molecules

Article
Polyphenol Rich Ajuga bracteosa Transgenic Regenerants
Display Better Pharmacological Potential
Samina Rubnawaz 1, * , Waqas Khan Kayani 2 , Nosheen Akhtar 3 , Rashid Mahmood 4 , Asif Khan 5 ,
Mohammad K. Okla 6 , Saud A. Alamri 6 , Ibrahim A. Alaraidh 6 , Yasmeen A. Alwasel 6 and Bushra Mirza 1

1 Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University,

Islamabad 45320, Pakistan; [email protected]
2 Department of Biotechnology, Faculty of Sciences, University of Kotli,
Azad Jammu and Kashmir 11100, Pakistan; [email protected]
3 Department of Biological Sciences, National University of Medical Sciences, Rawalpindi 46000, Pakistan;
[email protected]
4 Drugs Control & Traditional Medicines Division, National Institute of Health, Islamabad 45320, Pakistan;
[email protected]
5 Institute of Biological Sciences, Faculty of Sciences, University of Malaya, Kuala Lumper 50603, Malaysia;
[email protected]
6 Botany and Microbiology Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia;
[email protected] (M.K.O.); [email protected] (S.A.A.); [email protected] (I.A.A.);
[email protected] (Y.A.A.)



* Correspondence: [email protected]



Citation: Rubnawaz, S.; Kayani, Abstract: Ajuga bracteosa Wall. ex Benth. is an endangered medicinal herb traditionally used against
W.K.; Akhtar, N.; Mahmood, R.; Khan, different ailments. The present study aimed to create new insight into the fundamental mechanisms
A.; Okla, M.K.; Alamri, S.A.; Alaraidh, of genetic transformation and the biological activities of this plant. We transformed the A. bracteosa
I.A.; Alwasel, Y.A.; Mirza, B.
plant with rol genes of Agrobacterium rhizogenes and raised the regenerants from the hairy roots. These
Polyphenol Rich Ajuga bracteosa
transgenic regenerants were screened for in vitro antioxidant activities, a range of in vivo assays,
Transgenic Regenerants Display
elemental analysis, polyphenol content, and different phytochemicals found through HPLC. Among
Better Pharmacological Potential.
Molecules 2021, 26, 4874.
18 polyphenolic standards, kaempferol was most abundant in all transgenic lines. Furthermore,
https://doi.org/10.3390/ transgenic line 3 (ABRL3) showed maximum phenolics and flavonoids content among all tested plant
molecules26164874 extracts. ABRL3 also demonstrated the highest total antioxidant capacity (8.16 ± 1 µg AAE/mg),
total reducing power, (6.60 ± 1.17 µg AAE/mg), DPPH activity (IC50 = 59.5 ± 0.8 µg/mL), hydroxyl
Academic Editor: Francesco Cacciola ion scavenging (IC50 = 122.5 ± 0.90 µg/mL), and iron-chelating power (IC50 = 154.8 ± 2 µg/mL).
Moreover, transformed plant extracts produced significant analgesic, anti-inflammatory, anticoagu-
Received: 23 June 2021 lant, and antidepressant activities in BALB/c mice models. In conclusion, transgenic regenerants of
Accepted: 7 August 2021 A. bracteosa pose better antioxidant and pharmacological properties under the effect of rol genes as
Published: 11 August 2021
compared to wild-type plants.
Corrected: 5 May 2022

Keywords: Ajuga bracteosa; genetic transformation; antioxidants; polyphenols; metabolic profiling;

Publisher’s Note: MDPI stays neutral
RP-HPLC; pharmaceutical properties; BALB/c mice
with regard to jurisdictional claims in
published maps and institutional affil-
iations.

1. Introduction
Medicinal plants have been used in traditional medicines for thousands of years
and they contain a wide variety of biologically active plant products called secondary
Copyright: © 2021 by the authors.
metabolites. In the last decade, the potential toxicity of synthetic drugs led to a resurgence of
Licensee MDPI, Basel, Switzerland.
This article is an open access article
the use of these metabolites as a facile and economical alternative approach [1]. According
distributed under the terms and
to the World Health Organization (WHO), approximately, 80% population of developing
conditions of the Creative Commons countries depends on plant products to alleviate and treat serious ailments [2].
Attribution (CC BY) license (https:// Free radicals, such as reactive oxygen and nitrogen species, produced in the human
creativecommons.org/licenses/by/ body can be a root cause of aging, rheumatism, malignancies, diabetes, cardiovascular,
4.0/).

