Phytomedicine Plus 1 (2021) 100020

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Phytomedicine Plus
journal homepage: www.elsevier.com/locate/phyplu

Phytomedicinal properties of Cynodon dactylon (L.) pers. (durva) in its

traditional preparation and extracts
Vandana Singh a, Anita Singh a, Inder Pal Singh b, B. Dinesh Kumar a,∗
a
ICMR – National Institute of Nutrition (NIN), Indian Council of Medical Research (ICMR), Ministry of Health & Family Welfare, Govt. of India, Jamai- Osmania PO,
Hyderabad 500007, Telangana, India
b
National Institute of Pharmaceutical Education and Research (NIPER) S.A.S. Nagar, Sector 67, S.A.S. Nagar Mohali 160062, Punjab, India

a r t i c l e i n f o a b s t r a c t

Keywords: Background: Pharmacotherapy with Cynodon dactylon (L.) Pers. (CdL) (Sanskrit name durva) has been described in
Phytomedicine Ayurvedic text. The traditional preparation of Cynodon dactylon, called swaras (juice), has rarely been evaluated
Chemical markers for its phytochemical quality and phytomedicinal activities.
Cynodon dactylon (L.) Pers.
Immunomodulation Purpose: In this study, we aimed to perform the characterisation of phytochemicals, quantification of chemical
Cytokines markers, and evaluation of in vitro and ex vivo biological activity of CdL juice (durva swaras).
Quality medicinal plant
Methods: The cultivated CdL (durva) was authenticated by a botanist, and macro–microscopic examination was
performed before bio-deposition. Fresh green juice (S) of CdL was prepared and further lyophilised (JL); addition-
ally, water (W), hydroalcoholic (HA), and alcoholic (A) extracts were prepared. The phytochemical constituents
in all extracts were qualitatively analysed using liquid chromatography–mass spectrometry. The chemical mark-
ers in JL were identified and quantified using high performance liquid chromatography. To optimise biological
activity, the age of the plant was evaluated as a factor influencing the antioxidant activity of S using an in vitro
DPPH assay. In addition, an ex vivo proliferation assay of mouse splenocytes was performed after treatment with
the extracts, and the effects of the extracts on immunomodulation and inflammatory cytokines were assessed.
Results: The specific morphological characteristics of CdL, namely narrowly linear leaves with light-green stem,
along with its macro–microscopic features were confirmed, and the plant specimen was bio-deposited. The phy-
toconstituents (range: 62–72 compounds) in all extracts were qualitatively confirmed. The chemical markers
p-coumaric acid (0.48%) and trans-ferulic acid (0.35%) in JL were quantified. Plants aged 6 months and above
showed the most potent activity. The free radical-scavenging activity of the extracts ranged from 60% to 75%; the
IC50 of the extracts (range: approximately 450–650 𝜇g/mL) was higher than that of vitamin C (2.8 𝜇g/mL). The
effective concentration for proliferation of splenocytes treated with JL and W (0.45–62.5 𝜇g/mL) varied widely
compared with that of splenocytes treated with A and HA (0.45–1.95 𝜇g/mL). Significant reductions in the levels
of pro-inflammatory (IFN-𝛾 and TNF-𝛼) and anti-inflammatory (IL-4) cytokines were observed after JL treatment
compared with those after concanavalin A treatment.
Conclusions: Our findings are of biological significance because we quantitatively confirmed the chemical mark-
ers in CdL. Moreover, the antioxidant and immunomodulatory activities of JL as well as its ability to reduce
inflammatory cytokines were determined.

Abbreviations: A, Alcoholic extract; ANOVA, Analysis of variance; BSI, Botanical Survey of India; CBA, Cytometric bead array; CdL, Cynodon dactylon (L.) Pers.;
DMSO, Dimethyl sulfoxide; DPPH, 2,2-diphenyl-1-picrylhydrazyl; EC, Effective concentration; FBS, Foetal bovine serum; GACP, Good agricultural and collection
practices; GC, Gas chromatography; HA, hydroalcoholic extract; HPLC, High Performance Liquid Chromatography; IAEC, Institutional Animal Ethics Committee;
IL-1𝛽, interleukin-1𝛽; IL4, interleukin-4; IL10, interleukin-10; INF-𝛾, Interferon-𝛾; JL, Lyophilised juice extract; LC–MS, liquid chromatography–mass spectrometry;
MTT, [3-(4, 5-di-methylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; S, Green fresh juice; SD, Standard deviation; SOP, Standard operating procedure; TNF-𝛼,
tumour necrosis factor-𝛼; TPL, The Plant List; W, Water extract.
∗
Corresponding author at: Scientist-G, HOD Drug Toxicology Division and NIN Animal Facility, ICMR - National Institute of Nutrition (NIN), Indian Council of
Medical Research (ICMR), Ministry of Health & Family Welfare, Govt. of India, Jamai-Osmania PO, Hyderabad, 500007, Telangana, India.
E-mail address: nindineshpct@gmail.com (B. Dinesh Kumar).

