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Phenolic acid profiling and antiglycation studies of leaf and fruit extracts of
tyrosine primed Momordica charantia seeds for possible treatment of diabetes
mellitus

Article in Pakistan Journal of Pharmaceutical Sciences · November 2018

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Phenolic acid profiling and antiglycation studies of leaf and fruit
extracts of tyrosine primed Momordica charantia seeds for possible
treatment of diabetes mellitus

Nabila Farah1, Shazia Anwer Bukhari1, Muhammad Ali2,

Syed Ali Raza Naqvi3* and Saqib Mahmood4
1
Department of Biochemistry, Government College University, Faisalabad, Pakistan
2
Department of Zoology, Government College University, Faisalabad, Pakistan
3
Department of Chemistry, Government College University, Faisalabad, Pakistan
4
Department of Botany, Government College University, Faisalabad, Pakistan

Abstract: The increasing risk of variety of fatal diseases including diabetes mellitus is imposing serious challenge to
chemist, biologists and clinicians. Due to the side effects of the chemotherapy, worldwide it is thinking that phyto-
medicine are more effective to cope continuously increasing risk of fatal diseases without any side effect. Seed priming
is a strategic pre-sowing semi-bioengineering technique which has ability to improve the growth rate and biologically
active compounds in short time. Among seed priming techniques, tyrosine seed priming most frequently used because
amino acids provide best growth media for nutritional food crops. Seeds of Momordica charantia were subjected to
the pre-sowing tyrosine solution. Different growth parameters including growth emergence rate, seedling vigor, growth
and weight of root, shoot and leaf were studied. The results showed positive effect on Momordica charantia seed growth
and phenolic acids production i.e. ferulic acid – 43.95 ppm and sinapic acid – 18.39 ppm. The antiglycation assay
showed 23.45±1.23% antiglycation activity of primed-seed fruit extract as compare to control seed fruit extract
(0.87±0.03%). On the basis of the results, it is concluded that tyrosine primed seed fruit extract could effectively be
further tested for pre-clinical and clinical studies to manage diabetes mellitus disease.

Keywords: Tyrosine priming, antiglycation activity, diabetes mellitus, antioxidants, phenolic acids.

INTRODUCTION and canning processing is used to maintain the quality of

bitter gourd for a long time (Kumar et al., 2016). It is
Seed priming is a strategic pre-sowing semi- effectively used for the treatment of metabolic disorder,
bioengineering technique used for the improvement of infections and diabetes mellitus diseases that render the
germination speed, uniform growth and chemical attention of scientists to explore other medicinal
composition (Khalil et al., 2010). In recent years lot of utilizations (Kumar et al., 2016). Chemical priming at
scientific exercises are being done to explore the different stages of plant life was previously investigated
medicinal values of flora all over the world which and was found positive impact on biochemical and
explored numerous chemicals for therapy of infectious biological profile of plants which in some cases appear to
and malignant diseases, promisingly. However, the improve antiglycation activity (Kosseva, 2017), however,
isolation rate of these medicinal compounds from the the technique uses variety of sets of chemical
plants depends on their production in plants. To manage compositions which produce side effect along with the
the ever-increasing cases of infectious and malignant aforementioned roles (Zhang. et al., 2015).
diseases it is priority order challenge for chemists,
biochemists, technologists and biologists to develop semi- The aim of this study is to investigate antiglycation
bioengineering techniques to improve the uniform growth activity of Momordica charantia by seed priming with
rate with increased production of biologically active tyrosine solution Tyrosine (4-hydroxyphenylalanine) is
compounds. one of the 20 standard amino acids, has its role in protein
synthesis, photosynthesis, signal transduction processes. It
Bitter gourd (Momordica charantia) is a medicinal plant is hypothesized that the priming with naturally occurring
belonging to family Cucurbitaceae and is a good source tyrosine amino acid will enhance the antiglycation
of minerals, vitamins, protein and biologically active activity of the bitter gourd remarkably.
ingredients. Different processing techniques are in
practice to make the bitter gourd in use for long time in MATERIALS AND METHODS
order to take the advantage of its mineral profile and
biological active ingredients. Minimal processing, drying Collection of sample
Taxonomically identified and confirmed seeds of
*Corresponding author: e-mail: [email protected] Momordica charantia were obtained from Ayub
Pak. J. Pharm. Sci., Vol.31, No.6(Suppl), November 2018, pp.2667-2672 2667
Phenolic acid profiling and antiglycation studies of leaf and fruit extracts of tyrosine primed

