Lonchocarpus Cyanescens Benth (Fabaceae) Plant Using Liquid: Exploring Natural Dye and Bioactive Secondary Metabolites in

Exploring Natural Dye and Bioactive Secondary Metabolites in
Lonchocarpus Cyanescens Benth (Fabaceae) Plant Using Liquid

Chromatography-High-Resolution Mass Spectrometry and Compound
DiscovererTM Software
Tanvir A. Amit1; Luis A. Colón*
Department of Chemistry, University at Buffalo, State University of New York, Buffalo, NY, USA
1
tamit@buffalo.edu; *lacolon@buffalo.edu
*Corresponding author:
Luis A. Colón E-mail:
lacolon@buffalo.edu
Abstract
Lonchocarpus cyanescens Benth is a native African plant commonly known as "African
Indigo" or "Indigo Vine" because locals extract dyestuff from its leaves that gives it a
brilliant indigo-like hue. Several scientists have reported this plant to contain indigo dye;
however, there has been no evidence to justify their claim. Therefore, the primary dye
component(s) responsible for the indigo-like hue has been scientifically unexplored. In this
study, we utilized UV-Vis and HPLC-DAD-ESI-HRMS (OrbitrapTM) techniques to identify
the main chemical compound(s) responsible for the color of the dyestuff. To our surprise, the
presence of indigo dye was not detected in the isolated dye extract; we discovered that crystal
violet dye and crystal violet-like molecules are present in the extract, which can be
responsible for the blue color observed. In addition to its natural dye-producing capability,
this plant is used by local traditional healers to heal ailments including ulcers, arthritis,
intestinal disorders, etc. Many scientists have proved that the effect of this plant extract on
many of these disease states was justified; however, the molecular identities of the
compounds responsible for the plant's medicinal value have been unexplored. Therefore, we
also analyzed the leaf extract of L. cyanescens and identified several chemical compounds
having structural similarities to active drug compounds. The details of our findings will be
the focus of our research work.
https://doi.org/10.26434/chemrxiv-2023-ggwtj ORCID: https://orcid.org/0000-0001-8849-3708 Content not peer-reviewed by ChemRxiv. License: CC BY-NC-ND 4.0
1. Introduction
With the ever-increasing interest in "green" products, low carbon footprint lifestyles,
and eco-friendly consumerism, there is an escalating interest in natural products, including
natural dyes.1 There has been a great motivation and renewed interest among the scientific
community to adopt natural dyes for various applications over their synthetic counterparts.
Unlike synthetic dyes, natural dyes are much more benign as they are biodegradable and
have not been reported to pose any detrimental health issues.2-5 Furthermore, preparing
natural dyes involves raw materials from renewable sources, requires minimum chemical
processing steps, and barely causes disposal problems.6 Therefore, as the modern world is
continuously striving to adopt environment-friendly approaches in every sphere of our lives,
natural dyes continue to re-emerge as the alternative to synthetic dyes and a viable "green
chemistry" option.7, 8
Figure 1. Various contemporary applications of natural dyes.
During the last few decades, health and environmental concerns posed by synthetic
dyes have motivated scientists from around the globe to explore natural dyes for various
(scientific and commercial) applications. Figure 1 shows some of the contemporary
applications of natural dyes. The most common commercial application of natural dye is in
the food industries as food colorants.9 In addition to food colorants, there is a growing
interest in using natural dyes for textile and pharmaceutical products, dye-sensitized solar
cells, histological stains, and pH indicators.10-13 Another relatively new area of application
includes cosmetic industries, where natural dyes are increasingly utilized due to their UV
protection and anti-aging properties.33 Therefore, scientific research on identifying and
characterizing novel dyes in various natural sources is critical in establishing the dye at the
commercial level, expanding its applications, and maintaining long-term environmental
sustainability.
Lonchocarpus Cyanescens Benth (of the Fabaceae family) is a native plant to West
Africa. It is widely grown in the countries like Nigeria, Ghana, Cameroon, Ivory Coast,
Togo, Sierra Leone, Benin, and Guinea.14 Figure 2 shows a photograph of the dry leaves of
the Lonchocarpus plant. This plant is commonly known as the "indigo vine" or "West
African indigo." The word "indigo" was tagged along with its common name because local
people extract dyestuff from the leaves of this plant, which has a brilliant indigo-like hue.
The extracted dye is mainly used for various domestic purposes. Several scientists had
