Industrial Crops & Products 170 (2021) 113837

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Industrial Crops & Products

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Enhancing biomass and productivity of coumarins and essential oil in

ajwain (Trachyspermum ammi) sprouts via laser light treatment
Mansour A. Balkhyour a, *, Abdurazag M.S. Tammar a, Ahmed Saleh Summan a, b, Abdelrahim H.
A. Hassan c, *
a
Department of Environmental Science, Faculty of Meteorology, Environment and Arid Land Agriculture, King Abdulaziz University, Jeddah, Saudi Arabia
b
Centre of Excellence in Environmental Studies, King Abdulaziz University, Jeddah, Saudi Arabia
c
Department of Food Safety and Technology, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef, 62511, Egypt

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

Keywords: Ajwain, Trachyspermum (T.) ammi, is a coumarin-rich, highly nutritive, and medicinally important herb, how­
Laser irradiation ever, there is not enough data on the efficiency of laser irradiation in inducing such properties. Herein, the effect
Trachyspermum ammi of seed treatment with laser irradiations [helium-neon (He-Ne, 632 nm, 5 mW) and helium-cadmium (He-Cd,
Plant yield
460 nm, 16.2 mW)] at 500 mJ energy for 5 min on the biomass, photosynthesis, and the metabolism of cou­
Coumarins metabolism
marins, essential oil, and their precursor metabolites in two varieties of ajwain sprouts i.e., Desi and Chakwal was
Essential oil metabolism
Phenylpropanoid investigated for the first time. Laser irradiation increased sprout biomass accumulation by improving photo­
synthesis, chlorophyll content, and respiration. Additionally, laser treatment enhanced the metabolism of cou­
marins and essential oil, which was explained by the increased availability of their precursors (phenylalanine,
cinnamic acid, and shikimic acid). There were also increases in the activity of the key enzymes involved in the
biogenesis of phenylpropanoid and coumarins, i.e., 3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase
(DAHPS), cinnamate-4-hydroxylase (C4H), 4-coumarate: coenzyme A ligase (4CL), and p-coumarate 3-hydroxy­
lase (C3H). Furthermore, laser treatment induced the accumulation of total and individual phenolics and fla­
vonoids. Consequently, laser treatment significantly improved the antibacterial properties of ajwain sprouts
against four foodborne pathogens included Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, and
Salmonella spp. Ajwain variety-specific responses to laser light treatment confirmed by principal component
analysis (PCA) showed that Desi variety was more responsive. The efficiency of He-Ne was greater than He-Cd
laser, which suggests He-Ne laser treatment as a promising approach to boost the production of valuable pig­
ments, coumarins, and essential oils in ajwain sprouts.

1. Introduction ajwain [Trachyspermum (T.) ammi (L.)], belonging to the Apiaceae

Amongst the highly nutritional plant sources, sprouts have received a
high interest because of their valuable contents of health-boosting
phytochemical compounds. Sprouts are relatively richer in antioxi­
dants, fatty acids, amino acids, and vitamins than the mature plant,
which supports the significant role of sprouts as antioxidant, anti-
inflammatory and anticancer agents. In addition, sprouts have rela­
tively lower levels of anti-nutritional compounds compared with their
mature plants (Dal Bosco et al., 2015).
With reference to previous research results and conventional medi­
cine experiences, a lot of precious medicinal herbs have been presented
to the pharmaceutical and food industries. Out of these medicinal herbs,
family, is a valuable medicinally important seed spice (Seidemann,
2005). T. ammi, also known as Bishop’s plant, is a native herb of Egypt
that is grown also in Iraq, Iran, Afghanistan, Pakistan, and India, among
other places. T. ammi are valuable sources of protein, minerals, essential
oil, flavonoids and phenolic compounds (Hassan et al., 2021). It also
demonstrated many pharmaceutical properties including painkiller,
anxiolytic, antispasmodic, and anti-inflammatory effects. Also, their
seeds are commonly used as a food flavoring spice. Moreover, the seeds
showed many therapeutic effects and were used traditionally to combat
indigestion, flatulence, abdominal pain, diarrhea, and dyspepsia. The
seed oil of ajwain showed significant antibacterial and antiviral effects.
As well as it displayed good antioxidant, bronchodilatation, and

* Corresponding authors.
E-mail addresses: mbalkhyour@kau.edu.sa (M.A. Balkhyour), abdelrahim@vet.bsu.edu.eg (A.H.A. Hassan).