Molecules 2021, 26, 4874. https://doi.org/10.3390/molecules26164874 https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 4874 2 of 19

and neurodegenerative disorders [3]. A higher rate of incidence of these diseases trig-
gered the research of natural antioxidants and various studies have suggested that dietary
polyphenols are the most abundant antioxidants in nature [4]. These polyphenols can
directly scavenge free radicals, chelate metal ions, and inhibit their pro-oxidant activities,
thus reducing the risk of chronic metabolic diseases. Moreover, polyphenols also possess
anti-inflammatory, anti-thrombotic, and analgesic activities [5]. Likewise, different trace
elements not only improve plant immunity but also provide scaffolding for antioxidant
enzymes as cofactors in humans. Hence, it is a dire need to thoroughly investigate different
herbal extracts for their antioxidant properties to develop new drugs [6].
Ajuga bracteosa is considered an elixir to a variety of ailments and it is found in hilly
areas of Pakistan, Nepal, Kashmir, India, and the Himalayan region. An extensive litera-
ture survey reveals that different extracts of A. bracteosa have a variety of pharmacological
activities. It is used to cure skin infections, respiratory issues, digestive problems, malaria,
protozoal diseases, diabetes mellitus, hepatitis, arthritis, epilepsy, inflammation, neuro-
logical disorders, and cancer [7–10]. This medicinal importance is due to a repertoire of
metabolites such as essential oils, ecdysteroids, terpenoids, phenolics, flavonoids, and
withanolides characterized in this plant [11].
Root oncogenic loci (rol) genes are well known for the upregulation of secondary
metabolism [12]. Different rol genes have different induction capacities of secondary
metabolites [13] and a lot of the pharmacological activities are linked to the number
of secondary metabolites produced in the medicinal plants [14]. We hypothesize that the
amount of these secondary metabolites biosynthesized de novo in A. bracteosa could be
enhanced and hence the better pharmacological effects could be attained. Considering that,
we transformed A. bracteosa with rolABC genes of Agrobacterium rhizogenes, and hairy roots
were produced. To resolve the issue of organ specificity of certain metabolites, intact plants
(regenerants) were regenerated from these transgenic hairy root lines and their extracts
were screened for antioxidant activities. Multiple in vivo activities including analgesic,
anti-inflammatory, antidepressant, and anticoagulant activity was evaluated using BALB/c
mice. In the continuation of our previous study [15], estimation of essential elements of
transformed regenerated plants of A. bracteosa by atomic absorption spectrophotometry
and HPLC fingerprinting of polyphenol content was performed to support our results.

2. Results
2.1. Elemental Analysis
The elemental screening of transformed and untransformed A. bracteosa digested
extracts revealed the presence of 10 elements, commonly found in plants, as listed in
Table 1. Data shows that transgenic line 3 (ABRL3) is enriched with 3 macro-elements:
sodium 3.94 ± 2 µg/mg dry weight (DW), potassium 13.09 ± 2 µg/mg DW, and cal-
cium 1.95 ± 0.5 µg/mg DW. While magnesium is more abundant in transgenic line 1
(2.15 ± 0.3 µg/mg DW). Likewise, the highest amounts of all micro-elements are found in
ABRL3 except chromium which is more prevalent in ABRL1. On the other hand, untrans-
formed wild type (WT) plant extract has the least concentrations of all elements ranging
from 0.004 ± 0.01 µg/mg to 8.06 ± 0.3 µg/mg of dry sample. We also observed that
cadmium and lead are below the detection limit in all samples.

2.2. Qualitative Screening

Qualitative analyses showed the presence of medicinally important phytoconstituents
in the methanol: chloroform extracts of A. bracteosa summarized in Table 2. These findings
suggest that alkaloids, phenolics, flavonoids, and glycosides are present in all extracts in
a larger amount. Whereas tannins and saponins are found in moderate amounts. Antho-
cyanin, β-cyanins, coumarins, and sterols were absent in all extracts while terpenoids were
identified only in transgenic lines.
Molecules 2021, 26, 4874 3 of 19

Table 1. Quantity of different elements in aerial parts of Ajuga bracteosa.

