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Antioxidant activity of Flemingia
praecox and Mucuna pruriens
and their implications for male
fertility improvement
Shravan D. Kumbhare 1, Sanghadeep S. Ukey
1,2 & Dayanand P. Gogle
1,3*
Globally, 15–24% couples are unable to conceive naturally and 50% of cases of this problem are due
to infertility in males. Of this, about 50% of male infertility problems are developed due to unknown
reasons called as idiopathic infertility. It is well established that, reactive oxygen species (ROS) have
negative impact on male fertility and are involved in 80% of total idiopathic male infertility cases.
Medicinal plants are considered as an alternative approach for mitigating the health problems. The
plants with good antioxidant capacity can improve the male infertility symptoms generated by ROS.
Such medicinal plants can be used to alleviate the symptoms of male infertility with their diverse
phytoconstituents. Mucuna pruriens is a well-accepted herb, with its seeds being used to improve
the male fertility in various ways and one of the ways is by eliminating the ROS. In our eld survey,
another plant, Flemingia praecox, although less known, its roots are used in all problems related
to the male fertility by tribal people of the Gadchiroli district of Maharashtra, India. The study was
conducted to determine in vitro antioxidant potential of F. praecox and compared the results with
the well-established male fertility improving plant M. pruriens with special emphasis on medicinally
important roots of F. praecox and seeds of M. pruriens. The objective of the study was investigated by
studying their total phenol (TPC) and avonoid (TFC) content, antioxidant parameters (DPPH, FRAP,
ABTS, DMPD, β-carotene bleaching and TAA) and nally DNA damage protection capacity of the
plant extracts was studied. The plant parts used for the medicinal purposes have been investigated
along with other major parts (leaves, stem and roots of both the plants) and compared with synthetic
antioxidants, BHA, BHT and ascorbic acid. Moreover, the inhibition of two male infertility enzyme
markers, PDE5 and arginase by F. praecox root and M. pruriens seed extract was also studied in vitro.
The results showed that F. praecox possesses higher antioxidant activity than M. pruriens in the
majority of studies as observed in TFC, DPPH, TAA, ABTS and DMPD assays. However, M. pruriens
seeds showed best results in TPC, FRAP and DNA damage protection assay. F. praecox root extract
also gave better PDE5 inhibition value than M. pruriens seeds. This study will help to establish the
authenticity of F. praecox used by tribal people and will encourage its further use in managing the
male infertility problems.
It is evident from previous large scale surveys that sperm count had declined by 50–60% globally during the last
60-year1–3. Male infertility is associated with greater incidence of cancer4, obesity, diabetes5, metabolic syndrome6
and also with mortality and can even cause problems in the health of future progeny5. erefore, in a greater
perspective, male infertility should not be seen only as a medical condition aecting fertility, but also general
health and wellbeing7. e problem of male infertility is heterogeneous in origin which may be the consequence
of genetic or environmental factors or both. e genetic factor includes, microdeletions in Y-chromosome, auto-
somal deletions, X-linked gene copy number variations, mutation in Cystic Fibrosis Transmembrane Conduct-
ance Regulator (CFTR) gene, defects in DNA repair mechanisms, etc.8. Other factors contributing to this problem
includes environmental or occupational exposure to toxicants, lifestyle like smoking, alcohol consumption,
drugs, psychological stress9 and recreational drugs which acts at the level of hypothalamic–pituitary–gonadal
OPEN
1Post Graduate Teaching Department of Botany, RTM Nagpur University, Nagpur 440033, India. 2Department
of Botany, Lokmanya Tilak College, Yavatmal 445304, India. 3Post Graduate Teaching Department of Molecular
Biology and Genetic Engineering, RTM Nagpur University, Nagpur 440033, India. *email: dr.dayanand.gogle@
nagpuruniversity.nic.in
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axis or directly on spermatogenesis consequently causing infertility10. However, in most cases the causes of
male subfertility are poorly understood or not known, called idiopathic male infertility11. Recently, it has been
reported that the epigenetic modications, like abnormal DNA methylation, small non-coding RNA, histone tail
modication8, single nucleotide polymorphism12, etc. in reproduction-related genes are responsible factors for
idiopathic male infertility. However, many of these genetic problems are linked with antioxidant or ROS genes12
which might inuences the critical balance between antioxidant and ROS.
ROS are one of the most closely associated factors involved in deciding the male infertility. ROS or the
free radicals (FR) are the molecules with at least one unpaired electron. It is generated as a result of oxygen
metabolism. e unpaired electrons make ROS a highly reactive and damaging chemical13. Low levels of ROS are
required in various events of fertilization13 however, its excessive production, because of any reason and if it is not
counterbalanced by body’s own antioxidant defences like superoxide dismutase, catalase, glutathione peroxidase
and glutathione, etc.14 then it will lead to oxidative stress (OS). Consequently, it will cause oxidative damage to
spermatozoa by increasing lipid peroxidation in its plasma membrane and thus alter the sperm functioning15.
About 80% of the idiopathic infertile male16,17 and 30–40% of males with known causes has been reported to have
elevated levels of ROS called as male oxidative stress infertility (MOSI)18. is oxidative stress will results in the
protein, lipid and DNA damage in and around sperm atmosphere resulting in decline of fertility. e good thing
about OS is that it can be reversed by using oral antioxidants and thus provides a good opportunity for treatment9.
e plant based antioxidants can become a good alternative to mitigate the problem of MOSI. e major
antioxidant compounds in the plants are phenolics and avonoids which work by eliminating and preventing
the production of ROS19. Phenolic compounds have potential to scavenge major ROS and FR by dierent ways
(Fig.1). Moreover, the chemical structure of phenolics is more crucial than their concentration as it determines
the extent of their absorption in the plasma20. Flavonoids, in the same line, although having the better antioxi-
dant potential than phenols21 but due to their low absorption through intestinal route it was thought to work in
improving the male fertility in dierent ways. It is evident that avonoids improve male fertility preferably by
modulating the cell signalling pathways and improving22–26 (Fig.2). Hence, the plants with high concentration
of dierent phenolic compounds including avonoids can be used as a good source of antioxidants to alleviate
the problem of male infertility.
M. pruriens is a well-recognized plant and traditionally been used for improving the male fertility. It improves
male fertility by reducing ROS level, restoring mitochondrial membrane potential, regulating apoptosis27, con-
trolling unspecic generation of ROS28, reactivating the antioxidant defense system, managing stress29, reducing
lipid peroxidation30, acting on hypothalamus–pituitary–gonadal (HPG) axis and increasing levels of hormones28.
Moreover, it is also thought that the male specic hormone production is assisted by presence of an active compo-
nent, levodopa in its seeds28. However, a study showed that, apart from levodopa, other more superior bioactive
components must be present in its seeds31.
Genus Flemingia consists of 44 species and two varieties that are mostly distributed in old world tropics32. In
India, it is represented by 27 species and one variety33. e genus Flemingia is not only known for its high concen-
tration of avonoids but also contains its good diversity including avones, avanones, isoavones, isoavanone,
chalcones, dihydrochalcones, avonols, santalin avones, avanol, chromone and diavone34. Traditionally, genus
Flemingia have been used in the treatment of diseases like epilepsy, insomnia, ulcer, pain, swelling and regardless
of a long traditional use of some species, this genus has not been explored properly35. However, the selected taxon
for the study, F. praecox var. robusta (F. praecox hereaer) is endemic to India and has been reported in various
parts of India36,37 and its phytochemical study was not done before. Interestingly, the traditional medicinal prac-
titioners in Gadchiroli district of Maharashtra, India use this plant against male infertility problems. erefore,
we hypothesizing that F. praecox must be having chemical properties specic for improving the male fertility.
To check this hypothesis, we have conducted invitro antioxidant studies on both F. praecox (its leaves, stem
and medicinally important roots) and the well-recognized and traditionally used plant M. pruriens (its roots,
leaves, stem and medicinally important seeds) under the similar analytical conditions and compared the nd-
ings. e correlational studies were also performed to discuss probable action mechanisms of these plants on
ROS. Finally, inhibition of two male infertility markers, Phosphodiesterase 5 (PDE5) and arginase by the plant
extract were also studied invitro.
Based on recent literature reviews, it was observed that among various Flemingia species recognized for their
medicinal properties, the most important organ with medicinal use was its roots38–47 followed by leaves and
stems35,48. A very few studies have demonstrated the use of its seeds for medicinal purposes35. Moreover, none
of the work has shown its any organ with capacity to ameliorate the male reproductive health. Furthermore, the
traditional medicinal practitioners of Gadchiroli district were also denied the use of its seeds in male infertility
cases. Due to these reasons, and most importantly, the extremely limited availability, we have not included the
seeds of F. praecox in our studies.
