 5
LOD and LOQ values of the phenolic compounds.
Compound Concentration (ppm) Linear Equation R2 LOD (ppm) LOQ (ppm) Precison (RSD %)

Intraday Interday
P42 40–160 6274.3x + 23416 0.9983 4.98 15.10 0.13 0.70
SACL4 40–160 26788x + 79426 0.9987 4.26 12.92 0.07 0.62
PACL7 40–160 19493x + 1422.2 0.9992 3.37 10.23 1.25 0.68
MACL6 40–160 63408x + 120568 0.9985 4.63 14.04 0.30 0.52

where P42-catechin, SACL4-syringic acid, PACL7-p-coumaric acid and MACL6-morin

However, there were instances where trajectories did not converge, maximum simulation duration, indicating a stable ligand–protein com­
indicating potential loose connections between the ligand and the pro­ plex. Only a small portion displayed non-convergence between 70 and
tein. Nevertheless, points of convergence demonstrated strong and sta­ 80 ns, while the majority of the trajectories converging indicated the
ble interactions. In contrast, the trajectories of the MACL6-2DN1 formation of a stable and robust complex.
complex showed nearly complete convergence throughout the The interaction fraction diagram of P42-5U3A indicated that

21
F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Table 6 stable complex formation, were observed, with SER102 demonstrating

Percentage recovery of the phenolic compounds. the most significant hydrogen bond interaction, boasting an interaction
Compound Spiked concentration Recovery fraction exceeding 0.8, indicating substantial binding during 80 % of the
simulation time. Other hydrogen bonds involved HIS87, HIS58, and
%
P42 60 101.58 ASN97, each with noteworthy interaction fraction values. Minor con­
100 100.43 centrations of water bridges were also observed. Furthermore, SER102,
140 98.40 HIS87, and PHE108 contributed most to ligand binding. SER102 dis­
SACL4 60 101.54 played binding for almost 97 % of the simulation duration, while HIS87
100 100.33
140 98.85
and PHE108 exhibited binding durations of 67 % and 66 %, respectively.
PACL7 60 100.11 Intriguingly, the ligand’s intramolecular hydrogen bond interactions
100 100.71 with certain atoms may have limited their availability for binding,
140 99.76 potentially strengthening interactions if not present.
MACL6 60 100.59
All the findings collectively indicate the establishment of a robust
100 100.33
140 99.01 and stable ligand–protein complex between MACL6 and 2DN1. Notably,
the complex formed by MACL6 and 2DN1 exhibited greater stability
where P42-catechin, SACL4-syringic acid, PACL7-p-coumaric acid and MACL6-
compared to the complex formed by P42 and 5U3A. The observed
morin
flexibility of the compounds aligns with existing literature on poly­
phenols, confirming their dynamic nature (Ghosh, 2021).
hydrogen bonds were the predominant type of interaction. ASP197 and The optimization of the HPLC method for the simultaneous estima­
ASP356 exhibited the highest interaction fraction values, signifying tion of antidiabetic compounds from Tradescantia pallida involved the
significant hydrogen bond interactions, followed by HIS299, THR163, utilization of a gradient system analysis. The mobile phase consisted of
and ASP300. Although hydrophobic interactions were relatively scarce 0.1 % formic acid and acetonitrile. Notably, this specific mobile phase
in this complex, water bridges played a prominent role, involving composition has not been previously employed for the simultaneous
several amino acid residues not observed in molecular docking studies. estimation of the isolated polyphenolic compounds. Therefore, this
ASP356, for instance, formed multiple contacts with the ligand’s hy­ method stands as an innovative approach for the identification and
droxyl residue, with one hydrogen bond directly and another interaction quantification of catechin, syringic acid, morin, and p-coumaric acid.
mediated by a water bridge. Both interactions persisted significantly All the compounds designated for simultaneous analysis demon­
throughout the simulation duration. The second most notable interac­ strated complete solubility in the diluent acetonitrile. To ensure the
tion involved ASP197 and the hydroxyl residues of catechin, with appropriateness and effectiveness of the chromatographic setup for
interaction fractions of 98 % and 82 % during the simulation, suggesting subsequent analyses, various suitability parameters of the system were
the formation of a stable complex between P42 and 5U3A. The inter­ evaluated. These parameters encompassed the separation factor, ca­
action fraction analysis of MACL6-2DN1 revealed several amino acid pacity factor, resolution, tailing factor, and the number of theoretical
residues with substantial interactions with ligand atoms, some forming plates. The tailing factor, specifically, serves as an indicator of the peak’s
multiple contacts not observable in molecular docking results due to symmetry upon elution from the column (T) and increases proportion­
their static nature. PHE98 exhibited the most significant interaction ally with the elongation of the peak’s tail (Tesoro, 2022). The number of
fraction, surpassing 1.0, indicating more than one contact point with theoretical plates (N) serves as an indicator of the efficiency of the
more than one ligand atom. The primary interaction force with PHE98 column’s stationary phase. A low number of theoretical plates suggests
was hydrophobic in nature. Additionally, hydrophobic interactions poor separation effectiveness of the column (Li, 2020) therefore, to
involving LEU101, VAL132, VAL93, and LEU136 displayed noteworthy ensure effective separation, the number of theoretical plates should
interaction fraction values. Hydrogen bond interactions, crucial for ideally exceed 2000 (Mangla, 2020). In accordance with these criteria,