https://doi.org/10.1016/j.phyplu.2021.100020
Received 13 October 2020; Received in revised form 23 October 2020; Accepted 1 January 2021
2667-0313/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
V. Singh, A. Singh, I.P. Singh et al.
Introduction
Indian traditional medicine uses herbs and plant materials for the
treatment of chronic diseases. In traditional and modern medicine,
plants are used for their specific therapeutic activities, which depend
on the species, cultivar, parts, and age of the plant as well as the geo-
graphical location and agro–climatic conditions where it was cultivated
and collected. Moreover, the nature and preparation of plant extracts
affects their therapeutic activities (Moure et al., 2001; Charak Samhita
(Sastry, 2003a, 2006b); Ghasemzadeh et al., 2016; WHO, 2017). The
concept of rasayana from Ayurveda is generally considered to represent
the antioxidant activities of traditional preparations (Scartezzini and
Speroni, 2000). Nevertheless, there are no defined pharmacological
standards for these preparations or a scientific basis for their therapeutic
potential. Validation of the therapeutic effects of traditional plant-based
preparations is an emerging technical challenge in their development as
alternative medicines.
The therapeutic effect of plants is generally attributed to the ac-
tivity of phenolic compounds, flavonoids, tannins, lignins, or other
phytochemical constituents that can be extracted using polar and/or
non-polar solvents (Altemimi et al., 2017; Zhang et al., 2015).
WHO (2017) has established guidelines for maintaining the quality of
herbal medicines. In these guidelines, the presence of chemical markers
is a prerequisite.
Curcumin and bisdemethoxycurcumin in turmeric (Li et al., 2011),
anolide A in ashwagandha (Sangwan et al., 2008), and eugenol in clove
(Yun et al., 2010) are some examples of chemical markers used to de-
termine the quality of herbal medicines. It is important to record the
quality of herbal medicines and various ingredients in complex prepa-
rations to allow reproducibility of preparations and comply with regu-
latory requirements. Chromatographic techniques are considered ideal
for determining phytoconstituents and identifying chemical markers.
Among numerous plant species in India, durva or Cynodon dactylon
(L.) Pers. (CdL), which has variants such as neel, shwet, and gand, is
known to have therapeutic properties, as described in the Indian Ma-
teria Medica (Bhavprakash Nighantu) (Chunekar, 2009) and the book
written by Sivrajan and Balachandran (1994). In an ancient text de-
scribing Ayurvedic plants, written by Bhavmishra (1500–1600 AD),
the pharmacological properties of CdL are described as sheetaveerya
(anti-inflammatory), jeevaniya (antioxidant), vranya (wound-healing),
and aasrahrit (haemostatic) (Chunekar, 2009). Some of these proper-
ties have been validated to be anti-arrhythmic (Najafi et al., 2008), im-
munomodulatory (Mangathayarua et al., 2009), and diuretic activities
(Sadki et al., 2010). The Ayurvedic Pharmacopoeia of India (2004) rec-
ommends the traditional formulation of swaras (juice) owing to its po-
tential therapeutic activity; however, information on the therapeutic ac-
tivity of CdL juice is limited.
There are many approaches to evaluate the biological activities of
plants, including in vitro and ex vivo assays. The antioxidant activities of
the traditional plants Aegle marmelos (Raja and Khan, 2017), Aristolochia
longa (Omari et al., 2019), and Terminalia arjuna (Viswanatha et al.,
2010) have been confirmed by determining their free radical-scavenging
activity in vitro. In addition, the immunomodulatory activity of tradi-
tional formulations is gaining importance. Ex vivo models of splenocytes
have been used to assess the immunomodulatory potential of traditional
plants, including Terminalia bellerica, Gentiana kurroo, and Ocimum sanc-
tum (Saraphanchotiwitthaya and Ingkaninan, 2014; Mubashir et al.,
2017; Hemalatha et al., 2011).
Recently, it has been shown that in assessing the safety and efficacy
of a formulation, it is important to correlate the results obtained from
in vitro and ex vivo experiments (Moleiro et al., 2017). Therefore, in the
present study, specific chemical markers, phytochemical constituents,
in vitro (free radical-scavenging) and ex vivo (splenocyte proliferation)
activities, and the ability of CdL juice to reduce the levels of inflamma-
tory cytokines were evaluated and compared to those of conventional
extracts.
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Phytomedicine Plus 1 (2021) 100020
Materials and methods
Chemicals
High-purity 2,2-diphenyl-1-picrylhydrazyl (cat-D913-2), phosphate-
buffered saline (pH 7.4) (cat 806552), dimethyl sulfoxide (DMSO)(cat