Results and discussion
Preliminary phytochemical availability test
We have tested the availability of phenols, avonoids, glycosides, alkaloids, terpenoids, tannins, steroids and
saponins (Table1). ese tests were performed because we did not nd any previous studies in literature on
phytochemistry of F. praecox. e results obtained were compared with the M. pruriens. Multiple tests were per-
formed for various categories of secondary metabolites. All parts of F. praecox have shown positive results in all
the tests performed for phenols, avonoids and also to some extent tannins. However, terpenoids and glycosides
are almost absent in the F. praecox extracts which is also the case with the M. pruriens extract. M. pruriens also
showed presence of phenols and avonoids except its seed and root which showed negative results in some of
the tests. Alkaloids were present in the leaves of both the plants. e roots of F. praecox and leaves of M. pruriens
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also give positive results for the presence of steroids. M. pruriens seeds also possessed good foaming capacity
indicating the presence of saponins.
Alkaloids are produced by the plants mainly for deterring herbivory by vertebrates which are also observed
to have a negative role in pharmacological context49. Except phenolic and avonoid glycosides all other glyco-
sides are known to have adverse eects on50,51. Hence, the absence or low levels of glycosides and alkaloids in
medicinally important plant parts i.e. seeds of M. pruriens and roots of F. praecox eliminates its possible side
eects on the health. Previous data indicated the absence of alkaloids in aqueous and methanolic extract of M.
pruriens leaf however, in our analysis all the tests performed showed its presence. Moreover, the terpenoids
were absent in our analysis but they were found by other workers52. Previously alkaloids were isolated from M.
pruriens leaves53 and seeds54 and its bioactivity was also studied which validates our positive results for alkaloids
in M. pruriens55. Our results of saponins, tannins and avonoids in M. pruriens were in accordance with results
obtained by previous workers52. Steroids were reported in M. pruriens seeds56 however, in our analysis it was not
detected in seeds but observed in the leaf.
Work has been done on various species of Flemingia like F.57–59, F. macrophyla60, F. chappar61, F. philippinen‑
sis62, F. faginea48, F. grahmiana63 F. stricta64, etc. but no phytochemical work has been found in the literature on
the species F. praecox. However, these species have shown the presence of phenols, avonoids, steroids, tannins,
Figure1. Phenol (blue) with its dierent mechanism of action against ROS (red).
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glycosides, alkaloids and saponins and in most of the species the terpenoids were not64,65 our preliminary analysis
also, the terpenoids were not detected in F. praecox.
Quantication of phenolics
In the plant parts
Phenols and avonoids are major groups of secondary metabolites that are known to have maximum shares in
total antioxidant potential of any plant19. TPC and TFC were calculated rst in all the plant parts (Fig.3a and b)
and then the plant parts that gave the highest values were fractionated by using various solvents with increasing
polarity and again TPCs and TFCs of these fractions were estimated (Fig.4a and b).
In our analysis, the signicantly highest phenolic content was observed in M. pruriens seeds which is
327.48 ± 3.81mg of gallic acid equivalent per gram of plant extract (mg GAE/g) followed by F. praecox roots
containing 199.00 ± 5.96mg GAE/g. However, leaves of both the plants showed statistically similar concentra-
tions of phenol that is 154.78 ± 1.98 and 160.23 ± 5.85mg GAE/g in F. praecox and M. pruriens respectively. e
roots of M. pruriens and stem of F. praecox contain its minimum concentration (91.38 ± 0.88 and 90.71 ± 0.77mg
GAE/g respectively).
Unlike phenols, the highest level of avonoids was estimated in F. praecox root that is 360.93 ± 8.49mg of
quercetin equivalent per gram of extract (mg QE/g) followed by M. pruriens seeds containing 277.59 ± 16.14mg
QE/g. F. praecox leaves also have shown the better levels of avonoid (216.48 ± 8.49mg QE/g) than other remain-
ing plant parts. F. praecox stems contain lowest avonoid content among the rest of the plant parts of both the
plants (36.85 ± 3.21mg QE/g).
In the fractions
On the basis of results obtained, we have selected M. pruriens seeds and F. praecox roots which are also the parts
that are being used for improving the male fertility and attempted to quantify the phenols and avonoids from
their serial fractions made in dierent solvents with increasing polarity (Fig.4a and b). e serial fractiona-
tion was done in the following sequence, n-hexane → ethyl acetate → chloroform → acetone → methanol. In
our study, acetone extracted the highest fraction of phenol from F. praecox roots (175.87 ± 9.536mg GAE/g).
Figure2. Flavonoids contribute in improvement of male fertility by dierent mechanisms.
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Methanol also dissolved signicantly higher phenols from F. praecox roots (131.65 ± 34.90mg GAE/g) than the
remaining solvents. Also ethyl acetate fraction of F. praecox root has shown good phenolic value (67.77 ± 2.830mg
GAE/g). Among the M. pruriens seed fractions, the highest phenolic value was obtained in its methanolic frac-
tion (115.99 ± 0.270mg GAE/g) which is also statistically similar to phenols obtained in the methanolic fraction
of F. praecox root.
e TFC analysis of dierent solvent fraction showed that F. praecox root contain signicantly higher avo-
noids that are soluble in acetone (522.96 ± 6.55mg QE/g) followed by methanolic fraction (281.914 ± 1.07mg
QE/g). The ethyl acetate also extracted considerable flavonoids from F. praecox roots (194.26 ± 6.68 mg
QE/g). Among M. pruriens, its methanolic fraction contains a maximum avonoid than other fractions
(265.86 ± 12.05mg QE/g) indicating methanol as the best solvent for avonoid extraction from M. pruriens.
Previously, the phenol content of M. pruriens was quantied by using dierent extraction solvents and meth-
ods. Due to its medicinal property, phenols were quantied mostly from seed extracts made in water, ethanol
and methanol and it was found in the range of 3.9 to 230mg GAE/g66–70, clearly showing that solvent inuences
the phenolic extraction. ese levels were much lower than our quantied results in crude methanolic seed
extract. Some studies however showed a considerably high level of phenolics up to 3730mg GAE/g of extract71.
As stated earlier, the species F. praecox was not studied in the context of its phytochemistry. is may be
due to its very low population size or its rarity in nature. However, we studied its phytochemistry for the rst
time from its restored population in our experimental eld. In literature, we found that most of the work was
done on F. philippinensis72. In its leaves, the phenols were 40mg GAE/g and the roots showed 49mg GAE/g.
Other species of Flemingia expectedly showed varied amounts of phenols which ranges from 12mg GAE/g in
F. strobilifera and F. vestita to 280mg GAE/g in F. f ag inea 47,48,57,73,74 In our studies on F. praecox the phenols were
found in good concentration i.e. 199mg GAE/g in roots and also its leaves contain considerably higher phenols
than in the leaves of F. philippinensis72. us our observation indicates that F. praecox can be the better source of
phenolic antioxidants among its other species.
Table 1. Phytochemical availability tests in major plant parts of F. praecox and M. pruriens; presence of the
phytochemical is indicated by ‘ + ’ and its absence indicated by ‘–’. A1: Hager’s test, A2: Dragendor’s test, A3:
Mayer’s Test, A4: Wagner’s test, F1: Lead acetate test, F2: Shinoda test, F3: Alkaline reagent test, G1: Keller-
kiliani test, G2: Legal’s test, G3: Liebermann’s test, S1: Foam test, S2: Olive oil test, T1: Bramer’s Test, T2: Lead
acetate test, T3: Potassium dichromate test, T4: Gelatin Test, Ter1: Acetic anhydride test, Ter2: Chloroform test.
F. praecox M. pruriens
Plant parts Leaf Stem Root Leaf Stem Root Seed
Phenols
Ferric chloride test + + + + + – +
Flavonoids
F1 + + + + + + +
F2 + + + + + – –
F3 + + + + + + –
Alkaloids
A1 + – – + – – +
A2 + – – + – – –
A3 + + – + + + +
A4 + – – + – – –
Steroids
Salkowski test – – + + – – –
Tannins
T1 + + + + + + +
T2 + + + + + + +
T3 + + + + + + +
T4 – + + – + – +
Saponins
S1 + + – – – – +
S2 – – – – – – +
Glycosides
G1 – – – – – – –
G2 – – + – – – +
G3 – – – – – – –
Terpenoids
Ter1 – – – – – – –
Ter2 – – – – – – –
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e methanolic extract of M. pruriens seeds has signicantly higher concentration of phenols than its other
organs which may be the reason for using its seeds for improving the male fertility. On the other hand, methanolic
extract of F. praecox roots also showed considerably higher levels of phenols than its other organs which here as
well can be the reason for its use in alleviating the male infertility problems by conventional medicinal practi-
tioners. Moreover, acetone and methanol fractions of F. praecox give signicantly higher phenol estimates even
better than the similar fractions of M. pruriens seeds. us it can be concluded that F. praecox and M. pruriens
are both relatable in the context of its phenolic content and medicinal property.