https://doi.org/10.1016/j.indcrop.2021.113837
Received 20 April 2021; Received in revised form 1 July 2021; Accepted 14 July 2021
Available online 20 July 2021
0926-6690/© 2021 Elsevier B.V. All rights reserved.
M.A. Balkhyour et al.
antitumor properties (Bairwa et al., 2012). In context, Krishnamoorthy
and Madalageri (1999) reported that the seeds of ajwain are bitter,
pungent, however, they were a good treatment for abdominal tumors,
abdominal pains, and piles. Furthermore, Sivropoulou et al. (1996)
recognized various pharmacological effects of T. ammi such as antibac­
terial, antifungal, antioxidant, antinociceptive, hypolipidemic, antihy­
pertensive, anti-lithiasis, antitussive, anthelmintic, and anti-filarial
actions.
Previous pieces of literature exposed the occurrence of many sig­
nificant phytochemicals in T. ammi, including carbohydrates, glyco­
sides, saponins, phenolic compounds, volatile oils, lipids, fibers, and
minerals (Nagalakshmi et al., 2000; Choudhury et al., 1998). One of the
most significant bioactive phenolic compounds that abundant in T. ammi
are coumarins. They are known as benzopyrones and exist in high
amounts in plants, also coumarins have been found in microorganisms
and some animal sources. They can be divided into many categories
starting from simple coumarins to many other kinds of polycyclic cou­
marins (Borges et al., 2005). Coumarins have many significant biolog­
ical activities such as anticancer, antiviral, anti-HIV, anticoagulant,
anti-inflammatory activities. Moreover, coumarins have been reported
as significant antibacterial, antifungal, and antioxidant agents (Borges
et al., 2005; Borges Bubols et al., 2013). Ajwain is also rich in essential
oils, which were mainly composed of aromatic compounds (Vitali et al.,
2016). Among the former, the phenol thymol, carvacrol, p-cymene, and
γ-terpinene were undoubtedly the predominant components (Vitali
et al., 2016). The ajwain essential oil demonstrated antibacterial, anti­
fungal, antioxidant, nematocidal, scolicidal, and antitermitic activities.
Coumarins are derived from the phenylpropanoid pathway in higher
plants (Harborne, 1999). Essential oils also are mainly composed of
phenylpropanoid compounds, where cinnamic acid and p-coumaric acid
have been important secondary metabolites in essential oil biosynthesis
in plants (Wang et al., 2020). Phenylpropanoid biogenesis originates
from cinnamic acid and shikimic acid pathways (Maeda and Dudareva,
2012). The enzyme 3-Deoxy-D-arabino-heptulosonate-7-phosphate
synthase (DAHPS) catalyzes the aldol condensation of the glycolytic
intermediate phosphoenol pyruvate (PEP) and the pentose phosphate
pathway intermediate erythrose-4-phosphate (E4P) to 3-dexoy-D-arabi­
noheptulosonate-7-phosphate (DAHP), which is the first enzymatic step
in the pathway of shikimic acid (Sangwan et al., 2001; Tzin and Galili,
2010). Chorismate mutase (CM) then catalyzes the biosynthesis of
phenylalanine and additionally serves as a main axis leading to the
branch of phenylalanine biosynthesis. Consequently, phenylalanine
ammonia-lyase enzyme (PAL) catalyzes phenylalanine to trans-cinnamic
acid, which is then converted by cinnamate-4-hydroxylase (C4H) to
p-coumaric acid (Vialart et al., 2012). These reactions also involve the
hydroxylates of p-coumaryl-CoA by p-coumarate 3-hydroxylase (C3H)
(Rastogi et al., 2013). Then 4-coumarate: coenzyme A ligase enzyme
(4CL) converts p-coumaric acid to p-coumaroyl CoA. The CoA-ester in­
termediate is then hydroxylated by 4-coumaroyl CoA 2′ -hydroxylase
(C2’H) to form umbelliferone, a coumarin derivative biosynthetic
pathway intermediate (Sadeghi, 2013). The development of p-coumaric
acid from cinnamic acid occurs next. Therefore, PAL, DAHPS, C4H, 4CL,
and C3H are the main enzymes aiding the phenylpropene precursor
biosynthesis.
From the previous pieces of literature, we assume that T. ammi is a
significant source of therapeutically bioactive substances, which de­
serves a deeper investigation, especially in the sprout stage that has not
been widely studied. Particularly, the application of novel methods to
increase the levels of bioactive phytochemicals, chiefly coumarins
biosynthesis in T. ammi.
In context, researchers are working to find eco-friendly, simple, and
affordable methods that induce positive impacts on the physiology,
biochemistry, growth rate, germination, and yield abilities in plants to
enhance their nutritive value and health-promoting properties. Elec­
tromagnetic radiation, particularly laser treatment of plant seeds has
proven to be an efficient way to promote the antioxidant activity of
2
Industrial Crops & Products 170 (2021) 113837
plants, which eventually improves seed germination and induces fast
seedling growth and great biomass accumulation (Chen et al., 2005a).
Therefore, laser irradiation has been widely used to increase plant
nutritive value and productivity. There are two types of laser light:
pulsed and continuous lasers. The former, such as Nd : YAG and XeCl
lasers, is primarily exploited in medical applications, while the latter,
such as helium-neon (He-Ne), helium-cadmium (He-Cd), and CO2 lasers,
is used to boost crop production (Chen et al., 2005b). It was reported
that the alterations in plant properties as a result of laser light treatment
could be ascribed to the light effect, electromagnetism, and heat (Ri
et al., 2019). Certain doses of laser light improve seed germination,
enzymatic activities, chlorophyll content, water use competence, and
photosynthetic rate along with seedling growth and eventually plant