276855), p-coumaric acid (C9008), trans-ferulic acid (128708), 3,4-
dihydroxybenzoic acid (37580), 4-hydroxybenzoic acid (240141), and
ascorbic acid (A-7506) were purchased from Sigma Aldrich (USA).
Methanol (Qualigens, 32407), concanavalin A RM4157, 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (CAT-
TC191), foetal bovine serum (FBS), and RPMI 1640 were purchased
from HiMedia (Mumbai, India). Anti-Anti (100X) was obtained from
Gibco, Thermo Fisher Scientific (India). Mouse cytokine cytometric bead
array (CBA) kits were purchased from BD Biosciences (San Jose, CA).
Trypan blue was obtained from Merck (India).
Identification, re-cultivation, and collection of test materials
Durva is listed as the Sanskrit name of Cynodon dactylon (L.) Pers.
in the Plant List (TPL). This was identified based on the morpho-
logical features described in the Ayurvedic text Bhavprakash Nighantu
(Chunekar, 2009). The macro and microscopic features were confirmed
based on the Ayurvedic Pharmacopoeia of India (2004) and the study
by Vandana (2013). The plant material was collected from its wild nat-
ural habitat around Hyderabad, a city in southern India. CdL was iden-
tified and confirmed by a taxonomist at the Botanical Survey of India
(BSI), Hyderabad. A sample of the test material was deposited at the
BSI, Hyderabad. The material was re-cultivated in autumn in a garden
(contamination-free soil) at the institute. The plants were re-cultivated
in the designated area with adequate sunlight and water supply, and
they were protected using fertiliser spray according to the good agricul-
tural and collection practices (GACP). The CdL was collected at different
time points (30, 60, 120, 180, 240, 300, and 360 days) to investigate
antioxidant activity as a function of plant age.
Standard operating procedures (SOPs) were developed to prepare
the juice (swaras) in accordance with traditional texts. Briefly, the clean
whole plant was soaked in water for 15 min to remove any leftover
mud or other particles, followed by soaking in 0.9% normal saline to re-
move any microorganisms. After repeated washing with distilled water,
the whole plant was chopped and dried on a blotting paper (Whatman
460 × 570 mm, thickness 0.33 mm). Freshly chopped material was used
to prepare the juice. The remaining chopped material was dried at room
temperature (30°C) in shade for 5 days and then ground into a powder
using a cutting mill (Retsch SM 100, Germany). The aperture size of the
sieve was 0.5 mm. The powdered plant material was used for extract
preparation.
Traditional swaras preparation
Dried (250± 10 g) green plant material was ground with 400 ±20
mL distilled water for 10 min in a mixer to obtain a homogenous paste.
The paste was strained through a nylon double net (size PP3) to obtain
the juice. The resultant green fresh juice (S) was also lyophilised (JL),
and both preparations were used for subsequent experiments.
Water extract (W)
Powdered plant material (5 g) was hydrated in 100 mL of MilliQ
water for 60 min and then heated at 50°C to reduce the volume to 25
mL. This extract was lyophilised (Scanvac cool safe 110-4, Denmark)
and used in subsequent experiments.
V. Singh, A. Singh, I.P. Singh et al.
Alcoholic extract
The alcoholic extract (A) was prepared by soaking 5 g of powdered
plant material in 100 mL of absolute alcohol for 48 h, followed by evap-
oration (Buchi R-205 Rotavapor System, Switzerland).
Hydroalcoholic extract
To prepare the hydroalcoholic extract (HA), 5 g of powdered plant
material was soaked in 100 mL of hydroalcoholic solution (ethanol: wa-
ter = 1:1) for 48 h, followed by evaporation and lyophilisation.
Phytochemical profile assessment
The phytochemical profile of the extracts was assessed using liquid
chromatography–mass spectrometry (LC–MS), and the chemical mark-
ers were determined using high performance liquid chromatography
(HPLC).
Total chemical composition profile
The total chemical constituent profiles of the crude extracts (JL, W,
A, and HA) were determined using LC–MS. Briefly, each extract was
dissolved in methanol, and 10 μL of each sample was injected into an LC–
MS system (Shimadzu, Japan) equipped with an X Select CSH Phenyl-
hexyl column. The gradient solvent system consisted of mobile phase A
(0.1% formic acid with water) and B (0.1% formic acid with acetonitrile)
at ratios of 0.01:5, 2.50:5, 7:40, 12.50:60, 20:95, 24.0:95, 25.50:5, and
32.0:5. The MS spectra were acquired in the positive or negative ion
mode to detect metabolites, including polar, semipolar, and non-polar
compounds, using an X-Bridge C18 column (250 × 4.60 mm, 5.0 μm).
Chemical markers
The active chemical markers of JL were analysed qualitatively and