Previous studies have reported the high levels of avonoids and also new avonoids have been discovered
from time to time from dierent species of Flemingia. Moreover, invitro and preclinical properties of these
avonoids have also been reported by various researchers42,75–79. Some researchers also worked on isolation and
invitro properties of new avonoids from leaves of other F. praecox63,80,81.
In M. pruriens, previous studies indicated its avonoids are in the range of 63 to 807mg QE/g of aqueous
extract70,71 and 423mg QE/g of ethanolic extract82 again indicating the place of origin of the plant and extraction
procedure aecting the quantied values. In our analysis we observed maximum avonoids in the M. pruriens
seeds than its other organs studied which again signifying the use of its seeds for the medicinal purpose.
Studies on the avonoids in dierent species of Flemingia showed that its value was ranging from 0.75 to
52.76mg rutin equivalent per g aqueous47,73 and from 7.69 to 30.58mg QE/g of hydro-alcoholic74,83. In one more
study on the F. f aginea leafy shoot found to contain 33.31mg QE/g avonoids in its aqueous extract. However,
our study on F. p raecox showed signicantly high concentration of avonoids in both its roots and leaf. Moreover,
a.
b.
154.78c
113.16d
91.38e
327.48a
160.23c
90.71e
199b
0
50
100
150
200
250
300
350
M. Leaf M. Stem M. Root M. Seed F. Leaf F. Stem F. Root
mg/g of GAE
Total phenolic content of plant parts
138.09d
108.46e
80.06f
277.59b
216.48c
36.85g
360.93a
0
50
100
150
200
250
300
350
400
M. Leaf M. Stem M. RootM. Seed F. Leaf F. Stem F. Root
mg/g of QE
Total flavonoid content of plant parts
Figure3. Total phenolics in methanolic extract of organs of M. pruriens (M.) and F. praecox (F.) (a) and total
avonoids in methanolic extract of parts of M. pruriens and F. praecox (b). Values are presented as means of
three readings ± SD (standard deviation). Highest to lowest values are shown in alphabetical order. Means with
the dierent letter are signicantly dierent at 95% condence interval (p < 0.05) according to Tukey’s multiple
range test.
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the analysis of avonoid content in dierent solvent fractions of its roots shown even higher avonoids specially
in acetone fraction followed by methanolic fraction, indicating the acetone as a better solvent for avonoid
isolation from F. praecox roots which is also specied earlier by Pisoschi etal.84 Comparison of avonoids in M.
pruriens and F. praecox indicates that F. praecox species under study is far better source of avonoids than its
counterpart M. pruriens. F. praecox contain at least about more than two folds of avonoid concentration in its
acetone fraction than any fractions of M. pruriens and about 30% more in its medicinally important organ root
than M. pruriens seeds. Even the leaves of F. praecox contain considerably good concentration of avonoids.
Flavonoids are important group of secondary metabolite consist of cyclized diphenylpropane structure19 and
are secreted in plants in the form of pigments in owers, fruits, seeds, and leaves for recruiting pollinators and
seed dispersers, in defence as feeding deterrent and antimicrobial agents, and in UV protection85. However, due
to its diverse structure it is also found to have important medicinal applications. It was observed that in contrast
to simple phenols which have mainly antioxidant properties (Fig.1)22,86. Flavonoids show antioxidant activity
mostly by chelating free radical forming metal ions like Fe2+ by formation of coordinate bonds with them by its
–C=O and –OH groups87. Along with its major property of modulation of cell signalling pathway it was reported
that avonoids have capacity to improve vascular endothelial function by increasing the production of nitric
oxide (NO) through endothelial nitric oxide synthase (eNOS)88. Flavonoids also have a neuroprotective role as it
stimulates neuronal nitric oxide synthase (nNOS)89,90 and also possesses antidiabetic properties due to its insulin
production capacity therefore improving the diabetes mediated vascular dysfunction91. Moreover, another group
of avonoids, isoavones, have prostate cancer inhibition capacity by hormone dependent signalling pathway92
a.
b.
3.06d 10.47d 0.95d
14.16d
115.99b
4.01d
67.77c
19.79d
175.87a
131.65
b
0
20
40
60
80
100
120
140
160
180
200
mg/g of GAE
Total phenolic content of plant fractions
59.7de 79.4d59.1de
13.4f
265.86b
72.65d
194.26c
39.3ef
522.96a
281.91b
0
100
200
300
400
500
600
mg/g of QE
Total flavonoid content of plant fractions
Figure4. Total phenolics (a) and avonoids (b) in dierent fractions made by sequential extraction of
M. pruriens seeds (M.) and F. praecox roots (F.) in dierent solvents. Dierent letters represent signicant
dierences at the p < 0.05 level.
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along with various spermatogenesis promoting eects93 (Fig.2). is male fertility improving role of avonoids
can be met by its high levels in the F. praecox.
Phenol and avonoid detection in plant fractions by HPLC–MS/MS analysis
In F. praecox root, higher number of phenolic compounds were detected compared to the seeds of M. pruriens
(Tables2 and 3). Many of these compounds have been reported to have a positive impact on male fertility through
various mechanisms. Some of these compounds possess antioxidant properties and may contribute to the reduc-
tion of oxidative stress21 or they are acting at dierent levels of male reproductive system.
For instance, the epigallocatechin and other catechins from F. praecox have been shown to reverse testicular
damage94 possibly due to their active antioxidant95 or DNA damage protection properties96. Similar positive
eects on male fertility have been reported for compounds such as Lespenefril24, Chrysin97 and rutin98, all found
in F. praecox. Spermidine derivatives like N1, N5, N10-Tricoumaroyl spermidine have been associated with
ameliorative eects on sperm disorders in diabetic mice99. Other compounds in F. praecox, including Ononin100,
Procyanidin B7, Curcumin II101 and Mulberranol102 have spermatogenic eect by modulating testosterone and
other sex hormone levels. Moreover, avonoids like Isoliquiritigenin have been reported to ameliorate sexual
dysfunction103, while Licocoumarin A has been identied as an estrogen modulator104. Lastly, Xanthohumol has
demonstrated the capacity to inhibit the growth and invasion of prostate cancer cells105.
On the other hand, in M. pruriens seed extract, the reported phenolic compound 5-(3′,4′,5′-Trihydroxyphenyl)-
gamma-valerolactone which has been reported to possess neuroprotective properties106 and thus might have
implication in the psychogenic male infertility. Another compound, the isoavonoid, Luteolin have a well-known
positive role in the process related to steroidogenesis, apoptosis and in stress response107. However, Beclometha-
sone dipropionate another compound detected in the M. pruriens seeds has been associated with negative eects
on the reproductive function of male rats108. ese ndings collectively suggest that the presence of these phenolic
Table 2. Phenolic compounds detected in F. praecox root methanolic fraction by HRLC-MS/MS analysis.
Sr. no Compound name CID m/z Phenolic class
1 Epigallocatechin 72,277 307.0799 Flavonoids
2 Daidzein 5,281,708 255.0647 Isoavonoids
3 N1, N5, N10-tricoumaroyl spermidine 14,777,879 584.2731 Cinnamic acids and derivatives
4 Hellicoside 5,281,778 657.194 Cinnamic acids and derivatives
5Xanthohumol 639,665 355.1528 Linear 1,3-diarylpropanoids
6 Licocoumarin A 5,324,358 407.1838 Isoavonoids
7 Glycyrrhizaisoavone B 10,546,844 367.1163 Isoavonoids
8 4ʹ-O-methylkanzonol W 131,751,269 351.1211 Isoavonoids
9Licoisoavone A 5,281,789 355.1164 Isoavonoids
10 Kanzonol K 131,753,069 437.1938 Isoavonoids
11 Kanzonol L 131,753,032 489.2245 Isoavonoids
12 Isoliquiritigenin 638,278 257.0798 Linear 1,3-diarylpropanoids
13 Curcumin II 5,469,424 367.1524 Diarylheptanoids
14 Osajin 95,168 405.1677 Isoavonoids
15 Kuwanon Z 21,594,954 593.1443 Flavonoids
16 Lespenefril 5,486,199 577.15 Flavonoids
17 2-Methyl-5-(8-pentadecenyl)-1,3-benzenediol 6,452,209 331.2592 Phenols
18 Metaxalone 15,459 222.112 Phenol ethers
19 2″,4″,6″-triacetylglycitin 131,751,611 595.143 Isoavonoids
20 Camellianin A 5,487,343 643.1785 Flavonoids
21 Mulberranol 71,438,979 439.1742 Flavonoids
22 Kanzonol Z 10,319,154 407.1842 Flavonoids
23 N1,N5,N10-tris-trans-p-coumaroylspermine 10,908,386 641.3444 Cinnamic acids and derivatives
24 Kuwanone G 5,281,667 693.231 Flavonoids
25 2,2-dimethyl-3,4-bis(4-methoxyphenyl)-2H-1-benzopyran-7-ol acetate 255,270 431.1776 Isoavonoids
26 Procyanidin B7 13,990,893 579.1482 Flavonoids
27 Ononin 442,813 431.1327 Isoavonoids
28 Chrysin 5,281,607 255.0645 Flavonoids
29 Rutin 5,280,805 609.1403 Flavonoids
30 Isorhamnetin 3-glucoside 4′-rhamnoside 44,259,360 623.1558 Flavonoids
31 [Gallocatechin(4alpha- > 8)] 2catechin 14,890,508 897.2087 Flavonoids
32 Catechin-(4alpha- > 8)-gallocatechin-(4alpha- > 8)-catechin 131,752,348 881.2134 Flavonoids
33 Iriomoteolide 1a 16,723,501 505.3181 Phenylpropanoids
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and avonoid compounds in both F. praecox roots and M. pruriens seeds may contribute to the improvement of
male fertility, although through dierent mechanisms and at various levels within the male reproductive system.