Agriculture Research Institute (AARI), Faisalabad, solvent A (100%) from 30-35 minutes. Twenty microliter
Pakistan. Germination experiment was conducted in the sample was loaded through HPLC sample port using
fields of Government College University, Faisalabad. microsyringe. Detection was carried out with built in UV-
visible spectroscopic detector at 275 nm. Identification of
Seed priming with tyrosine and germination parameters phenolic acids was performed by correlating their relative
studies retention times with those of standard mixture
The Momordica charantia seeds were primed with water chromatogram. The amounts of individual compounds
which taken as control (non-primed seeds) and tyrosine were measured on the basis of the area under peak,
solution of three different concentrations i.e. 0.1%, 0.2% relative to the corresponding standard phenolic acid
and 0.3% for 12 hours (primed-seeds). After priming, concentration peak area relation.
seeds were washed with distilled water, covered with
filter paper and left to dry in air. Primed and non-primed Antiglycation activity assay
seeds were sowed in field of botanical garden at GC Took 1g sample and add 1.5mL methanol (50%),
University, Faisalabad, at different distances. Experiments centrifuged at 1500 rpm for 10 min. Now supernatant
were designed with three replicates. Seed germination used for the analysis of antiglycation activity following
data were recorded after 7th day of sowing the seeds using the procedure reported previously (Matsuda et al., 2003).
following parameters; Briefly, the reaction mixture comprises of 150µL D-
glucose, 150µL bovine serum albumin (BSA in 1mL
 Germination percentage was calculated at the end of sodium phosphate buffer pH 7.2) and 150µL samples
7th day by using the formula given below reported were incubated at room temperature for 7 days. The
earlier (Ijaz et al., 2012). absorbance was measured using a spectrophotometer at a
Gp = (Ng/Np) ×100 wavelength of 440 nm. The reaction mixture without D-
“Ng” is the last number of emerged seeds and “Np” is glucose was used as a blank solution. Measurements were
the total number of seeds sown. performed in triplicate. The percent inhibition of
glycation was calculated with following expression:
 Mean growth time (MGT) was calculated according to
as follow. Inhibition of Absorbance of control – Absorbance of sample
×10
glycation (%) = Absorbance of control
MGT = ∑(Dn) / ∑n
“
n” is the number of seeds germinated on day D, and D
is number of days counted from the beginning of the STATISTICAL ANALYSIS
germination test. Seedling vigor was calculated by All determination was made in complete randomize
following the formula described earlier (Vashisth and experiments. The presence or absence of significant
Nagarajan, 2010). difference among different factors was as curtained with
Vigor index I= germination (%) × seedling length (root the analysis of variances (ANOVA). The means were
+ shoot) compared to final significant difference using LSD testing
Vigor index II= germination (%) × seedling dry weight the cases of all. Overall interaction of all the factors was
(root + shoot) checked for significance using computer software
COSTAS (Cohort software, 2003).
 Similarly, primary root length, shoot length, leaf area,
total fresh weight of root, shoot and leaf was also RESULTS
measured.
Tyrosine priming effect on seed growth
Phenolic profiling through high-performance liquid Growth emergence rate and seedling vigor
chromatography The rate of final growth emergence of primed Momordica
HPLC was done for the investigation qualitative and charantia seeds was studied at different tyrosine
quantitative phenolic acids profile following the concentrations such as 0.1, 0.2 and 0.3% solution. The
procedure reported by Hussain et al., (2012) with slight growth emergence rate was calculated as mean of
modification. Varian HPLC using ODS (C18) reversed triplicate experiments and standard deviation (mean ±
phase column was used for the identification of phenolic S.D) as shown in fig. 1a. The significant difference in
acids present in extracts. In order to separate different treatment using different tyrosine solutions was calculated
phenolic acids two solvents i.e. solvent A (70% at p value <0.05. The mean growth emergence rate using
acetonitrile in methanol) and solvent B (0.5% glacial different parameters i.e. root, shoots and leaves,
acetic acid) were used as mobile phase with a constant calculated from the data obtained from growth emergence
flow rate of 1 mL/min in gradient mode. The gradient rate as shown in the fig. 1b. Similarly, seedling vigor also
scheme is as follow; solvent A (100%) for first 5 minutes, studied by treating the Momordica charantia seeds with
solvent A (95%) and solvent B (5%) for 5-10 minutes, 0.1%, 0.2% and 0.3% tyrosine solution. The seedling
solvent A (5%) and solvent B (95%) for 10-30 minutes, vigor of primed-seeds was compared with control groups.
2668 Pak. J. Pharm. Sci., Vol.31, No.6(Suppl), November 2018, pp.2667-2672
 Nabila Farah et al

Fig. 1: Treatment of Momordica charantia seeds with Tyrosine solution of different concentration and control solution.
Where a: final growth emergence rate, b: mean emergence rate and c: seedling vigour