quantitatively. To prepare the JL extract, 1 g of JL was mixed with 20
mL of ethyl acetate. The mixture was sonicated for 20 min three times,
and the solvent was evaporated to obtain 30 mg of the extract. The resul-
tant extract was then dissolved in 1 mL of methanol for HPLC using an
LC-20 AD system (Shimadzu Corporation, Kyoto, Japan) fitted with an
auto sampler and a photodiode-array detector. The sample was filtered
through a 0.22 μm filter and separated using an LC–gas chromatography
(GC) column (5 μm C18, 250 × 4.6 mm; InertSustain; GL sciences Inc.,
Japan) with a gradient mobile phase comprising acetonitrile (A) and wa-
ter containing 0.1% formic acid (B) at a flow rate of 1 mL/min at 30° C.
The standard retention times were 33.6 and 36.2 min for trans-ferulic
acid and p-coumaric acid, respectively. The extracts were evaluated for
the presence of 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, cat-
echin, p-coumaric acid, and trans-ferulic acid.
The chromatograms obtained for S and reconstituted JL were as-
sessed similarly using the described methodology, with the exception
that JL was dissolved in water.
Antioxidant activity
The free radical-scavenging activities of the extracts were deter-
mined using the method described by Blois (1958). JL, A, HA, and W
3
Phytomedicine Plus 1 (2021) 100020
Fig. 1. Morphological features of CdL (durva) in Ayurvedic
literature: golomi, shwetadanda, sahasraparva (Sanskrit
terms). Sanskrit name: durva; Hindi name: doob; English
name: couch grass; botanical name: Cynodon dactylon (L.)
Pers.; Family: Poaceae
were prepared at concentrations of 50–400 μg/200 μL and mixed with
50 𝜇L of 0.2 mM DPPH in methanol. Ascorbic acid (0.2–3 μg/200 μL)
was used as a standard. Each extract mixture was incubated in the dark
for 30 min at room temperature (30°C). The absorbance was measured
at 540 nm using a multimode reader (Synergy H1 hybrid reader; Biotek,
Canada) and calculated using the following equation:
DPPH radical-scavenging activity (%) = ([A0–A1/A0] × 100)
where A0 is the absorbance of the control, and A1 is the absorbance
of the extract or standard sample to determine IC50 . The free radical-
scavenging activity of S was considered a potential biological marker;
thus, this activity was determined in S prepared from plants collected at
30, 60, 120, 180, 240, 300, and 360 days of cultivation.
Immunomodulation activity
Splenocytes from healthy, naive, female Swiss albino mice were used
as explants. The splenocyte proliferation assay was conducted using an
established method (Snyder and Valle, 1991).
Splenocyte proliferation assay
The spleens were collected from healthy, 8- to 10-week-old, naive
female Swiss albino mice weighing 18–22 g and placed in RPMI
1650 medium. All experimental procedures involving animals were
approved by the IAEC (P28F/III-IAEC/NIN/12/2016/BDK/C57BL/6-
21+21/Swiss-60F). Under aseptic conditions, the spleen samples were
teased, dispersed, and passed through a fine steel mesh to obtain ho-
mogenous cell suspension/explants. The explants were harvested in
RPMI 1640 medium to prepare a single-cell suspension. The suspension
was centrifuged at 500 × g and 4° C for 5 min to obtain a cell pellet,
which was then incubated in a medium containing 1% antibiotic solu-
tion and 10% FBS. The cells at a density of approximately 14 × 106
cells/mL were assessed for viability with a haemocytometer using try-
pan blue staining and observed using an inverted microscope (EVOS FL
colour imaging system; AMEFC4300, Thermo Fisher scientific, Europe).
An MTT assay was performed in accordance with a method previously
described by Mossman (1983). The maximum concentration of mito-
gen (concanavalin A) was determined using the MTT assay. Spleen cell
proliferation was indicated by the presence of formazan after reaction
with DMSO and determined by measuring the absorbance of the culture
wells at 570 nm using a multimode reader Synergy H1 hybrid reader
(Biotek, Canada). The maximum effective concentration (EC) was deter-
mined using the dose–response curve of each extract (JL, W, A, and HA)
at concentrations between 0.025 and 500 μg/mL. The control samples
were without any test extracts. All samples were measured in triplicates.
Cytokine profiles
A multiplex flow cytometry assay was performed using a murine
CBA kit to measure pro-inflammatory (IFN-𝛾, TNF-𝛼, and IL-1𝛽) and
anti-inflammatory (IL4 and IL10) cytokines. Splenocytes (approximately
4 × 106 /mL) were added to each well of a microtiter plate and then
treated with the T-cell mitogen concanavalin A (10 μg/mL). Each test
material (JL, W, A, and HA; all at a concentration of 0.45 μg/mL) was
V. Singh, A. Singh, I.P. Singh et al. Phytomedicine Plus 1 (2021) 100020