Elements λmax Slit Width Samples

(µg/mg) (nm) (nm) WT ABRL1 ABRL2 ABRL3
Sodium 589.0 0.5 2.86 ± 0.2 3.54 ± 0.8 b 3.07 ± 1 b 3.94 ± 2 ab
Potassium 766.5 1.0 8.06 ± 0.3 12.00 ± 1 bc 12.62 ± 3 b 13.09 ± 2 a
Calcium 422.7 0.5 0.50 ± 0.06 1.69 ± 0.5 a 1.32 ± 0.4 a 1.95 ± 0.5 a
Magnesium 285.2 0.5 0.91 ± 0.1 2.15 ± 0.3 a 1.12 ± 0.2 b 2.12 ± 0.7 a
Zinc 213.9 1.0 0.25 ± 0.05 0.40 ± 0.1 b 0.24 ± 0.04 c 0.47 ± 0.03 a
Iron 248.3 0.2 0.25 ± 0.1 0.33 ± 0.2 c 0.46 ± 0.1 a 0.44 ± 0.2 a
Manganese 279.5 0.2 0.004 ± 0.01 0.02 ± 0.01 a 0.01 ± 0.03 b 0.02 ± 0.01 a
Nickel 232.0 0.2 0.09 ± 0.02 0.18 ± 0.1 b 0.19 ± 0.1 b 0.23 ± 0.2 a
Copper 324.8 0.5 0.005 ± 0.01 0.01 ± 0.02 b 0.01 ± 0.03 b 0.02 ± 0.01 a
Chromium 357.9 0.2 0.16 ± 0.03 0.45 ± 0.1 a 0.43 ± 0.2 a 0.36 ± 0.1 b
WT = in vitro grown untransformed Ajuga bracteosa plant extract; ABRL1–3 = crude extracts of transgenic lines 1,
2, and 3 of A. bracteosa. Data are represented as mean ± SD (n = 3). The values with different superscript (a–c)
letters show significantly (p < 0.05) different means.

Table 2. Phytochemical constituents of Ajuga bracteosa.

Samples
Phytochemicals
WT ABRL1 ABRL2 ABRL3
Alkaloids ++ ++ +++ +++
Glycosides + +++ ++ +++
Flavonoids + ++ ++ +++
Phenols ++ ++ ++ ++
Tannins + + + +
Saponins + + ++ ++
Terpenoids − + + +
Coumarins − − − −
β-cyanins − − − −
Anthocyanin − − − −
Sterols − − − −
(+) present, (++) moderate concentration, (+++) high concentration, and (−) absent. WT = in vitro grown
untransformed Ajuga bracteosa plant extract; ABRL1–3 =crude extracts of transgenic lines 1, 2, and 3 of A. bracteosa.

2.3. Quantitative Analyses

2.3.1. Determination of TPC and TFC
TPC and TFC were calculated as µg of gallic acid and quercetin equivalents/mg dry
extract by using calibration curves of gallic acid (y = 0.0163x − 0.0130, R2 = 0.9983) and
quercetin (y = 0.0517x + 0.0172, R2 = 0.9991), respectively (Figure 1). In this study, ABRL3
has maximum quantity of phenolics (13.39 ± 2 µg GAE/mg DW) followed by ABRL1
(11.28 ± 1 µg GAE/mg DW), and ABRL2 (8.31 ± 1.5 µg GAE/mg DW). Whereas WT
contains minimum amount of phenolics (4.803 ± 0.04 µg GAE/mg DW). Similarly, TFC
ranges from the highest value in ABRL3 (4.75 ± 0.16 µg QE/mg DW) to the lowest value
in WT (1.55 ± 0.08 µg QE/mg DW).

2.3.2. RP-HPLC
Among the eighteen polyphenolic standards tested through RP-HPLC, twelve were
detected in all extracts of A. bracteosa. Kaempferol was most abundant in all transgenic
samples ranging from 78.6 ± 5 µg/mg in ABRL2 to 101.26 ± 6 µg/mg in ABRL3. WT
contained the highest amount of ferulic acid (75.55 ± 3 µg/mg) and the lowest amount of
rutin (0.63 ± 0.5 µg/mg). Cinnamic acid was least abundant in all transformed lines with
4.36 ± 0.5 µg/mg in ABRL1, 5.47 ± 0.2 µg/mg ABRL2, and 6.09 ± 0.3 µg/mg in ABRL3
(Table 3). Overall, ABRL3 has a predominantly higher content of all polyphenols screened
in this study as represented by chromatogram in Figure 2.
Molecules 2021, 26,
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20