yield. Also, laser treatment of seeds enhances the accumulation of
bioactive compounds such as proteins, carotenoids, phenols, ascorbates,
antioxidant enzymes, superoxide radical, oxidized glutathione, essential
oils, as well as numerous other microelements and macromolecules
(Govil et al., 2006; Wu et al., 2007). Additionally, some studies have
shown that laser irradiation had a significant impact on the number of
branches per plant, total biomass, growth rate, leaf area, seedling
appearance, root lengths, shoot lengths, mineral profile, crop yields, and
germination of various plants such as faba beans, Isatis indogotica,
fennel, and alfalfa (Chen et al., 2005b; Dziwulska et al., 2006).
Even though the availability of papers on laser applications in plant
productivity, to our knowledge, the laser light effect on the accumula­
tion of phenolics, flavonoids, and pigments, and biosynthesis of cou­
marins and essential oils in T. ammi sprouts have never been addressed.
Therefore, this study aimed at addressing the effect of two types of laser
light treatment (He-Ne and He-Cd) on plant yield, photosynthesis, levels
of phenolics and flavonoids, and metabolism of coumarins, essential oil,
and their precursor metabolites in two species of T. ammi (Chakwal and
Desi) sprouts. Moreover, in-depth analyses were performed to reveal the
biochemical bases of the noticed increases in measured metabolites.
Additionally, we assessed the antibacterial properties of treated sprouts
against four foodborne pathogens, i.e. Staphylococcus aureus, Listeria
monocytogenes, Bacillus cereus, and Salmonella spp.
2. Materials and methods
2.1. Experimental setup
Seeds of T. ammi (Chakwal and Desi species) were soaked for 2 h in
distilled H2O before laser illumination using a laser equipment system
(DMC Equipment Ltd.). Three groups of each species seeds were used,
each group contained 100 seeds: 1) control group (unirradiated), 2)
helium-neon (He-Ne) laser-irradiated group and 3) helium-cadmium
(He-Cd) laser-irradiated group. Ajwain seeds were irradiated by either
He-Ne laser (632 nm, 5 mW) or He-Cd laser (460 nm, 16.2 mW) with a
beam diameter of 1 mm at 500 mJ energy for 5 min from the side of the
embryonic area. The laser was applied perpendicularly at the distance of
12 cm between the laser source and the ajwain seeds. These laser con­
ditions were chosen following a pilot experiment estimating the opti­
mum laser conditions that induce the best effect on sprout growth and its
antioxidant capacity (FRAP). The whole experiment was repeated
several times. The sprouting of control and irradiated seed groups was
performed on trays contain vermiculite. T. ammi seeds were watered
with distilled water every 3 days. Afterward, the trays were transferred
to a controlled growth chamber under controlled growth conditions i.e.,
25 ◦ C, 16 h/8 h (light/dark) cycle, 400 μmol m− 2 s-1 photosynthetically
active radiation (PAR) and 60 % humidity. Nine-day-old ajwain sprouts
from each tray were weighed up to measure the fresh weight (FW). The
dry weight (DW) was determined after 72 h of drying in an oven at 75◦ C.
Then the ajwain sprouts were frozen (liquid N) and stored (− 80 ◦ C) for
further biochemical and biological analyses. Four biological replicates
were used. and each replicate was a pool of ten ajwain sprouts from a
single tray.
M.A. Balkhyour et al.
2.2. Determination of photosynthesis and pigments
Photosynthesis and respiration were measured as μmol CO2 m− 2 s-1
of treated and untreated groups using an (EGM-4 infrared gas analyzer,
Zinta et al., 2018). The rates of ajwain sprout photosynthesis and dark
respiration were estimated from about 3 min of net exchange of carbon
dioxide measurements.
T. ammi sprouts were extracted in acetone solvent at a dark condition
and room temperature. Extracts were centrifuged (14,000 g, 4 ◦ C, and
for 25 min). Filtered supernatants (GHP filter, 0.24 μm) were measured
by HPLC (Shimadzu SIL10-ADvp) at 4 ◦ C and a reversed-phase was
applied. T. ammi extracted pigments were separated on a C18 column
(silica-based of 5 μm ODS1 and 4.6 × 250 mm and the temperature of
the silica-based column was maintained at 40 ◦ C. The applied mobile
phase contained 81 % acetonitrile, 9% methanol, and 10 % water (sol­
vent A) and 68 % methanol, and 32 % ethyl acetate (solvent B). The
mobile phase flow rate (1.2 mL/min) was applied at room temperature.
Different pigments were detected at different wavelengths i.e., 420, 440,
462, 660 nm by using a DAD detector. Pigment concentrations of ajwain
sprouts were determined using a standard calibration curve.
2.3. Determination of phenolic compound content
Total phenolic compounds were determined (Almuhayawi et al.,
2021; Al Jaouni et al., 2018). The concentrations of phenolics were
extracted by homogenizing 0.1 g of lyophilized ajwain sprouts in
aqueous ethanol (1 mL, 80 %, v/v). Extracts were centrifuged for 25 min
at 4 ◦ C and the clear supernatant was used. Phenolic acid and flavonoid
contents were measured using the Folin–Ciocalteu method and
aluminum chloride method, respectively.
To measure the individual phenolic acids and flavonoids, we used
HPLC (SCL-10 AVP, Shimadzu Corporation, Kyoto, Japan) (Hamad
et al., 2015; AbdElgawad et al., 2020). About 50 mg of freeze-dried
ajwain sprouts were extracted for a day by using 4:1 acetone-water so­
lution (v/v). The Shimadzu HPLC system was equipped with a diode
array detector and samples were separated on column (a Lichrosorb
Si-60 of 7 μm and 3 × 150 mm). The applied mobile phase was 90 %