Fig. 2. Liquid chromatography–mass spectrometry chromatogram of CdL whole-plant extracts (conventional extracts: W, water extract; A, alcoholic extract; HA,
hydroalcoholic extract. Traditional extract: JL, lyophilised powder of CdL juice)

Table 1
Qualitative phytochemical constituent profile of CdL extracts as determined by LC–MS
analysis. Some selected peak with % area and retention time (RT).

S. No W A HA JL
RT % area RT % area RT % area RT %area

1. 2.63 7.5 2.61 0.67 2.61 1.49 2.62 5.45

2 19.34 10.38 18.45 5.11 19.33 10.05 18.43 7.24
3. 19.73 4.82 19.34 10.68 19.72 6.49 18.53 3.88
4. 20.694 6.59 19.72 6.98 20.58 4.14 19.32 11.19
5 21.38 3.85 20.59 4.09 - - 19.71 5.25
6 22.02 13.26 21.95 9.29 - - 21.11 4.29
7. 22.78 2.26 22.31 4.71 - - 47.86 8.29
8. - - 47.87 6.46 - - - -

Conventional extracts: W, water extract; A, alcoholic extract; HA, hydroalcoholic ex-

tract. Traditional extract: JL of CdL juice.

added to six wells (4 × 6). Negative control samples (without test ex- for Windows (IBM, USA). The results are presented as the mean ± SD.
tract and mitogen) and positive control samples (mitogen only) were A P-value < 0.05 was considered to indicate statistical significance.
distributed equally in the other 12 wells (2 × 6). All cells were incu-
bated for 72 h. Next, they were centrifuged at 3000rpm for 15 minutes
Results
at 22°C, and the supernatant was collected from each group for cytokine
analysis using flow cytometry. All samples were analysed in duplicate.
Collection, identification, and authentication of CdL

Statistical analysis CdL (durva) was identified based on the following characteristics:
macroscopic confirmation viz., shwetadanda (light-green stem), golomi
One-way analysis of variance (ANOVA) and repeated ANOVA fol- (narrowly linear leaves), and sahasraparva (multiple nodes, Fig. 1). Mi-
lowed by post-hoc tests were performed using the SPSS 21.0 software croscopic observations revealed distinct endodermis and vascular bun-