Figure 1. (a) total phenolic content (TPC) and (b) total flavonoids content (TFC). WT = wild type untransformed A. bracteosa
Figure 1. (a) total phenolic content (TPC) and (b) total flavonoids content (TFC). WT = wild type untransformed A. bracteosa
plants; ABRL1–3 = transgenic lines 1–3 of A. bracteosa. Each value represents mean ± SD (n = 3). ** p < 0.01 and *** p < 0.001
plants; ABRL1–3 = transgenic lines 1–3 of A. bracteosa. Each value represents mean ± SD (n = 3). ** p < 0.01 and *** p < 0.001
statistical significance.
statistical significance.
Table 3. Polyphenolic composition of crude extracts of Ajuga bracteosa.
2.3.2. RP-HPLC
No.Among the eighteen
Compound
λmax
Name polyphenolic standardsExtracts (µg/mg Dry
tested through Extract) twelve were
RP-HPLC,
detected in all extracts of A.(nm) WT
bracteosa. Kaempferol ABRL1 ABRL2 in all transgenic
was most abundant ABRL3
samples
1 ranging from 78.6 ± 257
Vanillic Acid 5 μg/mg 8.98
in ABRL2
±1 to15.87
101.26
± 3±a 6 μg/mg in2ABRL3.
15.49 ± a WT
16.33 ± 1con-
a

tained the highest

Rutinamount of 257
ferulic acid
0.63(75.55
± 0.5 ± 3 μg/mg) c
4.49 ± 1 and the lowestb 2a
2 9.24 ± 2 amount
14.86of
±rutin
3 ± 0.5 μg/mg).
(0.63 Plumbagin Cinnamic 257 Nd abundantNd
acid was least Nd lines with
in all transformed Nd4.36 ±
0.54μg/mgThymoquinone
in ABRL1, 5.47 ± 0.2257μg/mg ABRL2,
Nd Nd± 0.3 μg/mg
and 6.09 Ndin ABRL3 (Table
Nd 3).
5 Gallic Acid 279 4.59 ± 0.3 14.99 ± 2 ab 15.01 ± 3 a 16.67 ± 1 a
Overall, ABRL3 has a predominantly higher content of all polyphenols screened in this
6 Catechin 279 Nd Nd Nd Nd
study as represented by chromatogram in Figure 2.
10.79 ± 0.8 b b 17.78 ± 3 a
7 Syringic Acid 279 13.93 ± 2 12.41 ± 1
8 Coumaric Acid 279 1.92 ± 0.7 15.39 ± 3 a 14.02 ± 1 ab 23.45 ± 2 a
Table
9 3. Polyphenolic
Emodin composition
279of crude extracts
Nd of Ajuga bracteosa.
Nd Nd Nd
10 Gentisic Acid 325 Nd Nd Nd Nd
λmax Extracts (µg/mg Dry Extract)
No.
11 Compound Name
Caffeic Acid 325 13.39 ± 2 25.51 ± 3 b 22.01 ± 2 b 30.18 ± 4 a
12 Ferulic Acid (nm)
325 WT
75.55 ± 3 ABRL1
77.17 ± 4 ABRL2
76.86 ± 4 ABRL3
78.05 ±3
1 Vanillic Acid 257 8.98
± ±0.71 15.87± ±0.5
3 c 15.49 ± 1 aa
13 Cinnamic Acid 325 3.19 4.36 a
5.47 ± ±0.2
2 b
a 16.33
6.09 ± 0.3
14
2 Luteolin
Rutin 325
257 0.63Nd± 0.5 4.49Nd
±1 c 9.24Nd
±2 b Nd± 2 a
14.86
15
3 Apigenin
Plumbagin 325
257 8.20
Nd± 2 ±5
20.84Nd bc 23.12 ±3
Nd
b 32.29Nd±4a
16 Myricetin 368 4.14 ± 0.7 13.66 ± 3 a 11.6 ± 2 b 13.37 ± 4 a
4 Thymoquinone 257 Nd Nd Nd Nd
17 Quercetin 368 4.68 ± 0.3 6.44 ± 0.8 c 7.52 ± 1 bc 9.19 ± 0.5 a
5
18 Gallic Acid
Kaempferol 279
368 4.59 ±
17.6 ± 20.3 14.99 ±
83.9 ± 42 b
ab 15.01 ±
78.6 ± 5 3b
a 16.67
101.26 ±± 61 aa
WT6= in vitro grown
Catechin 279 bracteosa plant
untransformed Ajuga Nd extract; ABRL1–3
Nd = crude extracts
Nd of transgenic
Ndlines 1,
7 3 of A.
2, and Syringic
bracteosa.Acid 279
Data being represented 10.79
as mean±±0.8
SD (n13.93 ± 2 values
= 3). The b 12.41 ± 1 b superscript
with different 17.78 ± 3(a–c)
a
letters show significantly (p < 0.05) different means.
8 Coumaric Acid 279 1.92 ± 0.7 15.39 ± 3 a14.02 ± 1 ab 23.45 ± 2 a