water contained 10 % formic acid (90:10, v/v) and 86 % acetonitrile, 9%
water, and 5% formic acid was applied (the flow rate was 0.75 mL/min).
Phenolic concentrations of ajwain sprouts were determined by
comparing with the peak areas of the corresponding standard.
2.4. Determination of coumarins, their precursors and related enzymes
The determination of coumarins and their precursor metabolites (p-
coumaric acid, umbelliferone, scopoletin, xanthotoxin, bergapten,
dehydrogeijerin, phenylalanine cinnamic acid, and shikimic acid) was
measured by using Waters UPLC (ultra-performance liquid chromatog­
raphy system). UPLC has contained a quadrupole mass spectrometer
equipped with an ESI source according to the method that was previ­
ously described in Wang et al. (2020).
Additionally, we analyzed the activities of five enzymes that catalyze
the reactions in the pathways of shikimic acid and cinnamic acid, which
are involved in the biogenesis of phenylpropanoid and thus biosynthesis
of coumarins. The measured enzyme activities were PAL, 4CL, C3H,
C4H, and DAHPS. The determination methods were according to the
techniques that were previously detailed in Wang et al. (2020). To
measure DAHPS activity, samples were homogenized in 3 mL 50 mM
Tris-HCL buffer. The assay buffer was Tris-HCl buffer (pH = 7.6 at a
concentration of 50 mM). The reaction mixture also contained
phosphoenol-pyruvate (0.2 mM), erythrose-4- phosphate (0.1 mM), and
MnSO4 (0.1 mM) / CoCl2 (0.1 mM). The reaction was stopped by adding
trichloroacetic acid (25 %, W/V) and DAHPS levels were measured (549
nm). PAL enzymes were extracted (0.2 M of Na-borate buffer at pH 8.8).
The activity was determined by measuring the production of
trans-cinnamic acid (290 nm). The activity of 4CL (4-coumarate:
3
Industrial Crops & Products 170 (2021) 113837
coenzyme A ligase) was monitored by estimating the increase in
p-coumarate (333 nm). Fresh samples were extracted in Tris-HCL buffer
(50 mM, pH 8.9). The activity was estimated as the increase in 4-hydrox­
y-trans-cinnamic acid (340 nm). One unit of 4CL activity equal to the
production of 1.0 nmol hydroxy-trans-cinnamic acid per min.
2.5. Determination of essential oils
The levels of essential oils in ajwain sprouts were measured (GC/MS)
as described in Hassanpour et al. (2014). Ajwain sprouts were dried at
room temperature and 10 g of the dried sample was used for essential oil
extraction. The dried sprouts were exposed to steam distillation for 4 h
using a Clevenger-type instrument. Sprouts were extracted with petro­
leum ether for 48 h at room temperature. The extract was evaporated to
dryness using rotary evaporation at reduced pressure. The essential oil
was passed over dark anhydrous sodium sulfate to remove moisture and
then measured using GC/MS with capillary column, HP-5MS (30 m
length, 0.25 mm diameter, 0.25 μm film thickness). Helium was carrier
gas at a flow rate of 1 mL/min and ionization energy 70 eV. The con­
centrations of essential oil were calculated as mg/g FW (fresh weight).
2.6. Determination of antibacterial activities
To investigate the antibacterial activities of laser-treated sprouts
versus untreated ones, T. ammi sprouts were extracted in ethanol and
evaluated by using the disc diffusion method. Five μg of sprout extracts
were loaded on sterilized discs (filter paper) and tested against bacterial
suspensions (106 CFU/mL) that were spread on Muller Hinton agar. The
negative control (ethanol) and sample discs were incubated at 37 ◦ C for
a day on the agar plates. Vernier caliper was used to measure the inhi­
bition zones.
2.7. Statistical analyses
One-Way Analysis of Variance (ANOVA) was conducted using SPSS
statistical package (SPSS Inc., Chicago, IL, USA). Tukey’s Test (P ≤ 0.05)
was used for averages separations. Also, the principle component anal­
ysis (PCA) was performed (XLSTAT software, 2011) to address variety-
specific responses and type-based laser efficiency.
3. Results and discussion
3.1. Enhancement of plant yield and photosynthesis by laser light
Laser light has long been thought to stimulate seed germination and
plant development. When a specific wavelength of laser light is absorbed
by phytochromes, it helps in increasing the seed internal energy by
transforming light energy into chemical energy, which enhances cell
pumping and increases the electro-potential of biological membranes
(Chen et al., 2005a). As a result, the generated energy speeds up cell
division and boosts enzymatic activities like amylase and protease, all of
which result in an increased development (Asghar et al., 2016; Chen
et al., 2005b; Perveen et al., 2011).
In order to investigate such a phenomenon on sprout biomass and
photosynthesis, two types of lasers (He-Ne and He-Cd) were applied to
the seeds of two species of T. ammi (Chakwal and Desi) (Table 1).
Apparently, laser light treatment induced the biomass of both species of
T. ammi sprouts, which was reflected in the fresh and dry weights of
treated groups. The effect of laser light treatment was more obvious in
the case of Desi species than Chakwal one, as both fresh and dry weights
were significantly (p < 0.05) higher, particularly in the treated groups of
Desi than control ones. Similar scenarios were noticeable in the case of
photosynthesis and respiration methods, as the influence of laser light
treatment was significant (p < 0.05) only in Desi species. Regarding laser
type, the greatest impacts were induced by He-Ne laser in almost all
parameters.
M.A. Balkhyour et al. Industrial Crops & Products 170 (2021) 113837