claimed that the blue color of the Lonchocarpus dye was due to the presence of the
"indigotin" compound (found in indigo dyestuffs).15, 16 However, as per our knowledge, there
are no such scientific reports available to date that show the identification of indigotin as the
dye component in the Lonchocarpus plant; thus, the claims are invalid. Therefore, the
identity of the chemical compound(s) responsible for the brilliant blue color of the dyestuff
extracted from the leaves of the Lonchocarpus Cyanescens plant is still unknown. The lack
of identification and characterization knowledge of the main dye component could explain
why dyestuffs obtained from this plant are yet to be utilized commercially. Therefore, more
scientific and systematic studies on natural dyes could be the steppingstone in their
commercialization and various applications. Herein, we utilized modern analytical
techniques such as UV-Vis and Ultra High-Pressure Liquid Chromatography-Diode Array
Detector (UHPLC-DAD)-Electrospray Ionization (ESI)-High-resolution Mass Spectrometry
(HRMS) to identify primary chemical compounds in a blue extract of Lonchocarpus plant
provided by Dr. Rosemary Bassey. The L. cyanescens plant specimens were collected from
Nigeria and authenticated by Dr. Bassey.
Figure 2 Dry leaves of Lonchocarpus Cyanescens Benth plant.
(https://vplants.org/portal/collections/individual/index.php?occid=21038619)
In addition to the natural dye, L. cyanescens is very popular among the local traditional
healers in Africa to treat several disease states. Leaves of L. cyanescens are traditionally used
as a part of the recipe for the remedy of ulcers and arthritis, intestinal disorder, diabetes,
dysentery, psychotic disorder, leprosy, and others.17, 18 There are quite a few scientific reports
available supporting these folkloric medicinal uses, and a few justifications have appeared in
the literature.19-27 For example, Samuel, et al., investigated the antioxidant property of its leaf
extract using bio-assay.14 Adeoye, et al., recently studied acute ulcerous pain in rats with
aqueous root extract of L. cyanescens, which exhibited both antiulcer and analgesic effects,
justifying the folkloric claim for the health benefits of this plant.16 Kazeem, et al., evaluated
an ethanolic extract of leaves on diabetes management (hypoglycaemic potential) in rats, i.e.,
leaf extracts of L. cyanescens inhibited the activities of diabetes-related enzymes such as α-
amylase and α-glucosidase activities.21 Akunne, et al., showed that the root powder of L.
cyanescens possessed insecticidal properties against Sitophilus zeamais on maize and wheat
grains.28 However, none of these studies were focused on identifying the bioactive
compound(s) or secondary metabolites responsible for the various healing effects of L.
cyanescens extract. Identification of these bioactive secondary metabolites in the
Lonchocarpus plant will provide more scientific grounds for this plant's use as folklore
medicine and, more importantly, make this plant a prosperous source of various potential
drug leads.
Secondary metabolites are the phytochemicals (compounds synthesized by plants) that
are not essential for plant survival but perform various plant-specific functions.29-33 Plant
secondary metabolites (also known as natural products) have historically been a diverse and
significant source for drug discovery due to their actions in pharmacological targets.34
Newman and Cragg reviewed the sources of new drugs from 1981 to 2006 and stated that
"natural product-derived drug" encompasses almost 50% of new drugs introduced in the
market during this period had a natural product origin.35 Therefore, A thorough screening of
the secondary metabolites in the L. cyanescens extract may open up a new avenue for drug
discovery research.
A preliminary report on the phytochemicals present in L. cyanescens extracts
employed thin-layer chromatography to show various classes of phytochemicals.17 Another
report utilized gas chromatography-mass spectrometry (GC-MS) to analyze volatile oils from
the leaf and stem of this plant.15 However, in this study, we adopted an untargeted screening
approach to identify natural dye components and secondary metabolites by utilizing a
modern analytical technique such as UHPLC-HRMS. Such a study will reveal the unknown
dye component in the extract provided and information on other molecular entities present,
which may be responsible for the reported physiological/biological activities in the L.
cyanescens plant.
HRMS(/MS) identification alone can often be considered definitive for an untargeted
experiment.36 The following key features are essential for an unknown screening: (i) an
automated peak detection by exact mass filtering from the chromatographic run; (ii) an