In vitro antioxidant capacity
e high concentrations of phenols and avonoids in the medicinally used M. pruriens seeds and F. praecox roots
also indicated the possibility of having high antioxidant values. Antioxidants are a major primary defence system
against ROS and FR. It is well established from previous research that ROS and FR are the important contributory
factors in various diseases including male infertility17,109–111. To check this hypothesis we studied the antioxidant
properties of the plant parts of M. pruriens and F. praecox by DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS
(2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) acid) and DMPD (N, N-dimethyl-p-phenylenediamine)
free radical scavenging assay, β-carotene bleaching, FRAP (Ferric ion reducing antioxidant power) and phos-
phomolybdenum antioxidant assay. e results obtained were compared with articial antioxidants, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and ascorbic acid.
DPPH⋅ scavenging activity
e DPPH radical scavenging activity is based on the reduction of purple coloured DPPH⋅ to its yellow hydrazine
product (DPPH-H) by hydrogen or electron donating capacity of the plant compounds19,112. We have studied
DPPH⋅ scavenging activity of plant parts (Fig.5a) and the dierent fractions of M. pruriens seeds and F. praecox
roots (Fig.5b). e study revealed that F. praecox root has best scavenging activity among all the plant parts with
IC50 (half-maximal inhibitory concentration) value 7.34 ± 0.315µg and is also statistically similar in its scaveng-
ing activity to articial antioxidants, ascorbic acid (5.24 ± 0.29µg) and BHA (4.3 ± 0.12µg) and even better than
BHT (13.7 ± 0.31µg). Other workers studied DPPH⋅ scavenging activity in dierent species of Flemingia, mostly
in its roots47,48,57,74,83 and leaves63. In leaves of F. grahmiana, Gumula etal. showed IC50 value 5.9µg whereas oth-
ers studies on roots of various species of Flemingia, the IC50 for DPPH⋅ scavenging was ranged from best in F.
faginea (15.04µg)48 to the least in F. vestita (287µg)74. However, among M. pruriens, its seed possesses the highest
scavenging activity with IC50 value 18.34 ± 0.182µg which is statistically similar to BHT. In previous studies on
alcoholic and hydro-alcoholic extract of M. pruriens seeds, the DPPH⋅ scavenging activity in terms of IC50, was in
range of 5.1 to 61.02µg67–70. Among the fractions of M. pruriens seed and F. praecox root, best DPPH⋅ scavenging
activity was observed in methanol and acetone fractions of F. praecox root with IC50 values 7.21 ± 0.26µg and
8.15 ± 0.83µg respectively followed by methanol fraction of M. pruriens seeds having IC50 value 11.79 ± 0.51µg.
ese all values are statistically similar to standards used at p < 0.05. Hexane and chloroform fraction of M.
pruriens seeds did not show any scavenging activity at the used concentration.
ABTS⋅+ scavenging activity
ABTS⋅+ scavenging activity demonstrates the capacity of the phytochemicals to neutralize ROS by hydrogen
atom transfer (HAT) or single electron transfer (SET) mechanism19. e best HAT or SET capacity again shown
by F. praecox roots having ABTS•+ scavenging IC50 value 3.63 ± 0.112µg (Fig.6a). Statistical tests shows that
ABTS•+ scavenging potential of F. praecox root is statistically similar (p < 0.05) to ascorbic acid (2.50 ± 0.125µg)
and BHT (3.10 ± 0.832µg). e articial antioxidant, BHA have shown the best ABTS•+ scavenging activity with
IC50 value 2.14 ± 0.066µg. Previous work on other species of Flemingia like F. faginia48 and F. vestita74 found
the ABTS⋅+ scavenging IC50 value 67.33µg and 11.49µg respectively. ese activities shown by other species
are much lower than our studied species F. praecox. In the case of M. pruriens, seeds (8.64 ± 0.149µg) and stem
(9.43 ± 0.196µg) have shown better scavenging activity than its other organs but signicantly lower than its
counterpart F. praecox root. At the end, M. pruriens root (14.60 ± 0.273µg) and F. praecox stem (14.00 ± 0.482µg)
have shown lowest capacity to scavenge ABTS radicals. However, a large range of ABTS⋅+ scavenging values were
observed by other workers in M. pruriens which is 6.009µg as found by Njemuwa etal.69 to 137µg which was
observed by Chittasupho etal.70.
Table 3. Phenolic compounds detected in M. pruriens root methanolic fraction by HRLC-MS/MS analysis.
Sr. no Compound name CID m/z Phenolic class
1 (Z)-N-feruloyl-5-hydroxyanthranilic acid 10,087,955 330.097 Cinnamic acids and derivatives
2 MS 3 100,450 411.141 Phenylpropanoids
3 Senkirkine 5,281,752 366.19 Phenylpropanoids
4 Dipivefrin 3105 352.213 Phenol esters
5 5-(3′,4′,5′-trihydroxyphenyl)-gamma-valerolactone 44,389,277 223.061 Phenols
6 2,6-Dihydroxyphenylacetate 440,944 167.035 Flavonoids
7Beclomethasone dipropionate 21,700 563.235 Flavonoids
8 10-Acetoxyligustroside 102,117,098 641.211 Flavonoids
9 Apimaysin 194,566 559.147 Flavonoids
10 Luteolin 5,280,445 285.041 Isoavonoids
11 LysoPE(18:2(9Z,12Z)/0:0) 52,925,130 476.282 Phenylpropanoids
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DMPD⋅+ scavenging
Another radical scavenging activity investigated with DMPD radical cation (DMPD•+) which is based on the HAT
and SET mechanism of radical scavenging. is assay is less sensitive to hydrophobic and more specic for the
hydrophilic antioxidants. is is opposite to DPPH⋅ and ABTS⋅+ scavenging assay19. e best scavenging results
were again observed in F. praecox roots (Fig.6b) with IC50 value 62.86 ± 0.64µg which is signicantly better than
BHA (114.58 ± 4.93µg). However, we found poor scavenging activity in all other plant organs of both F. praecox
and M. pruriens. Among M. pruriens, the better scavenging capacity was shown by its leaf (228.79 ± 35.25µg).
Previous studies on other M. pruriens species have reported 41% DMPD⋅+ scavenging at 40µg113 and about 85%
DMPD⋅+scavenging at 100µg aqueous extract of its raw seeds114. We did not nd any previous study on other
organs of M. pruriens and on any species of Flemingia with respect to DMPD⋅+ scavenging. In our study, the
obtained highest DMPD⋅+ scavenging activity of F. praecox root indicates that it contain abundant hydrophilic
antioxidants as compared to M. pruriens which showed signicantly lower DMPD⋅+ scavenging activity. is
indicates that the antioxidant capacity of M. pruriens is mostly governed by hydrophobic antioxidants and less
by hydrophilic antioxidants.
β‑carotene bleaching protection assay
Lipid peroxidation protection or peroxyl radical (ROO⋅) scavenging property of the plant extracts was assessed
by β-carotene bleaching assay. In this assay, the ROO⋅ generated by thermal autoxidation of linoleic acid reacts
5.24g 4.3g
13.7ef
33.04d
40.8bc46ab
18.34e
38.48cd
47.87a
7.34fg
0
10
20
30
40
50
60
IC50 (µg)
DPPH radical scavenging activity of plant parts
5.24c 4.30c 13.70cND
50.96b
ND
23.69c11.79c
213.96a
30.31c
133.62b
8.15c7.21c
0
50
100
150
200
250
IC50 (µg)
DPPH radical scavenging activity of fractions
a.
b.