Table 1 antioxidant capacity of that plant (Lee et al., 2016). Therefore, the effect
The effect of seed treatment with helium-neon (He-Ne) and helium-cadmium of seed pre-treatment with laser light on the phenolic and flavonoid
(He-Cd) lasers on the growth, photosynthesis, respiration and concentrations profiles of T. ammi species sprouts was investigated (Table 2). The most
of pigments in sprouts of Trachyspermum ammi Chakwal (TAC) and Trachy­ prevalent phenolic compounds in T. ammi were protocatechuic acid and
spermum ammi Desi (TAD) versus control conditions. The means ± standard p-coumaric acid, however, the two species of T. ammi were poor in
deviations of at least 3 replicates represent these data.
flavonoids. Laser treatment of seeds induced different degrees of
Trachyspermum ammi Chakwal Trachyspermum ammi Desi improvement in the levels of phenolics in T. ammi sprouts, such
Parameters Control He-Ne He-Cd Control He-Ne He-Cd improvement was particularly significant (p < 0.05) in p-coumaric acid.
laser laser laser laser The results of total phenolics and flavonoids were in harmony with that
Fresh weight (g) 8.72 ± 13.53 11.61 10.05 18.24 13.71 of individual phenolic and flavonoid findings, as T. ammi was richer in
0.72d ± ± 1.9c ± 0.35c ± ± total phenolics than total flavonoids. Interestingly, the efficiency of the
1.67b 3.04a 1.77b He-Ne laser was greater than the He-Cd laser in almost all measured
Dry weight (g) 0.68 ± 0.99 ± 0.92 ± 0.73 ± 1.29 ± 1.36 ±
parameters. These results coincide with some pieces of previous research
0.04c 0.073b 0.12b 0.05c 0.23a 0.33a
Photosynthesis 18.32 27.96 24.60 19.34 38.26 30.73 which stated the enhancing effect of laser on the phenolic and flavonoid
(μmol CO2 ± 2.50d ± ± ± 2.92d ± ± profiles in plants such as sunflower seeds (Perveen et al., 2011), soybean
m− 2 s-1) 3.61bc 3.17c 3.96a 6.78b (Asghar et al., 2016), and buckwheat sprouts (Almuhayawi et al., 2021).
Respiration 0.10 ± 0.18 ± 0.13 ± 0.10 ± 0.25 ± 0.19 ± The enhancement in the levels of phenolics and flavonoids in sprouts
(μmol CO2 0.01d 0.02b 0.034c 0.01d 0.032a 0.055b
− 2 -1
m s )
after laser pre-treatment of T. ammi seeds could be attributed to the
Pigments (mg/g FW) elevated net photosynthetic activity. Subsequently, the improved
Chlorophyll a 1.61 ± 1.13 ± 0.99 ± 0.98 ± 1.19 ± 1.08 ± photosynthetic activity boosted the carbon allocation and increased the
0.75a 0.12a 0.11a 0.02a 0.17a 0.13a non-structural carbohydrates, which could be exploited as precursors for
Chlorophyll b 0.37 ± 0.5 ± 0.44 ± 0.43 ± 0.58 ± 0.53 ±
secondary metabolite production (Li et al., 2017). Also, the improve­
0.05c 0.07abc 0.06abc 0.04bc 0.02a 0.04ab
α-Carotene 0.07 ± 0.09 ± 0.08 ± 0.07 ± 0.12 ± 0.11 ± ment in the contents of phenolics and flavonoids could be explained in
0.01c 0abc 0bc 0.02c 0.02a 0.02ab the light of that reported by Aladjadjiyan (2007) who stated that laser
β-Carotene 0.05 ± 0.06 ± 0.05 ± 0.06 ± 0.12 ± 0.11 ± light enhances the energy supply of seeds, which then converted to
0c 0.01bc 0.01c 0c 0.03a 0.03ab chemical energy that accelerates the vital processes and improves the
Lutein 0.17 ± 0.47 ± 0.41 ± 0.23 ± 0.45 ± 0.41 ±
production of secondary metabolites.
0c 0.13a 0.11ab 0.01bc 0.04a 0.06ab
Neoxanthin 0.01 ± 0.02 ± 0.02 ± 0.01 ± 0.02 ± 0.02 ±
0a 0a 0a 0a 0.01a 0.01a
Violaxanthin 0.06 ± 0.12 ± 0.11 ± 0.04 ± 0.11 ± 0.1 ± Table 2
0.01bc 0.02a 0.02ab 0c 0.02a 0.02ab
The effect of seed treatment with helium-neon (He-Ne) and helium-cadmium
Significant variations between means at p < 0.05 are shown by different small (He-Cd) lasers on the phenolic and flavonoid profiles of Trachyspermum ammi
letters (a, b, c) within a row. Chakwal (TAC) and Trachyspermum ammi Desi (TAD) sprouts versus control
conditions. The means ± standard deviations of at least 3 replicates represent
these data.
Consistent with our results, many researchers have previously re­
ported the positive effect of laser light on the growth rate and biomass of Phenolics and Trachyspermum ammi Chakwal Trachyspermum ammi Desi
various plants such as faba bean, maize, Isatis indogotica, alfalfa, flavonoids (mg/ Control He-Ne He-Cd Control He-Ne He-Cd
g FW)
greengram, spring barley, and radish (Chen et al., 2005b; Dziwulska laser laser laser laser
et al., 2006). Gallic acid 0.35 ± 0.64 ± 0.43 ± 0.36 ± 0.39 ± 0.32 ±
The positive effect of laser light on the growth rate of T. ammi sprouts 0.12a 0.21a 0.14a 0.12a 0.13a 0.11a
could be attributed to the enhancement of photosynthetic rate, chloro­ Caffeic acid 0.51 ± 0.96 ± 0.63 ± 0.43 ± 0.73 ± 0.47 ±
0.17a 0.32a 0.21a 0.09a 0.14a 0.16a
phyll content, and respiration as a result of laser treatment (Perveen
Chicoric acid 0.93 ± 1.11 ± 0.88 ± 0.42 ± 0.87 ± 0.57 ±
et al., 2011; Chen et al., 2005a). The high photosynthetic activity 0.31a 0.37a 0.29a 0.1a 0.29a 0.19a
enhanced the biosynthesis of sugars and organic acids, which are re­ Rosmarinic acid 1.21 ± 1.88 ± 1.33 ± 0.48 ± 1.09 ± 0.68 ±
flected in the total biomass of the plant (Li et al., 2017). Moreover, 0.4ab 0.63a 0.44ab 0.16b 0.36ab 0.23b
photosynthetic-produced sugars (mainly glucose) associate with oxygen Protocatechuic 3.86 ± 4.04 ± 3.4 ± 3.05 ± 4.69 ± 3.19 ±
acid 1.29a 1.35a 1.13a 0.5a 1.56a 1.06a
to produce cellular energy, C skeleton and energy required for biosyn­ Quercetin 0.04 ± 0.04 ± 0.03 ± 0.08 ± 0.14 ± 0.07 ±
thesis of bioactive primary and secondary metabolites through dark 0.01b 0.01b 0.01b 0.03ab 0.04a 0.02b
respiration (Bolton, 2009). In this regard, laser treatment enhanced Naringenin 0.05 ± 0.03 ± 0.03 ± 0.01 ± 0.03 ± 0.01 ±
photosynthesis processes (Govil et al., 2006) and upregulated its key 0.02a 0.01abc 0.01ab 0c 0.01abc 0bc
Kaempferol 0.06 ± 0.05 ± 0.05 ± 0.07 ± 0.07 ± 0.06 ±
genes (Qiu et al., 2017). Similarly, we observed a significant (p < 0.05)
0.02a 0.02a 0.02a 0.02a 0.02a 0.02a
increase in chlorophyll b, α-carotene, β-carotene, lutein, and viola­ Luteolin 0.06 ± 0.06 ± 0.05 ± 0.05 ± 0.07 ± 0.04 ±
xanthin pigment productions in both species as a result of laser light 0.02a 0.02a 0.02a 0.02a 0.01a 0.01a
treatment. Such data come in harmony with the aforementioned results Apigenin 0.01 ± 0.07 ± 0.03 ± 0.09 ± 0.06 ± 0.07 ±
of biomass, photosynthesis, and respiration. Likewise, laser treatment 0c 0.02bc 0.01c 0.02a 0.02abc 0.02ab
Rutin 0.01 ± 0.01 ± 0.01 ± 0.02 ± 0.02 ± 0.01 ±
was found to enhance chlorophyll a and b contents in plants like Isatis 0b 0b 0b 0ab 0.01a 0b
indogotica (Chen et al., 2005b), sunflower (Perveen et al., 2011), and Chlorogenic acid 0 ± 0b 0 ± 0b 0 ± 0b 0 ± 0b 0.01 ± 0.01 ±
soybean (Asghar et al., 2016). Additionally, since carotenoids are such 0a 0a
an important part of the photosynthesis process, light quality and in­ p-Coumaric acid 1.0 ± 1.56 ± 1.43 ± 0.66 ± 1.83 ± 1.35 ±
0.06b 0.07a 0.12a 0.09c 0.22a 0.18a
tensity are likely to have an effect on their levels (Wu et al., 2007).
Total phenols 8.93 ± 10.73 8.47 ± 5.1 ± 9.46 ± 6.42 ±
2.98a ± 2.82a 0.42a 3.15a 2.14a
3.58a
3.2. Laser light improved the accumulation of phenolic and flavonoids in Total Flavonoids 0.85 ± 0.92 ± 0.76 ± 1.24 ± 1.05 ± 0.97 ±
T. ammi 0.28a 0.31a 0.25a 0.32a 0.35a 0.32a