Table 7
Comparison of the resolutions of the compounds under normal and robust conditions.
Chromatographic Resolution
Parameters
Normal Condition Wavelength Flow Rate Oven Temperature
nm mL/min ◦
C

252 256 0.9 1.1 28 32

P42 – – – – – – –
SACL4 8.728 8.781 8.682 8.657 9.008 8.71 8.817
PACL7 5.533 5.599 5.456 5.350 6.057 5.633 5.594
MACL6 8.402 8.414 8.301 8.057 8.929 8.373 8.514

where P42-catechin, SACL4-syringic acid, PACL7-p-coumaric acid and MACL6-morin

Table 8
Comparison of the RSD and retention times under robust conditions.
Compound Chromatographic parameters

Wavelength Flow rate Oven temperature

252 nm 256 nm 0.9 mL/min 1.1 mL/min 28 ◦ C 32 ◦ C
Area RT Area RT Area RT Area RT Area RT Area RT

(RSD)
P42 0.49 0.87 0.53 0.74 1.24 0.95 1.14 1.98 1.04 0.92 0.84 0.88
SACL4 0.49 0.41 0.57 0.34 1.21 0.29 1.10 0.85 0.99 0.40 0.78 0.25
PACL7 0.49 0.28 0.71 0. 14 1.21 0.29 1.23 0.51 1.01 0.30 0.75 0.27
MACL6 0.38 0.20 0.90 0.28 1.21 0.37 1.08 0.31 1.15 0.34 0.78 0.40

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F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Fig. 14. Standard chromatogram at various chromtographic conditions (a) 252 nm (b) 256 nm (c) 0.9 mL/min (d) 1.1 mL/min (e) 28 ◦ C (f) 32 ◦ C where P42-
catechin, SACL4-syringic acid, PACL7-p-coumaric acid and MACL6-morin.

our findings demonstrated that each compound achieved more than capacity factor, illustrating the interaction duration between the active
2000 theoretical plates, coupled with a tailing factor of ≥ 1. Resolution, substance and the stationary phase, revealed favorable conditions for
a measure of the difference in peak heights between two distinct com­ efficient separation. The separation factor, crucial for peak resolution,
ponents eluting from the column at different retention times, was also underscored the distinct migration rates of the compounds (Freitag,
considered in our evaluation (Pérez-Cova et al., 2021). Additionally, the 2020). Overall, the analysis adhered to stringent system suitability