9 Total Antioxidant
2.3.3. Emodin 279 (TAC) and
Capacity NdTotal Reducing
Nd Power (TRP) Nd Nd
10 Gentisic Acid 325 Nd Nd Nd Nd
Total antioxidant capacity (TAC) and total reducing power (TRP) of samples against
11 Caffeic Acid 325 13.39 ± 2 25.51 ± 3 b 22.01
ascorbic acid equivalent are given in Figure 3. Results showed that ABRL3 exhibited ± 2 b 30.18 ±4a
12maximum
the Ferulic Acid
total antioxidant325 capacity
75.55 ± 3 ± 177.17
(8.16 ±4
µg AAE/mg 76.86
DW) ± 4while 78.05 ±3
minimum
13 Cinnamic Acid 325 3.19 ± 0.7 4.36 ± 0.5 c
antioxidant capacity was shown by WT (4.18 ± 0.16 µg AAE/mg DW). TAC was found 5.47 ± 0.2 b 6.09 ± 0.3 a

to14
decrease Luteolin
in the order ABRL3 325
> ABRL1 Nd > ABRL2 > WT. Nd All the transgenic
Nd lines Nd
showed
15
significantly Apigenin 325 activity
increased antioxidant 8.20 (p
± 2< 0.01)
20.84 ± 5 bc
compared 23.12
to wild± 3plants.
b 32.29
Data± also
4a
16 that Myricetin
shows ABRL3 exhibited the 368highest 4.14 ± 0.7 13.66
reduction power ± 1.17
± 3ofa 6.60 11.6 ± 2µg 13.37 ± DW
b AAE/mg 4a
followed
17 by 5.8 ±
Quercetin1 µg AAE/mg 368 DW in ABRL1. TRP
4.68 ± 0.3 6.44 ± 0.8followedc the as
7.52 ± 1 that
bc of9.19 ± 0.5all
TAC in a

plant
18 extracts. However, TRP 368
Kaempferol was significantly
17.6 ± 2 higher
83.9(p± <4 0.01) in
b transgenic
78.6 ±5 b regenerants
101.26 ±6a
than= wild
WT control
in vitro grownplants.
untransformed Ajuga bracteosa plant extract; ABRL1–3 = crude extracts of
transgenic lines 1, 2, and 3 of A. bracteosa. Data being represented as mean ± SD (n = 3). The val-
ues with different superscript (a–c) letters show significantly (p < 0.05) different means.
Molecules 2021, 26,
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Figure 2. Cont.
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Figure 2. RP−HPLC chromatograms for quantification

Figure 2. RP−HPLC of polyphenols
chromatograms in crude extracts
for quantification of Ajugainbracteosa.
of polyphenols (a) mixed
crude extracts of Ajuga
standard compounds. (b) crude extract
bracteosa. (a)ofmixed
transgenic of A. bracteosa(b)
line 3 compounds.
standard (ABRL3).
crude extract of transgenic line 3 of A. bracteosa
(ABRL3).
2.4. In Vitro Antioxidant Assays
2.4.1. Total
2.3.3. DPPHAntioxidant
Radical Scavenging
CapacityAssay
(TAC) and Total Reducing Power (TRP)
The percent scavenging activity of the extracts were evaluated using the DPPH free
Total antioxidant capacity (TAC) and total reducing power (TRP) of samples against
radical scavenging assay and IC50 values were calculated (Figure 4). IC50 values varied in
ascorbic acid equivalent are given in Figure 3. Results showed that ABRL3 exhibited the
a concentration-dependent manner however, all extracts showed higher IC50 values than
maximum total antioxidant capacity (8.16 ± 1 μg AAE/mg DW) while minimum antioxi-
ascorbic acid (IC50 39.3 ± 1 µg/mL), used as the positive control. The minimum IC50 values
dant capacity was shown by WT (4.18 ± 0.16 μg AAE/mg DW). TAC was found to decrease
were exhibited by ABRL3 (59.5 ± 0.8 µg/mL) followed by ABRL1 (97.64 ± 0.5 µg/mL).
in the order ABRL3 > ABRL1 > ABRL2 > WT. All the transgenic lines showed significantly
Overall, order of IC50 values was ABRL3 < ABRL1 < ABRL2 < WT. The DPPH radical scav-
increased antioxidant activity (p < 0.01) compared to wild plants. Data also shows that
enging activity of different extracts presented good correlation with TPC (R2 = 0.9627 ***,
ABRL3 exhibited the highest reduction power of2 6.60 ± 1.17 μg AAE/mg DW followed by
p < 0.001) and moderate correlation with TFC (R = 0.7627 **, p < 0.01) as shown in Table 4.
5.8 ± 1 μg AAE/mg DW in ABRL1. TRP followed the as that of TAC in all plant extracts.
However, TRP was significantly higher (p < 0.01) in transgenic regenerants than wild con-
trol plants.
 Molecules 2021, 26, x FOR PEER REVIEW 7 of 20