Significant variations between means at p < 0.05 are shown by different small
The level of phenolics and flavonoids, in a plant, is a key player in the letters (a, b, c) within a row.

4
M.A. Balkhyour et al.
3.3. Laser light improved the biosynthesis of coumarins, their precursor
metabolites, and associated enzyme activities in T. ammi
It is also well-known that T. ammi spp. are one of the richest natural
sources of coumarins (Matos et al., 2015). Coumarins are a group of
phenolic compounds formed by the linkage between β-pyrone rings and
benzene. Coumarins display a remarkable array of biochemical and
pharmacological activity such as antioxidant, antibacterial, and
anti-inflammatory (Matos et al., 2015). Moreover, the observed high
levels of bioactive p-coumaric acid can be obtained from the basic hy­
drolysis of coumarins. In our study, a variety of coumarins (umbelli­
ferone, scopoletin, xanthotoxin, bergapten, and/or dehydrogeijerin)
were found in T. ammi sprouts. Noticeably, He-Ne laser light treatment
of seeds significantly increased (p < 0.05) the accumulation of all
measured coumarins in sprouts of T. ammi Desi species. While He-Ne
laser treatment significantly improved (p < 0.05) the accumulation of
umbelliferone and scopoletin only in T. ammi Chakwal species. On the
other hand, the effect of He-Cd laser on coumarins was weaker than
He-Ne laser. Given that He-Cd laser induced a significant improvement
to the levels of umbelliferone and bergapten only in Desi, as well as
scopoletin and dehydrogeijerin only in Chakwal (Fig. 1).
5
Industrial Crops & Products 170 (2021) 113837
To understand the biochemical bases of the observed increase in
coumarins level, we subsequently analyzed the biosynthesis pathway of
coumarins, their precursors, and associated enzyme activities, besides,
the effect of laser light treatment on such pathway (Fig. 1). Coumarins
originate from the general phenylpropanoid pathway in higher plants
(Harborne, 1999). They are biosynthesized from phenylalanine via the
shikimic and cinnamic acids (Maeda and Dudareva, 2012). DAHPS is a
crucial enzyme in the biosynthesis of shikimic acid. Shikimic acid is
converted to chorismite, which is a precursor for phenylalanine
biosynthesis. Consequently, PAL catalyzes the conversion of phenylal­
anine to cinnamic acid, which is followed by the production of p-cou­
maric acid from cinnamic acid. The activation of p-coumaric acid to
4-coumaroyl CoA and 4-coumaroyl-CoA to feruloyl-CoA, the precursor
of coumarins, follows (Robe et al., 2020)
At the metabolic level, L-phenylalanine was significantly (p < 0.05)
enhanced in both T. ammi species after seed treatment with laser light.
While cinnamic acid was significantly higher (p < 0.05) in the sprouts of
both species of T. ammi treated with He-Ne laser light than He-Cd laser-
treated and control. Similarly, shikimic acid was also higher in both
species of T. ammi treated with He-Ne laser light than He-Cd laser-
treated and control ones, yet this rise was significant in Chakwal species
Fig. 1. The coumarin biosynthesis
pathway. The effect of seed treatment
with helium-neon (He-Ne) and helium-
cadmium (He-Cd) lasers on the
coumarin biosynthesis pathway in Tra­
chyspermum ammi Chakwal (TAC) and
Trachyspermum ammi Desi (TAD)
sprouts versus control conditions. The
means ± standard deviations of at least
3 replicates represent these data. Sig­
nificant variations between means at p
< 0.05 are shown by different small
letters (a, b, c) above bars. PEP: phos­
phoenol pyruvate; DAHPS: 3-Deoxy-D-
arabino-heptulosonate-7-phosphate
synthase; CM: chorismate mutase.
M.A. Balkhyour et al. Industrial Crops & Products 170 (2021) 113837