23
F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Fig. 14. (continued).

parameters, ensuring robust and reliable chromatographic performance employed, respectively. The calculated LOD values varied from 3.37 to
for the simultaneous estimation of the identified polyphenolic 4.98 µg/L, while the LOQ values ranged from 10.23 to 15.10 µg/L, as
compounds. determined from the calibration curves. These results affirm the sensi­
The correlation coefficients obtained in this study ranged from tivity and reliability of the developed HPLC method for the simultaneous
0.9909 to 0.9997 for all analytes, suggesting excellent linearity within estimation of the identified polyphenolic compounds in Tradescantia
the measured range. To determine the limits of detection (LOD) and pallida. The analytical method’s precision offers insight into random
quantification (LOQ), signal-to-noise ratios of 3.3 and 10 were errors, indicating the agreement among measurements obtained from

24
F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Fig. 14. (continued).

multiple samplings of a homogenous sample under specified conditions. retention times and peak areas of reference standard compounds and
This concept includes both repeatability (intra-day precision) and in­ calculating the percent relative standard deviation (% RSD). The data
termediate precision (inter-day precision) (ICH, Ich, 2017). The intra- demonstrated a favorable concordance among individual test results.
day variation was assessed through the analytical procedure within The six-sample percentage assay’s relative standard deviation (RSD)
the same laboratory, employing the same analyst, equipment, and day. should not exceed 2.0 (Vikas, 2020) and our results followed this
For inter-day precision, the procedure was replicated over three benchmark. Throughout the testing process in this study, every run
consecutive days (n = 6). The precision was determined by analyzing the exhibited a relative standard deviation (RSD) of less than 2 %, indicating

25
F. Imtiaz et al. Arabian Journal of Chemistry 17 (2024) 105703

Table 9 Funding.
Analytical solution stability analysis. This research did not receive any specific grant from funding
Compound Precision agencies in the public, commercial, or not-for-profit sectors.
Samples freshly prepared Samples after 24 h
Mean ± RSD CRediT authorship contribution statement
P42 99.34 ± 0.86 99.92 ± 1.48

SACL4 101.29 ± 1.57 99.55 ± 1.61 Fariha Imtiaz: Conceptualization, Methodology, Software, Valida­
PACL7 100.59 ± 0.69 100.35 ± 0.69 tion, Formal analysis, Investigation, Data curation, Writing – original
MACL6 99.96 ± 0.61 99.67 ± 1.58
draft, Writing – review & editing, Supervision. Muhammad Islam:
where P42-catechin, SACL4-syringic acid, PACL7-p-coumaric acid and MACL6- Conceptualization, Methodology, Formal analysis, Investigation, Data
morin curation, Writing – original draft, Writing – review & editing. Hamid
Saeed: Conceptualization, Methodology, Investigation, Writing – orig­
inal draft, Supervision. Muhammad Ishaq: Validation, Software,
Table 10 Formal analysis, Data curation, Writing – review & editing. Usman
Quantification of compounds from Tradescantia pallida Shareef: Methodology, Software, Formal analysis. Muhammad Naeem
extract. Qaisar: Methodology. Kalim Ullah: Methodology. Sibghat Mansoor
Compound Amount in extract Rana: Software. Anam Yasmeen: Methodology. Aneeqa Saleem:
mg/mL ± RSD Formal analysis. Romia Javaid Saddiqui: Writing – original draft. All
P42 0.072 ± 1.27 authors have read and agreed to the published version of the
SACL4 0.135 ± 2.00 manuscript.
PACL7 0.152 ± 0.91
MACL6 0.160 ± 1.12
Declaration of competing interest
where P42-catechin, SACL4-syringic acid, PACL7-p-cou­
maric acid and MACL6-morin The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
excellent precision of the developed HPLC method. Accuracy was the work reported in this paper.
assessed by spiking known quantities of standard substances with the
samples, and the percentage recoveries consistently exceeded 98 %. The References
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