Molecules 2021, 26, 4874 7 of 19

OR PEER REVIEW 8 of 20
Figure 3. (a) total antioxidant capacity (TAC) and (b) total reducing power (TRP). WT = wild type untransformed
Figure 3. (a) total antioxidant capacity (TAC) and (b) total reducing power (TRP). WT = wild type untransformed A. brac-
A. bracteosa
teosa plants;plants;
ABRL1–3ABRL1–3 = transgenic
= transgenic lines
lines 1–3 1–3
of A. of A. bracteosa.
bracteosa. Eachrepresents
Each value value represents (n =±3).
mean
mean ± SD SD**(n
p <= 0.01 p < 0.01
3). **statistical
statistical significance.
significance.

2.4. In Vitro Antioxidant Assays

2.4.1. DPPH Radical Scavenging Assay
The percent scavenging activity of the extracts were evaluated using the DPPH free
radical scavenging assay and IC50 values were calculated (Figure 4). IC50 values varied in
a concentration-dependent manner however, all extracts showed higher IC50 values than
ascorbic acid (IC50 39.3 ± 1 μg/mL), used as the positive control. The minimum IC50 values
were exhibited by ABRL3 (59.5 ± 0.8 μg/mL) followed by ABRL1 (97.64 ± 0.5 μg/mL).
Overall, order of IC50 values was ABRL3 < ABRL1 < ABRL2 < WT. The DPPH radical scav-
enging activity of different extracts presented good correlation with TPC (R2 = 0.9627 ***,
p < 0.001) and moderate correlation with TFC (R2 = 0.7627 **, p < 0.01) as shown in Table 4.

Table 4. Correlation of IC50 values of different antioxidant activities of A. bracteosa with total phe-
nolic and total flavonoid contents.

Antioxidant Activities Correlation R2

TPC TFC
DPPH Radical Scavenging Activity 0.9627 *** 0.7627 **
Hydroxyl Ion Scavenging Assay 0.9312 *** 0.8158 **
Iron Chelating Power 0.9159 *** 0.8243 **
Columns with different superscripts are significantly different: ** shows p < 0.01 while *** shows p
Figure 4. DPPH scavenging<assay
0.001.ofTPC
crude extracts
= total of A. content;
phenolic bracteosaTFC
along with flavonoid
= total IC50 values. WT = wild type untransformed
content.
A.Figure
bracteosa4.plants; ABRL1–3 = transgenic lines 1–3 of A. bracteosa; AA = ascorbic acid.
DPPH scavenging assay of crude extracts of A. bracteosa along with IC50 values. WT = wild
type untransformed A.Table
bracteosa plants;ofABRL1–3
4. Correlation IC50 values=of
transgenic lines 1–3activities
different antioxidant of A. bracteosa; AAwith
of A. bracteosa = ascorbic
total phenolic
acid. and total flavonoid contents.

Antioxidant Activities Correlation R2

2.4.2. Hydroxyl Ion Scavenging Assay
TPC TFC
All extracts of A. bracteosa scavenged •OH radicals while lowest IC50 values were rec-
DPPH Radical Scavenging Activity 0.9627 *** 0.7627 **
orded for ABRL3 (122.5 ± 0.90 Ion
Hydroxyl μg/mL) andAssay
Scavenging ABRL2 (129.7 0.9312 ± 2 μg/mL)
*** followed by ABRL1
0.8158 **
(138.4 ± 1 μg/mL), whereasIron theChelating
highestPower IC50 was observed0.9159 for WT*** (1056.9 ± 4 μg/mL). 0.8243 **IC50
Columns with different superscripts are significantly different: ** shows p < 0.01 while *** shows p < 0.001.
of all extract samples were significantly different from the standard gallic acid (81.1 ± 4
TPC = total phenolic content; TFC = total flavonoid content.
μg/mL) as given in Figure 5. A highly significant correlation was observed with TPC (R2 =
2.4.2.moderate
0.9312 ***, p < 0.001) and Hydroxyl Ion Scavenging
correlation Assay
with TFC (R2 = 0.8158 **, p <0.01) (Table 4).
All extracts of A. bracteosa scavenged • OH radicals while lowest IC50 values were
recorded for ABRL3 (122.5 ± 0.90 µg/mL) and ABRL2 (129.7 ± 2 µg/mL) followed by ABRL1
(138.4 ± 1 µg/mL), whereas the highest IC50 was observed for WT (1056.9 ± 4 µg/mL).
 Figure 4. DPPH scavenging assay of crude extracts of A. bracteosa along with IC50 values. WT = wild
type untransformed A. bracteosa plants; ABRL1–3 = transgenic lines 1–3 of A. bracteosa; AA = ascorbic
acid.