assignment of an elemental formula to the exact mass of interest; and (iii) a database search
of plausible structures for the determined elemental formula.37 The MS manufacturers offer
various software packages that use different peak detection algorithms for automated
compound detection.38 Therefore, an in-silico strategy for determining unknown chemical
structures by matching experimental fragmentation spectra with their computational
counterparts queried from chemical databases is a valuable tool.39, 40 We utilized Compound
DiscovererTM 2.1 (CD) to identify the probable unknown candidates in the Lonchocarpus dye
extract. The CD is a small molecule data processing software from Thermo Fisher
ScientificTM that uses its high-resolution, accurate-mass data (such as Full-scan MS1, delicate
isotopic pattern, dd-MS2, etc.) to search against the online and local library or databases to
detect and identify the unknown structures with confidence. The software performs online
database searches using various search nodes such as mzCloud (with ddMS2 data),
ChemSpider (exact mass or formula), and Predict Composition nodes.
2. Experimental
2.1.Reagents and materials
HPLC-grade acetonitrile and 88% v/v formic acid were purchased from Fisher
Scientific (Fair Lawn, NJ). Absolute ethanol (200 PROOF) was purchased from Decon
Laboratories, Inc. Water for the UHPLC-HRMS mobile phase was prepared by running in-
house deionized water through a Barnstead EasyPure II Ultra-Pure Water System with a 0.2
um filter. Each mobile phase was degassed for 20 minutes by sonication before use.
The L. cyanescens extract was provided by our collaborator, Dr. Rosemary Bassey,
from the Department of Botany, University of Uyo, Nigeria (now at Hofstra University). The
L. cyanescens leaves and extract were obtained as described below.
2.2.Collection of the Lonchocarpus Cyanescens plant
The L. cyanescens plant specimens were sourced from local farmlands between March
and December of the year and authenticated by Dr. Bassey of the Department of Botany,
University of Uyo, Nigeria, and deposited at the Herbarium of the University of Uyo. The
voucher specimen number of the L. Cyanescens plant was UUH 03/11. Duplicates of the
plant specimens were deposited in the Herbarium of the Department of Botany, University of
Lagos, Nigeria.
2.3.Extraction of dye from Lonchocarpus cyanescens Benth (Fabaceae)
The local master dyers prepared the dye from L. cyanescens in Ogun state, Nigeria. In
order to prepare the dye, fresh leaves of L. cyanescens were harvested and pounded in a
wooden mortar and pestle until they were pulverized, leaving a greenish-blue mass. Next, the
greenish-blue mass was rolled into fist-sized balls and sun-dried for a few days. The dried
balls of L. cyanescens were then crumbled into an alkaline solution, which was prepared
using a mixture of water and hardwood ash lye and left to stand for about three days, but with
continuous stirring for at least 30 minutes each day. The precipitate was then collected,
pressed into cakes, and dried. The dried dye material was shipped directly to us for further
analysis.
2.4.Dye sample preparation
A solid dye extract of L. cyanescens was dissolved in the same solvent to the
appropriate concentration (500 ppm) and filtered through a 0.2 µm nylon filter for the
subsequent analytical analyses.
2.5.UV-Vis spectroscopy
The UV-Vis spectrum of L. cyanescens Benth was recorded on a UV−vis
spectrophotometer (PerkinElmer UV/VIS Spectrometer Lamba 25) fitted with a
thermostatted 1 cm path length cuvette device. The scanning was performed in the 200-700
nm range to obtain the wavelength at the maximum absorbance (λmax) of the dye samples.
The other method parameters used are ordinate mode: A; slit width: 1 nm; scan speed: 480
nm/min; data interval: 1nm; cycle time: 1 nm; no. of cycles: 1; UV-Vis Lamp change at 326
nm.
2.6.Ultra high-pressure liquid chromatography (UHPLC) separation of L. cyanescens dye
extract
A Waters Acquity UPLC (from Milford, MA) was used for the separation of the
chemical compounds in the L. cyanescens extract. The separation was carried out using
Waters X-bridgeTM Shield RP C-18 column (100 x 2.1 mm id; 1.8 μm particle size). The
mobile phase consisted of acetonitrile (B) and water (A) (both acidified with formic acid to
0.1%, v/v). The flow rate was 0.4 mL/min throughout the run, and eluted peaks were
monitored at 590 nm. The injection volume was 5 μL. The mobile phase composition for the
chromatographic gradient run was kept very generic in an attempt to elute as many peaks as
possible. To this end, a shallow gradient of 5% B/ 95% A to 95% B/ 5%A was utilized in 60
minutes.
2.7.Liquid chromatography-mass spectrometry (LC-MS) of L . cyanescens extract
2.7.1. UHPLC conditions