Figure5. DPPH radical scavenging capacity of plant parts of M. pruriens and F. praecox (a) and the
sequentially extracted fractions (b) of M. pruriens seeds and F. praecox roots. Dierent letters represent
signicant dierences at the p < 0.05 level. ND not detected.
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with β-carotene and causes its discolouration. is discolouration is prevented when antioxidants in plants neu-
tralises the ROO⋅ to ROOH. is activity is an important indicator of capacity of plant extract to protect fragile
a.
2.51de 3.1de 2.14e
10.6b
9.43c
14.60a
8.64c9.52c
14a
3.64d
0
2
4
6
8
10
12
14
16
IC50 (µg)
ABTS Scavenging Activity
14.9a
114.6c
228.8d 279.5e 299.9g 293.1f
1866.1i
424.9h
62.9b
0
100
200
300
400
500
600
700
IC50 (µg)
DMPD Scavenging Activity
b.
c.
ND
73.01a
64.85b
8.51f
ND ND
14.85e 19.77d
8.52f
29.46c
0
10
20
30
40
50
60
70
80
% inhibition of bleaching
β-carotene bleaching inhibition activity
Figure6. ABTS (a), DMPD (b) radical scavenging and β-carotene bleaching inhibition activity (c) of plant
parts of M. pruriens and F. praecox. Dierent letters represent signicant dierences at the p < 0.05 level. ND not
detected.
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sperm membrane susceptible to lipid peroxidation by ROO⋅115. In our study we observed that at a concentration
of 30µg, F. praecox roots have maximum capacity to protect β-carotene from bleaching (29.46 ± 1.40%) followed
by F. praecox leaf (19.77 ± 2.71%) (Fig.6c). M. pruriens seed protected 14.85 ± 0.30% β-carotene from bleach-
ing. M. pruriens stem and root as well as ascorbic acid did not show any protection against the bleaching at this
concentration. β-carotene bleaching protection activity of M. pruriens was also reported previously where the
workers observed 5.4% bleaching protection activity by 100µg methanolic extract of M. pruriens116 and 59.35%
protection by 200µg processed extract of another species, M. gigantia117. No study was found on any F. praecox
species with respect to their invitro β-carotene bleaching protection activity. Among the standards, BHA showed
the maximum level of β-carotene protection from bleaching (73.01 ± 0.60%) followed by BHT (64.85 ± 0.38%).
Ferric ion reducing (Fe3+ → Fe2+) antioxidant power assay (FRAP)
e reducing capacity of the plant extracts was determined by FRAP assay which is based on the SET mecha-
nism. High reducing capacity of plant extract is an indicator of its potential antioxidant capacity19. e results
of TAA are presented in Table4 (SF1b and c). e highest FRAP value was observed in M. pruriens seed
(A700 = 0.194 ± 0.006 absorption units (AU)) followed by F. praecox root (A700 = 0.143 ± 0.003AU). Among stand-
ards, ascorbic acid showed the best reducing capacity (A700 = 1.148 ± 0.025AU). In previous studies on M. pruriens,
FRAP activity was found to be 561mg ascorbic equivalent/g68. However, in our analysis it was observed to have
155.90 ± 5.42mg ascorbic equivalent/g (SF1a). e FRAP studies on two species of Flemingia, F. vestita and F.
macrophylla showed FRAP values, although calculated dierently, 9.28mg GAE/g of hydroalcoholic extract74
and an IC50 of 23.05µg/mL of aqueous extract47 respectively. Stem and leaf of both M. pruriens and F. praecox
and roots of M. pruriens have shown minimum and statistically similar activity indicating their low antioxidant
capacity.
Total antioxidant activity (TAA)
To determine total antioxidant activity (TAA) of the plant extract phosphomolybdenum method was used.
This method evaluates both water-soluble and fat-soluble antioxidants from the plant extract118. In our
study we get the highest TAA value in F. praecox root (A695 = 0.251 ± 0.002AU) followed by M. pruriens seed
(A695 = 0.137 ± 0.006AU) as can be seen in Table5 (SF2a and b). However, among the standards used, the high-
est TAA value was found in ascorbic acid (A695 = 0.641 ± 0.005AU) followed by BHA (A695 = 0.568 ± 0.03AU)
and BHT (A695 = 0.317 ± 0.006AU). Previous studies were not found on M. pruriens and F. praecox in context
of the performed assay. is result shows that F. praecox root might contain both water-soluble and fat-soluble
antioxidants in abundance than M. pruriens seeds.
Any plant sample contains hundreds of compounds and its antioxidant property depends upon their phys-
icochemical properties. erefore, the antioxidant capacity of the plant extract or any sample should not be
concluded on the basis of any single antioxidant test model. To evaluate the overall antioxidant potential of the
plant extract thus required multiple antioxidant tests to be performed112. Our attempt of conducting multiple
Table 4. FRAP activity of plant parts of M. pruriens and F. praecox. Values in bold represent the highest
values. Signicantly dierent values are represented with dierent letters (n = 3; p < 0.05).
Concentration
(µg)
Abs700 ± SD
Standard M. pruriens F. praecox
Ascorbic acid BHA BHT M. seed M. leaf M. stem M. root F. root F. l ea f F. s te m
50.104 ± 0.011 0.045 ± 0.005 0.037 ± 0.003 0.018 ± 0.003 0.001 ± 0.001 0.011 ± 0.001 0.009 ± 0.002 0.012 ± 0.002 0.015 ± 0.001 0.002 ± 0.000
10 0.201 ± 0.004 0.088 ± 0.005 0.080 ± 0.001 0.029 ± 0.006 0.007 ± 0.001 0.016 ± 0.001 0.013 ± 0.002 0.024 ± 0.002 0.023 ± 0.001 0.007 ± 0.001
20 0.400 ± 0.005 0.155 ± 0.019 0.156 ± 0.001 0.058 ± 0.005 0.016 ± 0.001 0.024 ± 0.001 0.024 ± 0.002 0.042 ± 0.003 0.027 ± 0.002 0.015 ± 0.002
40 0.807 ± 0.013 0.333 ± 0.003 0.315 ± 0.008 0.133 ± 0.010 0.042 ± 0.002 0.049 ± 0.000 0.054 ± 0.002 0.095 ± 0.002 0.054 ± 0.004 0.035 ± 0.001
60 1.148 ± 0.025 0.487 ± 0.004 0.450 ± 0.006 0.194 ± 0.006 0.084 ± 0.003 0.074 ± 0.002 0.082 ± 0.002 0.143 ± 0.003 0.070 ± 0.002 0.057 ± 0.003
Signicance a b c d f fg f e fg g
Table 5. TAA activity of plant parts of M. pruriens and F. praecox. Values in bold represent the highest values.
Signicantly dierent values are represented with dierent letters (n = 3; p < 0.05).
Concentration
(µg)
Abs695 ± SD
Standard M. pruriens F. praecox
Ascorbic acid BHA BHT M. seed M. leaf M. stem M. root F. root F. l ea f F. s te m
20 0.104 ± 0.007 0.086 ± 0.007 0.067 ± 0.002 0.026 ± 0.001 0.027 ± 0.002 0.015 ± 0.003 0.021 ± 0.005 0.051 ± 0.001 0.018 ± 0.002 0.008 ± 0.005
40 0.228 ± 0.006 0.202 ± 0.010 0.136 ± 0.003 0.055 ± 0.002 0.044 ± 0.002 0.035 ± 0.002 0.037 ± 0.005 0.099 ± 0.004 0.032 ± 0.001 0.016 ± 0.001
60 0.356 ± 0.008 0.278 ± 0.015 0.196 ± 0.006 0.085 ± 0.004 0.066 ± 0.006 0.057 ± 0.001 0.064 ± 0.006 0.148 ± 0.005 0.048 ± 0.001 0.026 ± 0.001
80 0.487 ± 0.010 0.434 ± 0.038 0.255 ± 0.004 0.112 ± 0.008 0.094 ± 0.004 0.073 ± 0.004 0.084 ± 0.001 0.196 ± 0.013 0.066 ± 0.022 0.036 ± 0.003
100 0.641 ± 0.005 0.568 ± 0.030 0.317 ± 0.006 0.137 ± 0.006 0.115 ± 0.001 0.092 ± 0.010 0.113 ± 0.005 0.251 ± 0.002 0.079 ± 0.011 0.041 ± 0.000
Signicance a b c e f g f d g h
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tests revealed that F. praecox root and M. pruriens seed have very high antioxidant capacity as revealed by DPPH⋅,
ABTS⋅+, DMPD⋅+, FRAP, β-carotene bleaching and TAA assay. is property of F. praecox roots and M. pruriens
seeds might be the major contributing factor in improving male fertility. Moreover, the phytochemically unex-
plored plant, F. praecox which is used by the tribal people have shown exceptional antioxidant properties even
better than the conventionally used, M. pruriens. In some antioxidant aspects like DPPH, DMPD and β-carotene
bleaching assay, it is showing even higher activity than the well-known synthetic antioxidants such as BHA, BHT
and ascorbic acid (Table6) thus giving the promising alternative as a rich source of natural antioxidants to prevent
damage caused by oxidative stress to sperms and other important male reproductive physiological parameters.