Molecules 2021, 26, 4874 2.4.2. Hydroxyl Ion Scavenging Assay 8 of 19

All extracts of A. bracteosa scavenged •OH radicals while lowest IC50 values were rec-
orded for ABRL3 (122.5 ± 0.90 μg/mL) and ABRL2 (129.7 ± 2 μg/mL) followed by ABRL1
(138.4
IC50 of± all
1 μg/mL), whereaswere
extract samples the highest IC50 was
significantly observed
different fromfor
theWT (1056.9gallic
standard ± 4 μg/mL).
acid IC50
of all±extract
(81.1 4 µg/mL)samples were
as given in significantly different
Figure 5. A highly from thecorrelation
significant standard was
gallic acid (81.1 ± 4
observed
2 = 0.9312 ***, p < 0.001) and moderate correlation with TFC (R2 = 0.8158 **,
μg/mL)
with TPCas(Rgiven in Figure 5. A highly significant correlation was observed with TPC (R2 =
p < 0.01)
0.9312 (Table
***, 4). and moderate correlation with TFC (R2 = 0.8158 **, p <0.01) (Table 4).
p < 0.001)

Figure 5. Hydroxyl ion scavenging activity of A. bracteosa. WT = wild type untransformed A. bracteosa
Figure 5. Hydroxyl ion scavenging activity of A. bracteosa. WT = wild type untransformed A. bracte-
plants; ABRL1–3 = transgenic lines 1–3 of A. bracteosa; GA = gallic acid.
osa plants; ABRL1–3 = transgenic lines 1–3 of A. bracteosa; GA = gallic acid.
2.4.3. Ferrous Ion Chelating Activity
Molecules 2021, 26, x FOR PEER REVIEW 9 of 20
2.4.3.InFerrous Ionthe
this study, Chelating Activity
finest values for IC50 were exhibited by ABRL3 (154.8 ± 2 µg/mL)
followed by study,
In this ABRL2the
(179.2 ± 1values
finest µg/mL).for Overall,
IC50 wereorder of IC50by
exhibited ofABRL3
ABRL3 (154.8
< ABRL2
± 2 <μg/mL)
ABRL1 < by
followed WTABRL2
was observed
(179.2 ±(Figure
1 μg/mL). 6). The iron order
Overall, chelating activity
of IC of various
50 of ABRL3
extracts
< ABRL2
correlation with TPC (R 2 = 0.9159 ***, p < 0.001) and moderate correlation with TFC (R2<=ABRL1
showed good correlation with TPC (R2 = 0.9159 ***, p < 0.001) and moderate correlation
< WT was
0.8243 **, p observed (Figure
< 0.01) as given 6). The
in Table 4. iron chelating activity of various extracts showed good
with TFC (R2 = 0.8243 **, p < 0.01) as given in Table 4.

Figure 6.
Figure Iron chelating
6. Iron chelating power
power of A. bracteosa.
of A. bracteosa. WT
WT ==wild
wild type
type untransformed A. bracteosa
untransformedA. bracteosa plants;
plants;
ABRL1–3
ABRL1–3 ==transgenic
transgeniclines
lines1–3
1–3of
ofA.
A.bracteosa;
bracteosa;EDTA
EDTA ==ethylenediaminetetraacetic
ethylenediaminetetraaceticacid.
acid.

2.5. In Vivo Assays in BALB/c Mice

2.5.1. Analgesic Activity
The crude extracts of transgenic A. bracteosa plants showed a delayed latency period
and increased analgesic activity in BALB/c mice. Aspirin (positive control) and crude ex-
tracts displayed a time-dependent activity on a hot plate by suppressing nociceptor activ-
Molecules 2021, 26, 4874 9 of 19

Figure 6. Iron chelating power of A. bracteosa. WT = wild type untransformed A. bracteosa plants;
ABRL1–3 = transgenic lines 1–3 of A. bracteosa; EDTA = ethylenediaminetetraacetic acid.