A Dionex Ultimate 3000 ultra-high pressure liquid chromatograph (Thermo Scientific,
Sunnyvale, CA, USA) was used to separate unknown analytes. The conditions in the UPLC-
UV experiment were maintained for the UHPLC component of the UHPLC-MS, except the
flow rate was 0.3 mL/min.
2.7.2. High-resolution (OrbitrapTM) mass spectrometer

Q-ExactiveTM high-resolution tandem mass spectrometer (Thermo Scientific, Bremen,
Germany) equipped with heated electrospray ionization (HESI-II) was used to identify and
quantify unknown analytes. The mass spectrometer was operated in the full MS/data-
dependent MS/MS mode (full MS–dd-MS/MS) in positive ion mode. The following scan
parameters were used: sheath and auxiliary gas flow rates 45 and 3 arbitrary units,
respectively; spray voltage 3.5 kV; capillary gas heater temperature 300 ºC; capillary
temperature 320 ºC and S-lens RF level 60. The following parameters were used in full MS
mode: resolution 70,000 FWHM (defined for m/z 200; 3Hz); scan range 120–1000 m/z;
automatic gain control (AGC) target 1e6; maximum inject time (IT) auto and in the data
dependent-tandem MS (dd-MS/MS) mode: intensity threshold auto; resolution 17,500
FWHM (defined for m/z 200; 12Hz); scan range 120-1000 m/z; AGC target 1e5; minimum
AGC target 8.00 e3; maximum IT auto; normalized collision energy (NCE) / stepped (N)CE
10, 30, 60 eV; isolation window 4.0 m/z; dynamic exclusion of fragmented analyte auto. The
instrument was controlled by XcaliburTM 4.1 software (Thermo Scientific, San Jose, CA,
USA) was used.
10
2.8.Data analysis by Compound DiscovererTM 2.1 software
Full-scan MS1/DD-MS2 data obtained from the UHPLC-Orbitrap-MS were analyzed
using the ThermoScientificTM Compound DiscovererTM (CD) 2.1 software. CD software
offers an extensive set of data processing tools based on advanced algorithms called Nodes.
These nodes can be logically assembled into a workflow tree in many different ways
depending on the kind of experiment and/or according to the needs of the user. Figure 3
shows a CD data processing workflow that was created by assembling various nodes. The
workflow performs retention time alignments, unknown compound detection, and compound
grouping across all samples, also predicts elemental compositions for all compounds, fills
gaps across all samples, and hides chemical backgrounds using blank samples.
Figure 3. Data processing workflow in Compound DiscovererTM 2.1 software
11
3. Results and discussions
3.1.UV-Vis experiment
The UV-Vis spectrum of the L. cyanescens dye extract is shown in Figure 4 The
absorbance maxima (λmax) was found to be at 590 nm, which lies in the visible region of the
electromagnetic spectrum and corresponds to the observed blue color. The observed UV-Vis
absorbance pattern and λmax were found to be comparable to those of crystal violet dye in the
literature.41-43 The UV-Vis spectrum of crystal violet from literature is also presented in
Figure 5 for comparison.41
Figure 4. UV-Vis spectrum of the L. cyanescens dye extract.
12
Figure 5. UV-Vis spectrum of Crystal Violet dye obtained from the literature.41
At this stage, we suspected the presence of crystal violet in the sample, which may
suggest potential contamination of the Lonchocarpus dye extract since crystal violet is a
synthetic dye; however, further investigations were needed.
3.2.Chromatographic (UHPLC) separation of the L. cyanescens dye extract components
The Lonchocarpus dye extract was subjected to UHPLC analysis. Based on the λmax
observed in the UV-Vis experiment, the chromatographic peaks were monitored at 590 nm.
Various chromatographic peaks were observed during the UHPLC analysis, as shown in
Figure 6. The observation of multiple peaks implied the possibility of the presence of
various metabolites (phytochemicals) of the Lonchocarpus plant rather than just components
13
attributed to the dye. Such a possibility inspired us to perform a thorough unknown screening
of the dye extract using UHPLC-HRMS, followed by data analysis using Compound
DiscovererTM 2.1 software.
3.3.Identification of the dye component Compound DiscovererTM 2.1 software