only (p < 0.05). Likewise, the significant effect (p < 0.05) of He-Ne laser Table 3
light was noticeable in cinnamic acid values in both species of T. ammi The effect of seed treatment with helium-neon (He-Ne) and helium cadmium
sprouts. Conversely, He-Cd laser did not induce significant increases in (He-Cd) lasers on the concentrations of essential oils in sprouts of Trachy­
it. These findings clarify the weak He-Cd laser effect on the levels of spermum ammi Chakwal (TAC) and Trachyspermum ammi Desi (TAD) versus
coumarins in T. ammi sprouts (Fig. 1). Equally, Wang et al. (2020) re­ control conditions. The means ± standard deviations of at least 3 replicates
represent these data.
ported that exposure to full sunlight (100 %) significantly raised the
concentrations of phenylalanine, cinnamic acid, and p-coumaric acid in Trachyspermum ammi Chakwal Trachyspermum ammi Desi
Essential oils
Asarum heterotropoides. That improvement was attributed to the (mg/g FW) Control He-Ne He-Cd Control He-Ne He-Cd
enhancement in the photosynthetic activity of this plant. laser laser laser laser
In order to further explain these findings, we also determined five α-Piene 0.63 ± 0.75 ± 0.62 ± 0.48 ± 0.58 ± 0.5 ±
enzymatic activities that are included in the pathways of shikimic acid, 0.08ab 0.1a 0.08ab 0.06b 0.07ab 0.07b
L-phenylalanine, and cinnamic acid. Regarding PAL, we found signifi­ α-Thujene 8.86 ± 10.63 8.76 ± 6.6 ± 7.93 ± 6.91 ±
1.29ab 1.28ab 1.08b 1.29ab 1.13b
cant (p < 0.05) increases in its activities in both species of T. ammi ±
1.55a
sprouts treated with He-Ne laser. Whereas He-Cd laser caused a signif­ Sabiene 8.92 ± 10.71 8.82 ± 6.82 ± 8.19 ± 7.14 ±
icant (p < 0.05) effect in only Desi species. While 4CL enzymatic activity 1.21ab ± 1.2ab 0.86b 1.03ab 0.9b
was significantly (p < 0.05) improved in both species of T. ammi treated 1.45a
by either type of laser light. On the contrary, C3H enzymatic activity was β-Myrceme 14.99 ± 17.99 14.81 11.4 ± 13.68 11.93
2.07ab 1.46b
significantly (p < 0.05) boosted only in the case of Desi species treated
± ± ± ±
2.48a 2.04ab 1.75ab 1.53b
with either type of laser light. While C4H activity was significantly D-Limonene 3.6 ± 4.32 ± 3.56 ± 2.67 ± 3.2 ± 2.79 ±
enhanced (p < 0.05) only in Chakwal species treated with either He-Ne 0.53ab 0.64a 0.53ab 0.47b 0.57ab 0.5b
or He-Cd laser. Furthermore, DAHPS enzymatic activities were signifi­ α-terpinene 51.53 ± 57.59 47.42 35.72 ± 46.98 38.08
7.16ab 1.7b
cantly increased (p < 0.05) in both species of T. ammi treated with laser ± ± ± ±
8.01a 6.59ab 4.26ab 4.99b
light, where He-Ne laser had a more potent effect than He-Cd one Cymene 41.92 ± 40.7 ± 33.51 28.14 ± 34.13 30.2 ±
(Fig. 1). The obtained results of enzymatic activities associated with the 5.99a 5.68ab ± 3.01b ± 3.86ab
coumarin biosynthesis pathway coincide with the aforementioned re­ 4.68ab 3.78ab
sults of coumarins and their precursors in terms of the most effective α- terpinolene 11.78 ± 14.14 11.64 7.84 ± 11.61 9.25 ±
1.68ab 0.93b 1.37b
laser type and most responsive species. Correspondingly, high sunlight
± ± ±
2.02a 1.66ab 0.82ab
treatments of Asarum heterotropoides enhanced the enzymatic activities Thymol 1.31 ± 1.57 ± 1.29 ± 0.95 ± 1.52 ± 1.04 ±
associated with the pathways of shikimic acid and cinnamic acid, 0.18a 0.22a 0.18a 0.06a 0.48a 0.13a
namely DAHPS, 4CL, 4CH, and PAL (Wang et al., 2020). Carvacrol 0.78 ± 0.93 ± 0.77 ± 0.55 ± 0.79 ± 0.61 ±
0.11ab 0.13a 0.11ab 0.05b 0.07ab 0.09b
α-Thujone 0.26 ± 0.32 ± 0.26 ± 0.17 ± 0.47 ± 0.21 ±
3.4. Laser light boosted the levels of essential oils in T. ammi 0.04a 0.04a 0.04a 0.03a 0.27a 0.03a
Tricyclene 0.06 ± 0.08 ± 0.06 ± 0.05 ± 0.08 ± 0.05 ±
In higher plants, p-coumaric acid and cinnamic acid are important 0.01ab 0.01a 0.01ab 0.01b 0.02ab 0.01ab
metabolites for the biosynthesis of essential oils (Wang et al., 2020). β-Phellandrene 0.06 ± 0.03 ± 0.03 ± 0.04 ± 0.03 ± 0.02 ±
0.01a 0bc 0bc 0b 0.01bc 0c
High light treatment has been found to enhance the levels of phenyl­ Humuline 0.03 ± 0.02 ± 0.02 ± 0.02 ± 0.03 ± 0.02 ±
propanoid compounds and essential oils in Asarum heterotropoides var. 0ab 0ab 0ab 0b 0.01a 0b
mandshuricum fibrous root (Wang et al., 2020). Therefore, the laser light
Significant variations between means at p < 0.05 are shown by different small
effect on the essential oil content of T. ammi was addressed (Table 3).
letters (a, b, c) within a row.
α-Terpinene and cymene were the most common essential oils in
T. ammi, followed by β-myrceme and α- terpinolene. In this concern,
of T. ammi has not been recorded yet. So, we herein evaluated the effect
T. ammi fruit has been found to yield 25 % oleoresin containing 12 %
of both He-Ne and He-Cd lasers on the antibacterial activities of two
volatile oil including thymol, para-cymene, γ-terpinene, and α- and
species of T. ammi sprouts (Fig. 2). Under control conditions, similar
β-pinene (Nagalakshmi et al., 2000). Moreover, Choudhury et al. (1998)
inhibition rates were recorded by Chakwal and Desi species against
reported that the principal oil constituents of T. ammi are carvone,
Staphylococcus aureus and Listeria monocytogenes, while Desi showed
limonene, and dillapiole (46, 38, and 9%, respectively). Chakwal species
better inhibition rates against Bacillus cereus and Salmonella spp. than
was richer in essential oils than Desi species under control conditions.
Chakwal. Seemingly, T. ammi seed pre-treatment with He-Ne laser
Noticeably, the treatment with laser light led to different degrees of
enhanced all measured antibacterial activities in both species of T. ammi
elevations in the concentrations of essential oils in T. ammi, yet these
sprouts, whereas He-Cd laser did not affect the antibacterial activities.
improvements on a few occasions were significant (p < 0.05). Given that
Interestingly, the He-Ne laser significantly improved (p < 0.05) the
cinnamic acid and p-coumaric acid are vital metabolites for essential oil
antibacterial effect of both species of T. ammi against Staphylococcus
biosynthesis in higher plants (Wang et al., 2020), the improvement in
aureus and the antibacterial activity of Chakwal against Listeria mono­
the concentrations of essential oils in laser-treated sprouts could be
cytogenes (Fig. 2). The improvement in the antimicrobial effect of
attributed to the improved shikimic acid and cinnamic acid pathways.
T. ammi sprouts treated with laser light could be ascribed to the eleva­
Comparably, high light exposure enhanced the levels of phenyl­
tion in the levels of coumarins and related precursor metabolites, which
propanoid precursor compounds and consequently the essential oil
were reported as potent antibacterial, antiviral and antifungal agents
contents in Asarum heterotropoides var. mandshuricum fibrous root (Wang
(Hassan et al., 2016). Additionally, the increase in the essential oil
et al., 2020).
content as a result of He-Ne laser could be another reason. In this
concern, it was reported that using 0.5–4 g/mL of T. ammi essential oil to
3.5. Laser light boosted the antibacterial properties of T. ammi
prevent infections caused by Staphylococcus aureus, Escherichia coli,
Enterococcus, and Pseudomonas aeruginosa was effective (Moein et al.,
The antibacterial activity of T. ammi was previously reported (Gan­
2015). The authors attributed the antimicrobial activity of T. ammi
domi et al., 2014; Moein et al., 2015). Traditionally, T. ammi seeds have
species to the presence of thymol and carvacrol. Thymol destroys the
been used in many countries for the protection of food against microbial
bacteria which are resistant to the most common third-generation an­
spoilage (Dubey and Kashyap, 2015). However, to our knowledge, the
tibiotics, as well as multi-drug resistant bacteria (Nagalakshmi et al.,
effect of seed pre-treatment with laser light on this activity in the sprouts