Table 1: Phenolic acid profile of control and tyrosine primed Momordica charantia seed leaf and fruit extracts using
high performance liquid chromatography analysis

Concentration (ppm)
Retention
S No. Phenolic acids Control leaf Tyrosine treated Control fruit Tyrosine treated
time (min)
extract leaf extract extract fruit extract
1 Querecetin 2.82 3.77 2.73 2.96 2.22
2 Benzoic acid 14.65 1.64 × × ×
3 Chlorogenic acid 15.87 5.19 0.95 × ×
4 Syringic acid 16.55 3.31 × × 2.61
5 m-Coumeric acid 19.73 5.81 × × ×
6 p-Coumeric acid 17.64 X 0.77 × ×
7 Cinamic acid 24.71 16.25 15.29 11.58 ×
8 Ferulic aid 21.83 × 12.38 14.78 43.95
9 Sinapic acid 26.19 × × × 18.39
10 Vanillic acid 13.87 × × × 24.36
The results obtained from these investigations are shown phenolic acid profiling of control leaf and fruit extract are
in fig. 1c. shown in the HPLC chromatograms fig. 3 chromatogram
“a” and “c”, respectively. While in the fig. 3 the
Growth and weight study of root, shoot and leaf chromatograms “b” and “d” shows the HPLC results of
The growth attribute parameters such as growth of roots, primed-seeds leaf and fruit extracts, respectively. The
shoots and leaves, and weight of roots, shoots and leaves quantification was carried out by calculating the area
of control and primed-seeds were investigated. The results under each peak and comparing the standard phenolic
of these two parameters studying using roots, shoots and acid peaks as shown in the table 1.
leaves are shown in fig. 2(a-f). The effect of tyrosine
priming was expressed in term of significant difference (p
<0.05); the study points with similar signs, † or ‡,
indicates non-significant difference while with different
signs indicates significant difference and consequently
significant effect of treatment.

HPLC analysis for phenolic profiling

The qualitative and quantitative HPLC analysis of control
and primed-seeds leaf and fruit extracts was carried out
using gradient elution system. Prior to the sample elution,
the solution of standard phenolic acids i.e. querecetin,
benzoic acid, chlorogenic acid, syringic acid, m-coumaric
acid, p-coumaric acid, cinamic acid, ferulic acid, sinapic
acid and vanillic acid were eluted and leaf and fruit
extracts of control and primed-seed were also eluted Fig. 2: Effect of tyrosine seeds priming upon growth
under same conditions of temperature, pressure, flow rate attribute of Momordica charantia L. yield; a) root
and solvent system. The qualitative information about the growth, b) shoot growth, c) leaf area, d) root weight, e)

Pak. J. Pharm. Sci., Vol.31, No.6(Suppl), November 2018, pp.2667-2672 2669

Phenolic acid profiling and antiglycation studies of leaf and fruit extracts of tyrosine primed