Gentian violet was identified as the probable dye component in the L. cyanescens dye
extract. It is a triphenylmethane/hexamethyl pararosaniline dye having an indigo-like hue.44
It also possesses antibacterial, antifungal, anthelminthic, antitrypanosomal, anti-angiogenic,
and anti-tumor properties.45-47 Other applications of gentian violet include veterinary
medicine, textile dyeing, paper printing, biological stains, and dermatological agents.48, 49
Figure 7 shows an extracted ion chromatogram (EIC) of the protonated molecular ion
(M+H)+1 at m/z 372, which was proposed as the gentian violet by the Compound
DiscovererTM software. The blue (sample 1.A) and yellow (sample 1.B) traces correspond to
duplicates of the Lonchocarpus dye extract; a minor shift in retention time was observed of
about 0.09min. The two samples (1.A and 1.B) were obtained from the same dye-extraction
batch and were prepared the same way for the LC-HRMS analysis to avoid false-positive
findings.
14
Figure 6. Chromatogram of L. cyanescens dye extract. The separation of the dye extract was
carried out on the Waters Acquity UPLC instrument. UPLC conditions:
Column: Waters X-bridgeTM Shield RP C-18 column (100 x 2.1 mm id; 1.8 μm particle size);
Injection: 5 uL; Flow rate: 0.4 mL/min; Mobile phase: Acetonitrile (0.1% Formic acid) and
water (0.1% formic acid); Gradient: 5-95% acetonitrile in 60 minutes; UV detection: 590 nm.
15
Figure 7. EIC of the protonated ion (M+H)+1 at m/z 372. The blue trace corresponds to
sample 1.A and orange trace corresponds to sample 1.B.
The molecular ion at m/z 372 was identified with two peaks eluting at two different
retention times; at 22.5 and 23.0 minutes for sample 1.A and at 22.6 and 23.1 minutes for
sample 1.B. Observation of two peaks for a single ion (m/z 372) suggested the possibility of
isomers. Therefore, we further investigated the full-scan MS1 spectra and MS-MS spectra of
both peaks. Figure 8 shows full-scan MS1 spectra of the (M+H)+1 ion at m/z 372 of sample
1.A at RT 22.5 and 23.0. Both MS1 spectra have four isotopes, and all of them are annotated
with green rectangles in the figure. The green rectangles show isotopic pattern-match, as
automatically identified by the CD software. In this case, almost identical mass (MS 1) spectra
for the EIC peaks at 22.5 and 23.0 min were observed.
16
Figure 8. Full-scan MS1 spectra of (M+H)+1 372 of sample 1.A at RT 22.5 min (top) and
23.0 min (bottom). The green rectangles indicate an isotopic pattern match.
17
Next, we investigated the MS-MS fragmentation spectra of the (M+H)+1 ion at the RT
22.5 and 23.0 (see Figure 9). Both MS2 spectra generated the same fragmentation pattern,
including molecular ions and the major fragments at m/z 356 and 340. Such identical full-
scan MS1 and MS-MS data of both peaks suggest that both peaks belong to the same parent
ion, (M+H)+1 372, which may be present in the isomeric form.
Figure 9. MS2 spectra of the (M+H)+1 ion at RT 22.5 and 23.0 min (Sample 1.A).
Table 1 was prepared from the "Compound Table" generated by the CD software
during the compound identification process. Based on the data-dependent full-scan accurate-
mass MS1 and MS2 data, the Compound Table proposed a list of probable compounds for the
(M+H)+1 ion at m/z 372. Gentian violet was ranked as the number 1 candidate or the most
probable candidate because the proposed compound (gentian violet) had a full match with the
18
"Predict Compositions" node - meaning the predicted chemical formula (C25H29N3) is
accurate. The CD also showed a full match with the "mzCloud" search node for the proposed
gentian violet compound - meaning the experimental MS-MS fragmentation spectrum
matches with the mzCloud library (MS-MS) spectra corroborating the identity of the
compound. This is also indicated by the higher value of mzCloud Best Match Score (87-
88%) with sub-ppm mass errors.
Table 1. Chemical formula, molecular weight, retention time (RT), different library match
status, and mzCloud best match score for the most probable candidate, gentian violet.
Most mzCloud ΔMass