4
V. Singh, A. Singh, I.P. Singh et al.
Fig. 3. (A) HPLC chromatogram of chemical markers in JL of Cynodon dactylon (L.) pers., (durva) whole plant. (B) Superimposed HPLC chromatograms of CdL in S
and JL. S, green fresh juice of whole plant; JL, lyophilised powder of S.
dles arranged in a ring on different radials; wide pith was confirmed
in the root. The herbarium has been deposited in the BSI, Hyderabad
(BSI/DRC/17-18/Tech./212).
Extract yield
The optimum swaras concentration was obtained by using 200 g of
fresh CdL per 320 mL of distilled water. The yield of JL was 5%–7%.
The yields of the other extracts were as follows: 15.5% for W; 9.5% for
A; and 12.07% for HA.
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Phytomedicine Plus 1 (2021) 100020
Phytoconstituent profiles
The phytoconstituents of JL (72 compounds), W (62 compounds),
HA (64 compounds), and A (68 compounds) were identified using non-
polar, semipolar, and polar solvents based on molecular weight and re-
tention time. 3, 4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, cat-
echin, p-coumaric acid, and trans-ferulic acid were the most abun-
dant compounds. p-coumaric acid was not detected in JL, whereas p-
coumaric acid, and trans-ferulic acid were not detected in A & W (Fig. 2,
Table 1).
V. Singh, A. Singh, I.P. Singh et al.
Table 2
IC50 values of JL, W, A, and HA
W HA
IC50 (𝜇g/mL) 464.0 ± 25.99 a
447.5 ± 30.24
Values are expressed as the mean ± SD of triplicate experiments on three different occasions. Superscript
letters indicate significant differences at P<0.05, as determined using one-way ANOVA followed by mul-
tiple comparisons (post-hoc test). JL vs. W, HA, and vitamin C: P<0.001; JL vs. A: P<0.05. Conventional
extracts: W, water extract; A, alcoholic extract; HA, hydroalcoholic extract. Traditional extract: JL of
CdL juice
Fig. 4. Biological markers (antioxidant activity) in fresh juice (S) prepared us-
ing CdL of different ages [values are expressed as the mean ± SD of samples
measured in triplicate on two occasions, as determined by repeated ANOVA
test, followed by multiple comparisons (post-hoc test)]. ∗ Significant reduction
on day 60 compared with day 30.
Chemical markers
trans-ferulic acid (0.35%, 𝜆max = 323 nm) and p-coumaric acid
(0.48%, 𝜆max = 323 nm) were identified using HPLC in the ethyl acetate
extract of JL (Fig. 3A). The correlation coefficient (r2 ) was calculated as
0.99 for both the compounds. Superimposed HPLC chromatograms of S
and JL suggested an insignificant difference in their profiles (Fig. 3B).
Antioxidant potential of CdL at different ages
The IC50 of S consistently decreased (19–8 mg/mL) with increase in
the age of the plant. There was no significant reduction in IC50 (8.4.8.1
mg/mL) in S prepared using CdL older than 6 months to 1 year (Fig. 4).
Free radical-scavenging activity of CdL extracts
The most potent free radical-scavenging activity was shown by JL
(72%), followed by HA (69%), A (66%), and W (61%) at concentra-
tions of 0.7–1.5 mg/mL (Fig. 5). The free radical-scavenging activity of
5 μg/mL vitamin C was 76%. All extracts showed a dose-dependent inhi-
bition of free radicals at 0.25–1.5 mg/mL (Fig. 6). The IC50 of JL (651.9
± 19.18 𝜇g/mL), W (464.0 ± 25.99 𝜇g/mL), A (580.9 ± 60.51 𝜇g/mL),
and HA (447.5 ± 30.24 𝜇g/mL) was higher than that of vitamin C (2.8
± 0.08 𝜇g/mL)(Table 2). There was a significant (p<0.05) correlation
Phytomedicine Plus 1 (2021) 100020
A JL Vit C
a
580.9 ±60.51 b
651.9 ± 19.18 c
2.8 ± 0.08d
Fig. 5. Potential free radical-scavenging activity (%) of JL, HA, A, and W com-
pared with that of the standard, ascorbic acid (vitamin C). Values are expressed
as the mean ± SD of triplicate measurements on three different occasions, as de-
termined by one-way ANOVA followed by multiple comparisons (post-hoc test).
Values with different superscript letters are significantly different at P<0.05. JL
vs. W, P<0.001; JL vs. A and Vit C, P<0.05. Conventional extracts: W, water
extract; A, alcoholic extract; HA, hydroalcoholic extract. Traditional extract: JL,
CdL juice.
between the percentage of scavenging activity and the concentrations
of JL (r = 0.93) and HA (r=0.72; Fig. 7). These findings demonstrate the
potency of JL and HA extracts in vitro.
Determination of effective concentration using MTT assay
There were changes in cell proliferation after treatment with JL and
W at ECs of 0.45–62.5 μg/mL and with HA and A at 0.45–1.95 μg/mL
(Fig. 8). The spleen cell morphology is presented in Figure 9.
Cytokine estimation
Pro-inflammatory (IFN-𝛾, TNF-𝛼, and IL-1𝛽) and anti-inflammatory
(IL4 and IL10) cytokine levels were measured at an EC of 0.45 μg/mL.
TNF-𝛼 and IFN-𝛾 levels were reduced by approximately 34% after JL
treatment and by 25%–28% after treatment with W, A, and HA, com-
pared with that after concanavalin A treatment. IL-10 cytokine levels
decreased after treatment with JL, W, A, and HA when compared with
that after concanavalin A treatment but the decrease was not significant.
6
V. Singh, A. Singh, I.P. Singh et al.
Fig. 6. Free radical-scavenging activity (%) profile of JL, HA, A, and W at dif-
ferent concentrations. The values are expressed as the mean ± SD of triplicate
measurements on three different occasions. Conventional extracts: W, water ex-
tract; A, alcoholic extract; HA, hydroalcoholic extract. Traditional extract: JL,
CdL juice.
IL-1𝛽 levels reduced after treatment with JL, W, A, and HA, but it was
not significant (Fig. 10).
Discussion
Durva or CdL is a highly revered plant in India. Cynodon dacty-
lon (L.) Pers. has been described in the TPL with a high confi-
dence level. Its medicinal value has been recognized since the pre-
historic Vedic period (Dvivedi, 2009) and revisited more recently
by Mangathayaru et al. (2009) and Jazani et al. (2011). There is a
sloka (a classical Sanskrit description) that reads as follows: ‘Door-
vati hinasti visarpadeen rogan iti, hinsayam doorvyate hinsyate pashub-
hih iti doorva,’ which means that CdL relieves diseases such as visarpa
(erysipelas)(Shastri, 2001).
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Phytomedicine Plus 1 (2021) 100020
Fig. 8. Effective concentration (% splenocyte proliferation) of CdL extracts (JL,
HA, A, and W). Spleen cells were incubated with extracts at various concentra-
tions (0.45–62.5 μg/mL). Values are expressed as the mean ± SD of triplicate
measurements on three different occasions. Conventional extracts: W, water ex-
tract; A, alcoholic extract; HA, hydroalcoholic extract. Traditional extract: JL,
CdL juice.
According to WHO (2017) guidelines, the CdL identified in this
study was re-cultivated in compliance with GACP. The microscopic