6
M.A. Balkhyour et al. Industrial Crops & Products 170 (2021) 113837

Fig. 2. The effect of seed treatment with

helium-neon (He-Ne) and helium-cadmium
(He-Cd) laser light on the antibacterial proper­
ties against A) Staphylococcus aureus, B) Listeria
monocytogenes, C) Bacillus cereus and D) Salmo­
nella spp. of Trachyspermum ammi Chakwal
(TAC) and Trachyspermum ammi Desi (TAD)
sprouts versus control conditions. The means ±
standard deviations of at least 3 replicates
represent these data. Significant variations be­
tween means at p < 0.05 are shown by different
small letters (a, b, c) above bars.

2000). Furthermore, depending on their concentration, phenolic com­
pounds like thymol and carvacrol are believed to be either bactericidal
or bacteriostatic agents (Singh et al., 2004).
3.6. Species-specific responses to laser light
In order to investigate the variations among the two varieties
(Chakwal and Desi) as a result of laser light treatment, as well as the
efficiency of laser type (He-Ne versus He-Cd), we conducted a principal
component analysis (PCA) to all measured parameters (Fig. 3). The PCA

Fig. 3. Principle component analysis (PCA) of the average of all measurements in the two species of Trachyspermum ammi sprouts grown from either untreated seeds
or seeds exposed to laser treatment. The PCA is based on Z-score normalized data. Red, blue and green circles represent control, He-Ne laser and He-Cd laser
treatments, respectively. The circles were arbitrary. The means of at least 3 replicates represent these data.

7
M.A. Balkhyour et al.
shows two dimensions, dimension 1, which represents the effect of
treatment with laser on the measured parameters, confirms that 44.1 %
of the determined parameters were significantly affected by laser
treatment. On the other hand, dimension 2, which represents the sprout
species-specific responses, confirms that 31 % of the measured param­
eters significantly varied among the two species. Additionally, PCA
clearly indicated that He-Ne laser had a significantly greater efficiency
than He-Cd laser. Furthermore, Desi species showed significantly greater
responses to laser treatment than Chakwal. The ontogeny and species
diversity among the two species of T. ammi sprouts could explain the
differences in responses, which agrees with that previously stated by
Zhang et al. (2006) and Chaudhary et al. (2016).
4. Conclusions
We conclude that He-Ne laser irradiation could significantly enhance
the biomass, photosynthesis, and pigment accumulation in two varieties
of ajwain sprouts. Besides, He-Ne laser accumulated health-promoting
compounds including essential oils and coumarins in ajwain sprouts.
The induced antibacterial activities in He-Ne laser-treated sprouts might
be attributed to the elevation in the levels of phenolics, flavonoids,
coumarins and related precursor metabolites, and could make ajwain
sprouts, particularly the Desi variety, a valuable industrial crop. Our
findings could support the use of He-Ne laser irradiation in ajwain
sprouts as a promising method for increasing coumarin and essential oil
production.
CRediT authorship contribution statement
Abdelrahim H.A. Hassan and Mansour A. Balkhyour: planned
and designed the research. Abdelrahim H.A. Hassan and Mansour A.
Balkhyour: performed the experiments. Abdelrahim H.A. Hassan,
Abdurazag M. S. Tammar, Ahmed Saleh Summan and Mansour A.
Balkhyour: analysed the data. Abdelrahim H.A. Hassan and Mansour
A. Balkhyour: contributed to the reagents/chemicals. Abdurazag M. S.
Tammar and Ahmed Saleh Summan: Written the draft version of the
manuscript. Abdelrahim H.A. Hassan and Mansour A. Balkhyour:
revised and finalized the manuscript. All authors approved the submit­
ted version of the manuscript.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
This research work was funded by the Institutional Fund Projects
under grant number (IFPHI-206-155-2020). Therefore, the authors
gratefully acknowledge the technical and financial support from the
Ministry of Education and King Abdulaziz University, DSR, Jeddah,
Saudi Arabia.
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