RT Predict mzCloud ChemSpider
Peak probable Formula Mol. Wt. Best (ppm)
(min) Compositions Search Search
compound Match
1st 22.5 Gentian C25H29N3 371.23613 Full Full Partial 87.9 0.04
violet Match Match Match
2nd 23.0 Gentian C25H29N3 371.23628 Full Full Partial 88.3 0.02
violet Match Match Match
3.4.Compound identification by mzCloud search node
mzCloud is the primary search node of the CD software. It is an online fragmentation
spectral library that includes highly curated, chemically diverse, accurate mass MS/MS and
MSn data. These mzCloud spectra have been acquired at various collision energies and for
multiple adducts using collision-induced dissociation (CID) and higher-energy collisional
dissociation (HCD). The CD utilizes dd-MS2 data to search against the mzCloud library
spectra for MS fragmentation pattern match. The CD sends the stepped collision energy
query spectrum (data-dependent MS2 fragmentation spectra) to its library. The library
performs a search for possible candidates and pulls spectra from the database at close
matching energies to create an average spectrum to produce a final match score. Thus,
19
mzCloud library searches alone only produce putative candidates for identification. The
putative results can be ranked using the extensive fragmentation spectra in mzCloud using
the mzLogic algorithm,
The "mzCloud Results Table" (proposed compound name, chemical structure,
molecular weight, ΔMass (ppm), best match score, etc.) was generated by the CD software,
which is shown in Figure 10. mzCloud node proposed only one probable compound within
less than one ppm mass error (only 0.04 ppm) and provided a Best Match Score of 88.3 using
the mzLogic algorithm. The proposed compound, gentian violet, is shown by green
annotation, which indicates a full/valid match with the database.
Figure 10. mzCloud Table generated by Compound DiscovererTM 2.1 software.
20
Figure 11. mzCloud spectral comparison between query (top) and library spectra (bottom)
visualized in a mirrored plot for gentian violet. The query spectra were obtained using HCD
at a stepped collision energy of 10, 30, and 60 eV and compared with the library spectra
obtained using HCD at a stepped collision energy of 10, 30, and 50 eV.
For the structure proposed via mzCloud, the spectra comparison between the query
spectrum and library match spectrum is visualized in a mirrored plot (see Figure 11). The
library spectra included precursor ion at m/z 372.243, which found a match with the
precursor ion of the query spectra. This is called an identity match. The identity match
provides added confidence for unknown identification. In addition to the precursor ion
match, the fragments at m/z 356 and m/z 251 were found to match with the corresponding
fragments of the query spectra.
21
3.5.The Predict Compositions node
Orthogonal chemical databases such as Predict Compositions and ChemSpider
typically include additional compound information to aid in making identifications. The
Predict Compositions node predicts the elemental compositions of the unknown compounds.
The "Predict Compositions" node includes the latest spectral fit algorithm that uses fine
isotopic structures from high-resolution accurate-mass Orbitrap full MS (MS1) data and
MS/MS fragments to predict compositions. The green rectangles in Figure 8 mean isotope
pattern match for predicted composition C25H29N3 based on a resolution of Full-scan MS
(MS1) of the peak at 22.5 minutes (top) and 23.0 minutes). The green rectangles also imply
that the measured mass of isotopic peaks and their intensity fall within the user-specified
Figure 12. Predicted Compositions results table for gentian violet as provided by the Compound
DiscovererTM software.
mass tolerance window (+/- 5 ppm) and user-specified intensity tolerance window (+/-
30%); therefore, isotope pattern match was detected. Thus, the isotopic pattern match
corroborates the accuracy of the predicted chemical formula, C25H29N3, provided by the
Predict Compositions node. Figure 12 shows the result table generated by the compound
discoverer Predict Compositions search node, where two chemical formulas were generated
based on a single accurate mass, 371.23615 Da. The chemical formulas were ranked based
on ΔMass (ppm), number of matched isotopes, number of fragments, and Full-MS and MS-
MS coverage.
22
Again, the green annotation in the Predict Compositions result table indicates a full-
match with the database and implies that the predicted formula, C25H29N3, is the most
confident chemical formula given by the Predict Compositions node because of the superior
values of the identification parameters (ΔMass is only -0.04 ppm; MS-MS coverage is 73%),
whereas the red annotation means a very poor match or invalid match indicated by such a
high ΔMass value (-4.50 ppm) and low MS-MS coverage (only 3.00%) compared to the #1
candidate.
3.6.The ChemSpider search node
ChemSpider is a secondary or orthogonal search node that searches the predicted
compositions against online chemical databases and ranks the proposed compounds based on
the number of matched references from various data sources. In this case (see Figure 13),
CD provided two partial matches (yellow annotation) because Chemspider could only
correlate the predicted chemical formulas with the accurate mass, 371.23614 Da, but could
not find a valid structural match (proposed by mzCloud) based on that molecular weight and
chemical formula.
Figure 13. ChemSpider result table generated by Compound DiscovererTM software.
23
3.7. Identification of other bioactive secondary metabolites
Along with the identification of the probable dye component, several probable
candidates of phytochemicals with various bioactive/physiological properties were identified
based on the results obtained from the CD software. Identification of these secondary
metabolites using CD involved the same logic and parameters as those of the gentian violet
identification process. However, data analysis using CD software was challenging as the
software provided a comprehensive list of data for 960 candidates, especially narrowing
down these compounds to only seven potential probable candidates, including the dye
component (gentian violet) required to maintain strict identification criteria. In order to do
that, various identification parameters such as MS-MS fragmentation pattern match (using
mzCloud), mass error (< 5 ppm), blank subtraction, and isotopic pattern match, etc., were set
to screen for the probable candidates and eliminate any possibility of false positive.
However, the detailed findings of the CD results bioactive secondary metabolites are beyond
this report's scope. Nevertheless, a list of probable candidates with some important
identification parameters is shown in Table 2 for some separated compounds. MS2
Fragmentation pattern match of the experimental data against the mzCloud library data was
prioritized for the compound identification. All probable candidates had the isotopic-pattern-
match and full-match with the Predict Compositions nodes for the chemical formula. The
proposed probable compounds are known for various physiological effects, which are shown
in Table 3. The EIC and the chemical structures of the proposed compounds are shown in
Figure 14 and Figure 15, respectively.
24
Table 2. List of probable bioactive/phytochemicals present in the Lonchocarpus plant
Compounds Formula MW RT Mass Isotopic mzCloud Predict ChemSpider