DNA damage protection activity
DNA protection capacity of all the plant extracts against damaging agent, Fenton’s reagent was assessed by
agarose gel electrophoresis. Fenton’s reagent generates highly reactive, DNA damaging hydroxyl radical (HO⋅).
is radical is known to damage the DNA by oxidizing 2-deoxyribose to malonaldehyde116. erefore, the plant
antioxidants are used to assess their HO⋅ radical scavenging capacity and to protect DNA against the damage
caused by the radical (Fig.7a and b). e highest DNA protection was governed by M. pruriens seed extract
which protected 98.88% DNA at 50µg concentration followed by its stem and leaf which protected 90.92% and
87.73% respectively. In F. praecox, its roots protected the maximum 65.63% DNA followed by its leaf which
showed 18.34% protection (Fig.8 and SF3). e F. praecox stem did not show any protection against DNA dam-
age. Previous studies shows that M. pruriens have capacity to scavenge HO• radical and protect DNA in dose
dependent manner116. One study shows the methanolic extract of the M. pruriens has DNA protection capacity
at IC50 value of 38µg66. Not much work has been done before on DNA protection activity of F. praecox. However,
in one study where Kim etal. isolated bioactive compound, auriculasin form F. philippinensis which showed
90.9% DNA protection at 60µM concentration83.
Table 6. e assays performed are either specic in its mechanism to scavenge particular ROS that have a role
in male infertility or are used for assessing antioxidants in the samples with dierent solubility. Overall, our
results shows that, except FRAP assay, in all the assays F. praecox roots have better antioxidant capacity than M.
pruriens seeds. AA ascorbic acid.
Sr. no Assay Mechanism Analogy and function Role in male fertility Best results shown by
1DPPH⋅HAT and SET, assesses hydro-
phobic antioxidants Free radicals, reducing ability Involved in idiopathic
infertility119 BHA = AA > F. praecox
root > BHT > M. pruriens seed
2ABTS⋅ + HAT and SET, assesses
hydrophilic and hydrophobic
antioxidants Free radicals, reducing ability Involved in idiopathic
infertility119 BHA > AA = BHT > F. praecox
root > M. pruriens seed
3DMPD⋅ + HAT and SET, assesses hydro-
philic antioxidants Free radicals, reducing ability Involved in idiopathic
infertility119 AA > F. praecox root > BHA > M.
pruriens leaf
4 β-carotene bleaching protection Assesses lipid peroxidation
preventing antioxidants ROO⋅, membrane protection Damages sperms by lipid per-
oxidation of membranes120 BHA > BHT > F. praecox root > F.
praecox leaf > M. pruriens seed
5 FRAP SET, assesses metallic free radi-
cal scavenging capacity
Iron chelation and reducing
power that can work against
H2O2, 1O2, HO⋅, and O⋅−
Involved in sperm DNA damag-
ing HO• generation, idiopathic
infertility121
AA > BHA > BHT > M. pruriens
seed > F. praecox root
6 TAA HAT and SET, assesses total
antioxidants from broad spec-
trum of samples
Metal chelation and Reducing
power that can work against
H2O2, 1O2, HO⋅, and O⋅−
Involved in idiopathic
infertility119,121 AA > BHA > BHT > F. praecox
root > M. pruriens seed
Figure7. DNA damage protection activity of methanolic extracts of M. pruriens seeds (a) and F. praecox
roots (b). L1 = Plasmid DNA (pDNA), control; L2 = pDNA + Fenton’s Reagent. In (a) L3 = pDNA + FR + M.
pruriens Seed Extract; L4 = pDNA + FR + M. pruriens Leaf Extract; L5 = pDNA + FR + M. pruriens Stem
Extract; L6 = pDNA + FR + M. pruriens Root Extract. In (b) L3 = pDNA + FR + F. praecox Root Extract;
L4 = pDNA + FR + F. praecox Leaf Extract; L5 = pDNA + FR + F. praecox Stem Extract. Arrows indicate distinct
forms of plasmid DNA: OC (open circular); SC (supercoiled).
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Correlation study
e dierent antioxidant assays studied represent their dierent modes of action towards dierent ROS and
FR84. To know whether their modes of actions are correlated to their properties of eliminating ROS and FR, we
studied the correlation among their activity by using Pearson’s correlation (Fig.9 and SF4). e TPC was found
positively correlated to only TFC (r = 0.76) at p < 0.05 signicance level possibly because phenolics and avonoids
are structurally related. On the other hand, avonoids have shown strong positive correlation (p < 0.01) with
DPPH (r = 0.92) and ABTS (r = 0.90) showing their ability of HAT and SET to eliminate ROS and FR. Previously,
researchers have also found good correlation between total polyphenols (including avonoids) and DPPH, ABTS
activities but have noted comparatively lower correlation with DMPD radical scavenging activity122. Moreover,
avonoids also displayed good correlation with β-carotene bleaching, TAA and FRAP at p < 0.05 signicance
level representing that they are involved in lipid peroxidation protection. ese results indicate that avonoids
are better antioxidants with a wider spectrum of scavenging mechanisms than phenols which is also evident
from the result of previous work21. TAA is also strongly correlated with DPPH (r = 0.89) and DMPD (r = 0.88)
at p < 0.01 signicance level. is might be attributed to capacity of TAA assay to measure both hydrophobic
and hydrophilic antioxidants118 and thus showing cumulative activity based on the principle of both DMPD and
DPPH assay which are known to be more specic to hydrophilic and hydrophobic antioxidants respectively19.
Similarly, β-carotene bleaching and ABTS activity are strongly correlated (r = 0.90). is indicating the similar
mechanism of HAT might be required for scavenging ROO⋅ in β-carotene bleaching assay and ABTS assay19.
Finally, DNA damage protection although is positively correlated with all the assays except β-carotene bleaching
and ABTS, its correlation was found to be non-signicant. is suggesting that, DNA damage protection assay
which is mainly based on HO• scavenging property of the compound123 may be having quite dierent mechanism
of action towards scavenging HO⋅ than other assays tested. FRAP assay is based on ferric ion reducing capacity
100.00
ND
98.88
87.73 90.92
58.27
65.63
ND
18.34
0
20
40
60
80
100
120
Percent DNA Protected
DNA Damage Protection
Figure8. DNA damage protection activity of methanolic extracts of M. pruriens and F. praecox plant organs
(ND not detected).
Figure9. Similarity matrices (correlation study) between the antioxidant assays that were represented as
heatmap and hierarchical clustering tree.
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of antioxidants19 and under OS ferric ions can generate DNA damaging HO⋅ by Fenton’s reaction116. Our results
of FRAP assay showed best activity in M. pruriens seeds and similar results were also observed in DNA damage
protection assay indicating both the activities are correlated and M. pruriens seed’s metabolites may have active
involvement in improving male fertility by protecting sperm DNA from HO⋅.
Collectively, our results indicate that avonoids are the major contributing factor for the antioxidant capac-
ity of the plant extract. erefore, F. praecox roots being the most avonoid rich part of the plant can serve as
a better source of antioxidants than conventionally used M. pruriens seeds to protect from ROS and FR and to
repair and improve the male fertility.
PDE5 and arginase inhibition activity
Both PDE5 and arginase enzymes are considered as negative regulators of erection and their over activity or
expression can cause erectile dysfunction by independent mechanisms. PDE5 is known to terminate cyclic
nucleotide signalling required to mediate relaxation of smooth muscle necessary for the penile erection124. Medi-
cines like sildenal, vardenal and tadalal are eective inhibitors of PDE5 thus helping in the management of
erectile dysfunction125. Here we have attempted to study whether our plant extracts have any capacity to inhibit
the PDE5 activity (Fig.10). Our study revealed that F. praecox root extract at 100µg concentration inhibited the
PDE5 activity by 13.12% whereas at the same concentration M. pruriens showed inhibition activity of 4.85%.
is result suggests that F. praecox might contain more eective PDE5 inhibitors than M. pruriens. However,
sildenal citrate has shown nearly similar inhibition percent to M. pruriens at 100nM concentration (4.48%).
Another biomarker for erectile dysfunction studied is arginase which works by competing for the -argi-
nine, the substrate for the nitric oxide synthase (NOS) needed for the synthesis of nitric oxide (NO). NO is an
important molecule for penile cavernosal tissue relaxation and erection126. erefore, arginase inhibitors can
enhance L-arginine bioavailability to NOS. Our study showed that both M. pruriens seed and F. praecox root
extract have nearly similar arginase inhibition capacity with their IC50 value calculated to be 144.41 ± 46µg and
146.20 ± 29.68µg respectively (Table7).