observations were comparable to those of our earlier investigations
(Vandana, 2013). In the present study, to investigate the therapeu-
tic activities of CdL juice, traditional pharmaceutical preparations and
conventional extracts of CdL were prepared, and SOPs were devel-
oped for the collection of the whole plant and preparation of the
test materials. In addition, we ensured that the test materials were
free of contaminants, in accordance with the procedures described
in WHO standard guidelines. Besides plant identification, we also
identified the chemical markers among the complex phytochemical
constituents of CdL and evaluated the therapeutic activity of tradi-
tional formulations. Chromatographic similarities between S and JL
suggested that there was no loss of components that are respon-
sible for the therapeutic activities of S, which is the traditional
formulation.
Fig. 7. Pearson’s correlation coefficient (R) between free radical-
scavenging percentage and CdL extract concentrations (JL, HA, A,
and W). ∗ P<0.05, ∗ ∗ P<0.01. Conventional extracts: W, water extract;
A, alcoholic extract; HA, hydroalcoholic extract. Traditional extract:
JL, CdL juice.
V. Singh, A. Singh, I.P. Singh et al.
Fig. 9. Splenocyte proliferation observed under an inverted microscope at 4 × magnification (A) Spleen cells, (B) Spleen cells treated with CdL extracts, (C) Formazan
formation, (D) Dissolved formazan, (E) Spleen cells without mitogen, (F) Spleen cells with the mitogen concanavalin A.
The pharmacological properties of plants depend on their phyto-
chemical constituents. Min Lee et al. (2017) reported that the analy-
sis of phytochemical constituents using LC–MS can provide complete
information on metabolites and their isomers, although the number of
compounds that can be identified is limited in an untargeted analysis.
In the current study, by using untargeted LC–MS analysis, we identified
polar, semipolar, and non-polar phytochemical constituents; the num-
ber of compounds identified from the different extracts ranged between
62 and 72. Shabi et al. (2010) reported 22 compounds in the HA of
C. dactylon using GC–MS analysis. Bagewadi et al. (2014) reported 17
compounds in the A extract of Cynodon dactylon using LC–MS analysis.
Truong et al. (2019) observed that the bioactive compounds in plant ma-
terials varied according to their solubility in different solvents. Besides
the macroscopic and microscopic features of plants, Vandana (2013) re-
vealed the presence of various phytochemical constituents in CdL (e.g.,
phenols, alkaloids, and steroids) using qualitative analysis. We believe
that these data are useful in setting the standards for chemical mak-
ers and their relative abundance for the authentication of CdL prepara-
tions. As per the recommendations of international agencies, uniform
methodologies and sample quality are crucial indicators of potential
markers and pharmacological activities of plants intended for medicinal
use.
Mika et al. (2005) reported that plants belonging to the Poaceae fam-
ily have abundant phytochemical constituents, including phenolic com-
pounds. Among these compounds, p-coumaric acid, trans-ferulic acid,
3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, and catechin are
commonly reported (Verma et al., 2011). Cynodon. Dactylon also be-
longs to this family. Karthikeyan et al. (2015) isolated p-coumaric acid-
containing fractions of CdL. In the current study, the presence of these
constituents was qualitatively confirmed in various CdL extracts. A study
on the ratio of non-marker to marker compounds could provide further
insights into the identification of the plant source.
In view of the above findings, our study focused on the identifica-
tion of p-coumaric acid and trans-ferulic acid as standard chemical mark-
ers. We successfully identified p-coumaric acid (0.48%) and trans-ferulic
acid (0.35%) in the ethyl acetate extract of the traditional preparation S
and the lyophilised powder JL using HPLC analysis. This analysis has the
potential to be qualified to become a part of the regulatory requirements
for confirming the quality of CdL juice in future studies.
8
Phytomedicine Plus 1 (2021) 100020
The antioxidant activity of the HA and methanolic extracts of Cyn-
odon dactylon is well established (Poojary et al., 2016; Krishanti et al.,
2010). Our aim was to develop the juice of CdL as a standardized tra-
ditional pharmaceutical formulation for therapeutic use in the treat-
ment of chronic diseases. Significant correlations between free radical-
scavenging activity and extract concentration were observed only in the
traditional preparation and HA. However, contradictory findings on the
antioxidant activity of CdL extracted using various solvents have been
reported (Khlifi et al., 2013; Smitha et al., 2014). In the present study,
the most potent antioxidant activity was shown by JL, followed by HA,
A, and W.
Surinrut et al. (2005) classified the antioxidant potential of vari-
ous fruit juices (e.g., mango, grape skins, lichee, carambola mulber-
ries, and guava) based on their DPPH-scavenging activity (expressed as
IC50 values) as follows: strong (1.10–9.60 mg/mL), moderate (11.18–
32.80 mg/mL), and low (50.62–110.46 mg/mL). Based on this classi-
fication, S showed a strong antioxidant effect (IC50 : 8 mg/mL). This
finding suggested that the jeevaniya (antioxidant) property of CdL led to
the rasayana (immunomodulatory) outcome, thereby emphasizing the
therapeutic importance of CdL.
The free radical-scavenging activity of 1.7 mg/mL JL (approximately
72%–76%) was comparable to that of 5 μg/mL vitamin C. This result
further validated the biological activity of the lyophilised powder and
traditional formulation, supporting the intended medicinal benefit of
CdL.
We optimized the green fresh juice (S) by using 6-month-old CdL
plants, which showed the most potent free radical-scavenging activity.
The results of the study by Ghasemzadeh et al. (2016) supported our
finding that biological activity varied with the age and growth stage of
a plant.
The immunomodulatory (rasayana) (Mangathayaru et al., 2009) and
anti-inflammatory (Garg and Paliwal, 2011) activities of Cynodon dacty-
lon juice have been confirmed in mice and rat models. In the present
study, the EC for proliferation of spleen cells after treatment with JL
and W was 0.45–62.5 μg/mL without cell death. However, the ECs for
A and HA was 0.45–1.95 μg/mL, which resulted in cytotoxicity. Our
experimental procedure was consistent with that of Allen et al. (2005),
who assessed the toxicity profile in tissues before the analysis of cytokine
profile.
V. Singh, A. Singh, I.P. Singh et al. Phytomedicine Plus 1 (2021) 100020