error pattern match Composition match
(ppm) match match
Citral C10H16O 152.1201 35.46 -0.13 Yes Yes Yes Yes
Palmitoylethanola C18H37NO2 299.2821 17.42 1.03 Yes Yes Yes Partial

mide (PEA)
Azaperol C19H24FN3O 329.1889 18.20, 4.10 Yes Yes Yes Partial

19.01
Lysergol C16H18N2O 254.1419 35.98 0.02 Yes Yes Yes Yes
Cathinone C9H11NO 149.080 24.43 0.66 Yes Yes Yes Yes
Methyl-4-Boc- C12H22N2O4 258.1579 15.37 -0.87 Yes Yes Yes Yes

piperazine-2-
acetate
25
Figure 14. Stacked EIC of the probable secondary metabolites obtained from the UHPLC-
HRMS experiment.
26
Figure 15. Chemical structure of the probable candidates proposed by the Compound
DiscovererTM 2.1 software.
27
Table 3. Various physiological effects of the probable phytochemicals as obtained from
various literature.
Compounds Physiological properties
Citral - Used as a food additive and as a fragrance in cosmetics 50

- Antioxidant property 51
- Antimicrobial property 52
Palmitoylethanolamide (PEA) - Analgesic properties; used in the treatment of chronic pain 53,54,55
- Anti-inflammatory property 56
- Psychoactive property
Azaperol - Sedative and anti-emetic effects 57
Lysergol
- Used as hypotensive, psycho-tropic analgesic and uterus- and
intestine-stimulating drug 58, 59
Cathinone - Psychoactive property 60, 61
Methyl-4-Boc- - Antioxidant activity 62

piperazine-2-acetate - Antimicrobial property 63
4. Conclusions
Lonchocarpus cyanescens Benth is a very popular medicinal and dye-producing plant
in Africa. However, from the scientific perspective, this important plant is very much under-
explored and thus remains underrated. We explored an extract of this prevalent West African
medicinal plant for the probable identity of the dye component and various secondary
metabolites. The components identified in this study were based on the Compound
DiscovererTM software after LC-MS analysis of the extract in our possession. This software
proposed the probable candidates based on the online library and local database search.
28
However, a retention time and fragmentation pattern match using the standard reference
compounds will further confirm the identity of these proposed compounds.
The discovery of gentian violet in the plant extract provided to us suggests potential
contamination or adulteration of the extract sample since gentian violet (i.e., crystal violet) is
a synthetic dye and unlikely to be present in the Lonchocarpus cyanescens plant. Our study
of an extract of the L. cyanescences revealed some drug-like compounds that can account for
the use of the plant for medicinal purposes; we are the first group that proposes the
possibility of the existence of such drug-like molecules in this plant. However, given that we
also found crystal violet in the plant extract at our disposition, our findings must be
corroborated with a new study using a sample from a different source, where contamination
is unlikely. Positive corroboration of our findings may provide justification to explore L.
cyanescences further to potentially open up a new avenue of drug discovery using this plant.
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37

Lonchocarpus Cyanescens Benth (Fabaceae) Plant Using Liquid: Exploring Natural Dye and Bioactive Secondary Metabolites in