Conclusion
As it is evident from the previous work that ROS has a huge impact on the male fertility and its eect can be
reversed with the help of antioxidants from natural sources like M. pruriens. erefore, the aim of the present
investigation was set to examine a similar role of a less explored but traditionally eective and endemic plant, F.
praecox and its activity was compared with the activity of M. pruriens. is aim was investigated with the help
of examining their antioxidant parameters like phenolic, avonoid content, DPPH, ABTS, DMPD radical scav-
enging capacity, β-carotene bleaching protection, FRAP and TAA activity along with DNA damage protection
a
bb
0
2
4
6
8
10
12
14
16
Flemingia
100µg
Mucuna
100µg
Sildenafil
100nM
% inhibition
PDE5 inhibition activity
Figure10. PDE5 inhibition activity of methanolic extract of F. praecox and M. pruriens. Dierent letters
represent signicant dierences at the p < 0.05 level.
Table 7. Statistically similar values of arginase inhibition activity (at p < 0.05 level) of methanolic extract of F.
praecox and M. pruriens.
F. praecox M. pruriens
Arginase inhibition activity (IC50)146.20 ± 29.68µg 144.41 ± 46µg
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capacity. e second aim was to directly examine comparative inhibition potential of both the plants against
infertility markers PDE5 and arginase enzymes.
e study has identied F. praecox is having better antioxidant activity than M. pruriens in majority of the
antioxidant assays suggesting that antioxidant potential of F. praecox may be the contributing factor for its fertil-
ity improving activity. Another signicant observation about F. praecox is that its radical scavenging capacity is
better than articial antioxidants thus implicating its further use as a source of dietary antioxidants or can be
used in combination with available antioxidants for better synergistic eects for improving the male fertility. e
presence of a higher number of phenolic compounds in F. praecox roots compared to M. pruriens seeds, along
with the diverse mechanisms by which these compounds positively inuence male fertility, emphasises their
potential role in enhancing male reproductive health. Finally. the nding of better PDE5 inhibition activity and
similar arginase inhibition values of F. praecox in relation to its counterpart, M. pruriens is again encouraging
for its further preclinical and clinical trials to study its actual potential.
Materials and methods
Plant material collection and processing
For collection of plants, all relevant permits or permissions have been obtained. e study also complies with local
and national regulations. F. praecox C.B. Clarke Ex Prain was collected from Gadchiroli district of Maharashtra,
India and identied by D. L. Shirodkar, botanist from Botanical Survey of India (BSI), Pune and deposited in the
herbarium of BSI, Pune with identication No. BSI/WRC/Iden. Cer./2021/0911210004872. F. praecox is extremely
rare in the natural habitat therefore, very few seeds of it were collected from Gadchiroli district of Maharashtra,
India and then it was planted and grown for two years till its further successful seed setting has occurred. Later,
its leaf, stem and roots were harvested, cleaned, washed, chopped, dried in a hot air oven at 45°C and powdered
in a mechanical grinder. M. pruriens L. was collected from RTM Nagpur University Educational Campus, Nagpur,
India and identied by Prof. N. M. Dongarwar, taxonomist in Department of Botany, RTM Nagpur University,
Nagpur (identication No. 187). Its seeds, leaves, stem and roots were collected and processed in a similar way
like that of F. praecox. All samples were extracted in methanol by soxhlet. Also the seed of M. pruriens and roots
of F. praecox were extracted sequentially in dierent solvents like n-hexane, ethyl acetate, chloroform, acetone
and methanol with their increasing polarity then ltered and used for further analysis.
Preliminary phytochemical analysis
Preliminary phytochemical tests were done as per the standardized protocols127–129.
Test for phenols
Ferric chloride test. ree to four drops of 5% FeCl3 solution was added in 2mL of crude extract of plants.
Appearance of bluish black colour conrms the presence of phenols.
Test for avonoids
Lead acetate test. 1mL of 10% lead acetate solution was added to 1–2mL of plant extract. e appearance of
blue colour conrms the presence of avonoids.
Shinoda test. For this test, in the aqueous extract of plants some pieces of magnesium metal ribbons were
added followed by addition few drops of concentrated HCl which within a minute or two gives pink, crimson or
magenta colour that shows presence of avonoids.
Alkaline reagent test. For this test 2mL of 2% NaOH was added to 1–2mL of aqueous extract of plants that
give intense yellow colour. Addition of 3mL of 5% HCl to it turns reaction mixture colourless indicates presence
of avonoids.
Test for alkaloids
Hager’s test. Freshly prepared Hager’s reagent (1g picric acid in 100mL hot water) when added to plant extract
gives yellow precipitate indicating presence of alkaloids.
Dragendor’s test. Few drops of Dragendor ’s reagent were added to the plant extract which gives orange, red
or creamy precipitate conrms presence of alkaloids.
Mayer’s test. Mayer’s reagent (potassium mercuric iodide) when reacted with alkaloids in plant extract (2mL)
gives yellow, whitish or creamy precipitate.
Wagner’s test. 1mL of Wagner’s reagent added to 2mL of plant extract, reddish brown precipitate conrms
the presence of alkaloids.
Test for steroids
Salkowski test. In this test, 2mL of extract is used and 2mL chloroform and 1–2mL concentrated sulphuric
acid were added to it, the reddish brown colour at the junction of aqueous and chloroform layer indicates pres-
ence of steroids.
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Test for tannins
Bramer’s test. 2-3drops of 5% FeCl3 solution was added to diluted plant extract. Appearance of green or bluish
black precipitate indicates presence of tannins.
Lead acetate test. In this test, to the 2mL of extract 10% lead acetate solution was added. Appearance of white
precipitation conrms the presence of tannins.
Potassium dichromate test. In 2mL of plant extract, formation of red or dark coloured precipitate aer addi-
tion of 10% potassium dichromate conrms the presence of tannins.
Gelatin test. 1mL of 1% gelatin solution in 10% NaCl was prepared and added to 2mL of extract. Formation
of white precipitate indicates presence of tannins.
Test for saponins
Foam test. 5mL of aqueous extract or 500mg of dry extract was heated and shaken with 5mL distilled water.
Foam produced persisted for 10min indicates presence of saponins.
Olive oil test. In 5mL of extract a few drops of olive oil was added and the solution was shaken vigorously.
Formation of emulsion conrms presence of saponins.
Test for glycosides
Keller-kiliani test. To the 2mL of plant extract 1mL of glacial acetic acid was added followed by addition of
a few drops of FeCl3 and at the end 1mL of H2SO4 added slowly and the solution allowed to settle. A reddish
brown colour ring appears at the junction of two layers and the upper layer turns bluish green. ese results sug-
gest the presence of cardiac steroidal glycosides (aglycon).
Legal’s test. 2mL of concentrated extract mixed with 2mL of pyridine, few drops of 2% freshly prepared
sodium nitroprusside solution and few drops of 20% NaOH. Blue or pink coloration indicates presence of agly-
con moiety.
Liebermann’s test. 2mL of extract was heated with 2mL of acetic anhydride. Aer its cooling a few drops of
concentrated H2SO4 was added from the sides of the test tube. Appearance of the blue or green colour precipitate
indicates presence of glycosides.
Test for terpenoids
Acetic anhydride test. 2mL of acetic anhydride was added to 2mL of extract followed by addition of 2–3 drops
of concentrated H2SO4. e deep red coloration indicated the presence of terpenoids.
Chloroform test. In this test, to the 2mL of plant extract, 2mL chloroform was added and the solution was
evaporated in a water bath to make its concentrate. Later 3mL H2SO4 was added and the solution was boiled.
e grey colour will appear when the terpenoids are present.
Total phenol content (TPC)
TPC was estimated by Folin-ciocalteu method130. In brief, 2.5mL of 10% Folin-ciocalteu reagent and 2mL of
7.5% sodium carbonate were added to 500µg of extract. e reaction mixture was incubated at 45°C for 45min
and the blue coloured phosphomolybdic/phosphotungstic acid complex was measured at 760nm. e TPC value
was calculated using gallic acid standard and presented as mg GAE/g of extract.
Total avonoid content (TFC)
TFC was determined by aluminium chloride method131 with slight modication. 200µL of 5% sodium nitrite
was added to 200µg of extract and allowed to react for 5min. 300µL of 10% aluminium chloride was added to
the mixture and aer 5min, 2mL of 1M NaOH was added and the absorbance of the orange-red aluminium
complex was taken at 510nm. e TFC value was calculated using the quercetin standard and presented as mg
QE/g of extract.