Fig. 10. Cytokine (pg/mL) profiles determined using flow cytometry. A. TNF-𝛼; B. IFN-𝛾; C. IL-𝛽; D. IL-4; E. IL-10. Effect of CdL extracts (JL, HA, A, and W; 0.45
μg/mL) compared with that of 10 μg/mL concanavalin A (C-A) on the regulation of cytokine level. Values are expressed as the mean ± SD in the supernatant of two
pooled samples of six each. Values with different superscript letters are significantly different at P<0.05. All control vs. C-A: P<0.001, except for IL-𝛽.

The levels of pro-inflammatory (IFN-𝛾, TNF-𝛼,) and anti-
inflammatory (IL-4 and IL-10) cytokines were significantly reduced
after treatment with all extracts, except for those of IL-1𝛽 and IL-
10, which contradicts the findings of Ilkhanizadeh et al. (2016).
This contradiction may have occurred because cytokine levels were
measured in the serum of NMRI-REV1-challenged mice in the pre-
vious study. Based on IL-13, IL-6, IL-4, and TNF levels, Pripp and
Stanis (2014) reported that blood and haematoma showed differ-
ent pro- and anti-inflammatory reactions as local inflammatory
responses. Kowsar et al. (2019) reviewed the cross-regulation between
pro- and anti-inflammatory cytokines (IL-12 and IL-1𝛽 versus IL-4
and IL-10, respectively) and found that this cross-regulation was
due to unknown interactions between pro- and anti-inflammatory
genes.

9
V. Singh, A. Singh, I.P. Singh et al.
Notably, five types of pharmaceutical preparations are described in
Ayurvedic text as ‘ Panchavidha Kasaya Kalpana’ to assign potential ther-
apeutic properties based on the preparation method of the selected plant
(Sastry edt. Charak Samhita, 2003c). Jaziri et al. (2016) observed a sig-
nificant correlation between phenolic compounds (caffeic, ferulic, and
p-coumaric acid) and antioxidant and immunomodulatory activities. We
have conceptualized the traditionally described concepts and biolog-
ical activities of CdL by documenting chemical markers in juice and
its lyophilised powder. The study scope would have been increased if
the markers are analysed on LC-MS-MS. The in vivo biological activities
could have added further therapeutic potential CdL.
Conclusion
The juice (swaras) of CdL has potential antioxidant (jeevaniya),
wound healing (vranya), and anti-inflammatory (sheetaveerya) proper-
ties as per traditional pharmaceutical preparation described in classi-
cal Ayurvedic texts. p-coumaric acid and trans-ferulic acid as the main
chemical markers along with other phytoconstituents will determine the
quality of CdL. These findings will greatly benefit the development of
safe, effective, and economically viable therapeutic formulations of CdL.
Author contributions statement
Vandana Singh contributed to the innovation, planning, and exper-
imental procedures of this study, as well as manuscript writing. Anita
Singh supported the experimental procedures and reference work. Inder
Pal Singh provided analytical support for chemical profiling. B. Dinesh
Kumar, senior author, facilitated the design, planning, and experimental
procedures of this study, as well as manuscript writing. All the authors
have read and approved the final manuscript.
Funding
This work was supported by the Indian Council of Medical Research
(5/3/ITR-F/2016-ITR).
Declaration of Competing Interest
The authors declare that there are no conflicts of interest.
Acknowledgments
The authors are grateful to the Indian Council of Medical Research
for the fellowship of ICMR-SRF (5/3/ITR-F/2016-ITR). We thank Dr. R.
Hemalatha (Director of the ICMR-NIN) for her constant encouragement.
We would also like to acknowledge the technical support of Dr. K. Suresh
Babu (Principal Scientist) and Mr. Kiran Kumar Tatapudi (Technical As-
sistant of the CSIR-IICT, Hyderabad) for LC–MS, Mr. K. Prasanna (JTA,
NIPER-SAS Nagar) for providing technical support in performing HPLC,
Dr. B. Neeta Kumari (Executive Director, Industrial Graphite) for assist-
ing with LC–MS data interpretation, and Mr. Vijaya Bhaskar (ICMR-NIN)
for his support in statistical analysis.
ICMR-NIN communication number Lib/Pub/17/2020.
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