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Lonchocarpus Cyanescens Benth (Fabaceae) Plant Using Liquid: Exploring Natural Dye and Bioactive Secondary Metabolites in

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Exploring Natural Dye and Bioactive Secondary Metabolites in

Lonchocarpus Cyanescens Benth (Fabaceae) Plant Using Liquid

and eco-friendly consumerism, there is an escalating interest in natural products, including

continuously striving to adopt environment-friendly approaches in every sphere of our lives,

Figure 1. Various contemporary applications of natural dyes.

protection and anti-aging properties.33 Therefore, scientific research on identifying and

commercial level, expanding its applications, and maintaining long-term environmental

commercialization and various applications. Herein, we utilized modern analytical

techniques such as UV-Vis and Ultra High-Pressure Liquid Chromatography-Diode Array

Detector (UHPLC-DAD)-Electrospray Ionization (ESI)-High-resolution Mass Spectrometry

(HRMS) to identify primary chemical compounds in a blue extract of Lonchocarpus plant

Nigeria and authenticated by Dr. Bassey.

Figure 2 Dry leaves of Lonchocarpus Cyanescens Benth plant.

an ethanolic extract of leaves on diabetes management (hypoglycaemic potential) in rats, i.e.,

leaf extracts of L. cyanescens inhibited the activities of diabetes-related enzymes such as α-

compound(s) or secondary metabolites responsible for the various healing effects of L.

cyanescens extract. Identification of these bioactive secondary metabolites in the

Secondary metabolites are the phytochemicals (compounds synthesized by plants) that

A preliminary report on the phytochemicals present in L. cyanescens extracts

employed thin-layer chromatography to show various classes of phytochemicals.17 Another

approach to identify natural dye components and secondary metabolites by utilizing a

which may be responsible for the reported physiological/biological activities in the L.

HRMS(/MS) identification alone can often be considered definitive for an untargeted

compound detection.38 Therefore, an in-silico strategy for determining unknown chemical

structures by matching experimental fragmentation spectra with their computational

counterparts queried from chemical databases is a valuable tool.39, 40 We utilized Compound

ChemSpider (exact mass or formula), and Predict Composition nodes.

2.1.Reagents and materials

L. cyanescens leaves and extract were obtained as described below.

2.2.Collection of the Lonchocarpus Cyanescens plant

2.3.Extraction of dye from Lonchocarpus cyanescens Benth (Fabaceae)

2.4.Dye sample preparation

subsequent analytical analyses.

The UV-Vis spectrum of L. cyanescens Benth was recorded on a UV−vis

spectrophotometer (PerkinElmer UV/VIS Spectrometer Lamba 25) fitted with a

2.6.Ultra high-pressure liquid chromatography (UHPLC) separation of L. cyanescens dye

2.7.Liquid chromatography-mass spectrometry (LC-MS) of L . cyanescens extract

2.7.1. UHPLC conditions

flow rate was 0.3 mL/min.

2.7.2. High-resolution (OrbitrapTM) mass spectrometer

dependent-tandem MS (dd-MS/MS) mode: intensity threshold auto; resolution 17,500

USA) was used.

Full-scan MS1/DD-MS2 data obtained from the UHPLC-Orbitrap-MS were analyzed

using the ThermoScientificTM Compound DiscovererTM (CD) 2.1 software. CD software

Figure 3. Data processing workflow in Compound DiscovererTM 2.1 software

Figure 5 for comparison.41

Figure 4. UV-Vis spectrum of the L. cyanescens dye extract.

synthetic dye; however, further investigations were needed.

3.2.Chromatographic (UHPLC) separation of the L. cyanescens dye extract components

DiscovererTM 2.1 software.

3.3.Identification of the dye component Compound DiscovererTM 2.1 software

extract. It is a triphenylmethane/hexamethyl pararosaniline dye having an indigo-like hue.44

It also possesses antibacterial, antifungal, anthelminthic, antitrypanosomal, anti-angiogenic,

and anti-tumor properties.45-47 Other applications of gentian violet include veterinary

carried out on the Waters Acquity UPLC instrument. UPLC conditions:

sample 1.A and orange trace corresponds to sample 1.B.

ion, (M+H)+1 372, which may be present in the isomeric form.

gentian violet compound - meaning the experimental MS-MS fragmentation spectrum

88%) with sub-ppm mass errors.

Most mzCloud ΔMass

3.4.Compound identification by mzCloud search node

mzCloud is the primary search node of the CD software. It is an online fragmentation

multiple adducts using collision-induced dissociation (CID) and higher-energy collisional