Phenol and avonoid detection in plant fractions by HPLC–MS/MS analysis
One gram of dried Flemingia root powder and Mucuna seed powder were macerated in HPLC grade Methanol
for 48h. e extract was ltered by Whatman lter paper no. 1 and clear ltrate was used for the metabolome
analysis by HRLC-MS/MS. e metabolomics data generated was then searched for the phenol and avonoid
compounds. Detailed set up procedure for HPLC–MS/MS instrument for the analysis is given in supplementary
data le.
2, 2-Diphenyl-1-picrylhydrazyl radical (DPPH⋅) scavenging assay
DPPH⋅ scavenging assay was done as per the procedure explained by Tuba and Gulcin132 with some modication
as per Kedare and Singh133. Purple coloured DPPH⋅ solution was prepared in methanol till the absorbance was
achieved to 0.950 ± 0.025 at 517nm. 3mL methanol was added to 4, 8, 12, 16 and 20µg of plant extract followed
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by addition of 1mL DPPH⋅ solution. e reaction mixture vortexed and incubated at RT for 30min in the dark.
Absorbance of the pale yellow hydrazine product measured at 517nm with blank containing only methanol. IC50
values of samples were calculated along with the ascorbic acid, BHA and BHT standards.
2, 2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid radical (ABTS⋅+) scavenging assay
ABTS⋅+ scavenging activity of the plant extracts were determined by rst generating ABTS radical cation (ABTS⋅+)
by mixing 7mM ABTS and 2.45mM potassium persulfate in deionized water and kept at room temperature for
overnight (12–16h) and nally the absorbance of ABTS⋅+ was adjusted to 0.750 ± 0.025 at 734nm. Later 3mL
methanol and 1mL ABTS⋅+ solution was added to 2, 4, 6, 8 and 10µg of plant extract. Aer 10min of incuba-
tion at RT, the absorbance of decolorized/scavenged ABTS⋅+ was measured at 734nm with blank containing
only methanol134. IC50 values of samples were calculated along with the ascorbic acid, BHA and BHT standards.
N, N-dimethyl-p-phenylenediamine dihydrochloride radicle (DMPD⋅+) scavenging assay
DMPD cation radical (DMPD⋅+) generated by reacting DMPD with ferric chloride in acetate buer. For this
500µL of 100mM DMPD was added to 50mL of 0.1M acetate buer (pH 5.3) and then 100µL of ferric chloride
added to generate DMPD⋅+. Finally the absorbance of this solution was adjusted by using acetate buer or ferric
chloride to 0.900 ± 0.100 at 505nm. Now, 2mL of the DMPD⋅+ solution was added to 10, 20, 30, 40 and 50µL of
extract and incubated at RT for 10min and discoloration is noted at 505nm by using acetate buer as blank135.
IC50 values of samples were calculated along with the ascorbic acid, BHA and BHT standards.
Ferric ion reducing (Fe3+ → F e 2+) antioxidant power assay (FRAP)
e FRAP assay for formation of intense perl’s prussian blue complex of the Fe2+–ferricyanide complexes from
yellow coloured Fe3+–ferricyanide complexes by the reducing power of plant extract was also performed132.
Briey, dierent concentrations of plant extracts (5, 10, 20, 30, 40 and 60µg) was taken and reacted with 2.5mL
of 1% potassium ferricyanide in 2.5mL sodium phosphate buer (0.2M; pH 6.6) and incubated at 50°C for
20min. en 2.5mL of 10% trichloroacetic acid was added. 2.5mL of this reaction mixture was taken then
diluted with 2.5mL distilled water and 0.5mL of 0.1% ferric chloride was added. e absorption of the complex
was measured at 700nm.
β-carotene bleaching protection assay
A β-carotene bleaching assay was done by using protocol of Duan etal.136. Shortly, 1mg/mL β-carotene solution
was prepared in chloroform and 4mL of it was added to 45µL of linoleic acid and 365µL of tween-20. Chloroform
was evaporated and slowly 100mL oxygenated distilled water was added and vortexed to form emulsion and to
initiate the β-carotene bleaching. 4mL of it was added to 30µg of the plant extract and delay in discolouration
by plant extract was noted aer 60min of incubation for 45–50°C at 470nm.
Phosphomolybdenum method for total antioxidant activity (TAA)
In this method dierent concentration of plant extract (20, 40, 60, 80 and 100µg) was reacted with 5.4mL
phosphomolybdenum reagent made up of 28mM sodium phosphate, 4mM ammonium molybdate and 0.6M
sulfuric acid. e reaction mixture then incubated at high temperature of 95°C for 90min, cooled at room
temperature and subsequently the absorbance of green phosphate/Mo(V) complex formed noted at 695nm118.
DNA damage protection activity
DNA damage protection capacity of the plant extract from Fenton’s reagent was determined by using plasmid
DNA as explained by Kim83 with some modications. Briey, in the sequence, reaction mixture of 3µl of Plas-
mid DNA (0.35µg/mL), 9µl of 50mM sodium phosphate buer (pH 7.4), 2µl of 1mM FeSO4, 50µg of sample
and 3µl of 30% H2O2 was prepared. en the reaction mixture incubated at 37°C for 30min in the dark. 5µl
of it was loaded in 0.8% agarose gel with 1µl of 6 × DNA loading buer for electrophoresis for 60min at 85V
and 90mA. e bands generated were analyzed by using Image Lab soware and percent DNA protection was
calculated by comparing with control containing only plasmid DNA.
In vitro PDE5 inhibition activity
Rat lung homogenate was used as a source of PDE5 enzyme137. e homogenate (10% w/v) was centrifuged at
13,000rpm for 20min and supernatant was used as a source of enzyme for inhibition assay. e reaction mix-
ture was prepared in the following sequence. 100µg of plant extract in 5% DMSO was added to 2mL of 20mM
Tris–HCl (pH 8.0) containing 5mM MgCl2 followed by the addition of 100µL of enzyme extract. Finally, 100µL of
5mM 4-nitrophenyl phenylphosphonate substrate was added to initiate the reaction. Aer incubation of 60min
at 37°C, the absorbance of the hydrolysed product from substrate was noted at 400nm138 using 5% DMSO as
blank and compared with the sildenal as a positive control.
In vitro arginase inhibition activity
Arginase inhibition capacity of the plant extract was determined by using lung tissue homogenate as a source of
arginase by method developed by Iyamu etal.139. e reaction mixture including 100µL enzyme extract, 100µL
of 100mM MnCl2, 1mL of 50mM Tris–HCl (pH 7.5) and 50µL of 0.5M arginine substrate/ substrate with 50µL
plant extract (1mg/mL)/ substrate with 50µL DMSO (5%) incubated at 37ºC for 60min. en the reaction was
stopped by adding 1mL of 0.72M HCl, the solution was centrifuged for 5min at 5000rpm. 1mL of supernatant
was mixed with 2mL of 6% ninhydrin in ethanol. Finally, the solution was incubated at 60°C for 30min, cooled
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at RT and the formation of a reddish complex was noted at 505nm. e inhibition percent was calculated by
comparing the result of sample with control.
All experimental protocols were approved by the Institutional Animal Ethical Committee of Smt. Kishoritai
Bhoyar College of Pharmacy, Kamptee, Nagpur, Maharashtra (IAEC approval No. 853/IAEC/22-23/23). Quar-
antine procedures and animal maintenance followed the recommendations of CPCSEA (Committee for the
Purpose of Control and Supervision of Experiments on Animals) guidelines for laboratory animal facilities, and
the methods are reported in accordance with ARRIVE guidelines.
Statistical analysis
All the analyses were performed in triplicate experiments (n = 3). e results of TPC, TFC, TAA and FRAP were
calculated as mean of observations ± SD. Whereas for DPPH, ABTS and DMPD radical scavenging activities,
the means of IC50 ± SD was calculated. β-carotene bleaching assay, DNA damage protection assay and in-vitro
PDE5 and arginase inhibition capacity were calculated as mean of percent protection/inhibition ± SD. For den-
ing the statistical signicance between the observations, analysis of variance (ANOVA) and Tukey’s post-hoc
test was applied (p < 0.05) and for Pearson’s correlational studies between antioxidant tests, multcompview and
metan package in R were used.
Data availability
All data generated or analysed during this study are included in this published article and its supplementary
information le.
Received: 3 June 2023; Accepted: 3 November 2023
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Acknowledgements
We acknowledge the support from University Grant Commission, India for the fellowship received to SDK
(UGC-Ref. No.: 853/(OBC) (CSIR-UGC NET DEC. 2016)).
Author contributions
Conceptualization by D.P.G. and S.D.K., analysis by S.D.K. and S.S.U., interpretation of data and writing original
dra by S.D.K., reviewing and editing by D.P.G. and S.D.K. All authors have read and agreed to the published
version of the manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 023- 46705-9.
Correspondence and requests for materials should be addressed to D.P.G.
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