Fig. 3: HPLC analysis of controlled and primed-seed sample; a) controlled leaf extract sample, b) primed-seed leaf
extract, c) controlled fruit extract, and d) primed-seed fruit extract analysis of Momordica charantia L. for the phenolic
profile.
shoot weight and f) leaf weight different signs “† and ‡” The effect of tyrosine seed priming on growth was studied
show significant difference between the two results. using different concentrations of tyrosine amino acid i.e.
0.1, 0.2, and 0.3% solution. The results indicate that 0.3%
Antiglycation study solution of primer enhanced the growth rate of the seeds
Antioxidants have ability to show antiglycation potential significantly (p<0.05). The lower concentrations didn’t
and consequently the diabetes mellitus. As the primed- show the significant effect as compared to the control
seeds fruits showed promising increase in phenolic acid growth, however, the mean growth rate showed 0.1%
production – so the primed-seed fruit extracts was used to solution is more compatible (p<0.05) to gain uniform
study the antiglycation activity comparing to control fruit speed growth rate. The seedling vigor i.e. the strength of
extract activity. The results of the study are shown in fig. the growing plant, was more affected by 0.2 and 0.3%
4. The increased antiglycation level (23.45±1.23%) was tyrosine solution (p<0.05) as represented in fig. 1. The
noted with tyrosine primed-seed fruit extracts; which was priming effect was also further investigated using growth
significantly (p<0.05) higher than control seed fruit and weight parameters of root, shoot and leaf. In all cases
extract (0.87±0.03%). except root growth the priming effect remained
significant (p<0.05) as shown in fig. 2. The reverse effect
DISCUSSION was recorded in case of growth of root, where the growth
of root reduced significantly (fig. 2a). The similar effect
This study aimed to test the growth and phenolic was noted using the tyrosine for priming maize seeds
composition of Momordica charantia (a well-known plant (Mahmood et al., 2017).
for its medicinal values in broad range) by priming its
seeds using tyrosine amino acid solution prior to sowing. The basic aim of priming effect was to sort out the
The selection of tyrosine amino acid priming play criticle phenolic profiling and antiglycation potential of the
role on plant physiology and biochemistry in a number of controlled and primed-seed growing plants. The true fig.
ways; they act as buffers, synthesize other organic of phenolic composition with-out and with primed-seed
compounds like proteins, vitamins, enzymes, terpenoids growth plant was analyzed using state of the art HPLC
(El-Aziz, et al., 2007). It involves the stimulation of technique. The phenolic profile was assessed qualitatively
certain biochemical processes of seeds that play a key role and quantitatively by running standard phenolic acids
in dormancy breakdown and mobilization of reserved (querecetin, benzoic acid, chlorogenic acid, syringic acid,
food of seeds. It also includes better enzymatic activity m-coumeric acid, p-coumeric acid, cinamic acid, ferulic
leading towards the early emergence of the embryonic acid, sinapic acid and vanillic acid) prior to controlled and
part during germination with better synchrony. Tyrosine primed sample (leaf and fruit extracts) through HPLC
as priming amino acid has been reported for improved under similar conditions of temperature, pressure, solvent
growth of maize in adverse environmental conditions and system and flow rate. The results showed dual effect on
medicinal plant Trachyspermum ammi L. under normal phenolic profile i.e. suppression effect in case of primed-
field conditions (Mahmood et al., 2017). seed leaf and up-lifting effect in case of primed seed fruit.
The leaf is commonly less known for phenolic
2670 Pak. J. Pharm. Sci., Vol.31, No.6(Suppl), November 2018, pp.2667-2672
 Nabila Farah et al

compounds; however the fruits are considered the major that act as a precursor of phenylpropanoid pathway
source of phenolic components of medicinal plants. through which majority of medicinal plant secondary
metabolites including phenolics produces (Kallscheuer, et
al., 2017).

Natural products with antioxidant activity are often strong

antiglycating agents (Ghous, et al., 2015). Glycation, as
reported, is the major pathway to diabetes mellitus
(Neves, 2013). The diabetes mellitus can be avoided
through intake of antioxidant foods. The enhanced
phenolic acid concentration in primed-seed fruit extract
was subjected to antiglycation assay and it was found that
the fruit extract showed credible strength to suppress
glycation process. The controlled sample showed 0.87±
0.03% antiglycation activity while tyrosine primed-seed
fruit extract showed 23.45±1.23% antiglycation activity;
which was significantly (p<0.05) higher than control seed
fruit extract.
Fig. 4: Effect of tyrosine seeds priming upon
antiglycation activity of Momordica charantia L.- CONCLUSION
different signs “† and ‡” show significant difference
between the two results Tyrosine is a precursor of phenylpropanoid pathway
which is responsible for the production of plant’s
In present study phenolic acids; querecetin, benzoic acid, secondary metabolites, especially phenolic acid. Our
chlorogenic acid, syringic acid, m-coumeric acid, and study revealed the positive effect of tyrosine priming on
cinamic acid was found in controlled leaf extracts in the growth rate of medicinally positive plants and
range of (1.64 - 16.25 ppm) while querecetin, chlorogenic consequently on the enhanced production of phenolic
acid, p-coumeric acid and cinamic acid was found acids. From this study we concluded that seed priming
relatively in less concentration as compared to the with naturally occurring amino acid have potential to
controlled leaf extract sample. The p-coumeric acid and grow plants in uniform speed with increased
ferulic acid was not detected in controlled but primed- concentration of biologically active components that can
seed leaf extract showed 0.77 and 12.38 ppm, be isolated in good quantity to cope variety of
respectively. But in case of fruit extract analysis; the malignancies. At a spot, the difference in antiglycation
controlled extract sample showed querecetin, cinamic activity of controlled and primed extract reveals the
acid and ferulic acid 2.96 ppm, 11.58 ppm and 14.78 strategy is fruitful for enhancing the antiglycation activity
ppm, respectively. The primed-seed fruit extract sample, of medicinal plants and consequently to manage the rate
in contrast, showed querecetin-2.22 ppm, syringic acid - of appearance of diabetes mellitus. The data and approach
2.61 ppm, ferulic acid-43.95 ppm, sinapic acid-18.39 ppm of this study can also be used to further studies and pre-
and vanillic acid-24.36 ppm. The major breakthrough was clinical trials using animal model before jumping to
seen in case of ferulic acid, as it was detected 14.78 ppm clinical study.
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