2.5.InInVivo
2.5. VivoAssays
Assaysinin BALB/c
BALB/c Mice
Mice
2.5.1. Analgesic Activity
2.5.1. Analgesic Activity
The crude extracts of transgenic A. bracteosa plants showed a delayed latency period
The crude extracts of transgenic A. bracteosa plants showed a delayed latency period
and increased analgesic
and increased analgesic activity
activity in BALB/c
in BALB/c mice. mice.
AspirinAspirin
(positive(positive control)
control) and crudeand
ex- crude
extracts displayed a time-dependent activity on a hot plate by suppressing
tracts displayed a time-dependent activity on a hot plate by suppressing nociceptor activ- nociceptor
activity
ity in mice
in mice (Figure
(Figure 7). Maximum
7). Maximum activity
activity was observed
was observed after 1 after 1 h ofdose
h of oral oralindose in ABRL3
ABRL3
(87.3±±3%)
(87.3 3%) followed
followed byby aspirin
aspirin (85.66
(85.66 ± 4%).
± 4%). Whereas
Whereas normalnormal saline-treated
saline-treated mice (negative
mice (negative
control)produced
control) producedthe theleast
least significant
significant analgesic
analgesic effect
effect (13 ±(13 ± 4%).
4%). ABRL1 ABRL1 and ABRL2
and ABRL2 also also
demonstratedincreased
demonstrated increasedactivity
activity
(76(76 ± 3%
± 3% andand
74 ±74 ± compared
2%) 2%) comparedto theto the wild-type
wild-type controlcontrol
group (41±±
group(41 3%).
3%).

Figure7.7.InInvivo
Figure vivo analgesic
analgesic activity
activity of Ajuga
of Ajuga bracteosa
bracteosa crude crude extracts.
extracts. WT =WT wild=type
wilduntransformed
type untransformed
A.
A. bracteosa
bracteosaplants;
plants;ABRL1–3
ABRL1–3 = transgenic lines
= transgenic 1–3 1–3
lines of A.ofbracteosa. EachEach
A. bracteosa. valuevalue
represents mean ±mean
represents SD ± SD
Molecules 2021, 26, x FOR PEER REVIEW
(n
(n==5).
5).nsnsshows
shows non-significant values,
non-significant * p <* 0.05,
values, and **
p < 0.05, andp <**0.01
p <statistically significant
0.01 statistically 10 of 20
data.
significant data.

2.5.2. Anti-Inflammatory Activity

Pain and inflammation
2.5.2. Anti-Inflammatory are often linked to each other. Therefore, crude extracts were
Activity
alsoPain
tested
andfor anti-inflammatory
inflammation are oftenactivity
linked toagainst carrageenan-induced
each other. hind paw
Therefore, crude extracts wereedema
in BALB/c mice. Observations were made after 1 h, 2 h, and 3 h of oral
also tested for anti-inflammatory activity against carrageenan-induced hind paw edema dose for edema
treatment.
in BALB/c mice. Diclofenac potassium
Observations was after
were made used1as h, a2 positive
h, and 3 hdrug and
of oral showed
dose 77.6 ± 3%
for edema
activity after
treatment. 3 h post
Diclofenac dosage.was
potassium ABRL3
used asrevealed
a positivethe highest
drug anti-inflammatory
and showed 77.6 ± 3% activ-activity
ity
(82.3 ± 32 h%)
after post dosage.
while ABRL3 revealed
WT manifested the highest
the lowest activity (46.6 ± 2%) among
anti-inflammatory activityall
(82.3 ± 2 %)
tested samples
while WT8).
(Figure manifested the lowest activity (46.6 ± 2%) among all tested samples (Figure 8).

Figure8.8.InIn
Figure vivo
vivo anti-inflammatory
anti-inflammatory activity
activity of Ajuga
of Ajuga bracteosa
bracteosa crudecrude extracts.
extracts. DP = diclofenac
DP = diclofenac potas- potas-
sium;
sium; WT = wild type untransformed A. bracteosa plants; ABRL1–3 = transgenic lines 1–3 ofbrac-
WT = wild type untransformed A. bracteosa plants; ABRL1–3 = transgenic lines 1–3 of A. A. bracteosa.
teosa.
EachEach
valuevalue represents
represents meanmean
± SD± SD (n5).
(n = = 5).
* p* <
p <0.05,
0.05,and
and****pp<<0.01
0.01 statistically
statistically significant
significant data.
data.

2.5.3. Antidepressant Activity

The potential antidepressant activity of A. bracteosa crude extracts was evaluated in
mice by tail suspension test and results are displayed in Figure 9. All transgenic lines ex-
hibited enhanced antidepressant activity compared to wild-type and positive control
Molecules 2021, 26, 4874 Figure 8. In vivo anti-inflammatory activity of Ajuga bracteosa crude extracts. DP = diclofenac10 potas-
of 19
sium; WT = wild type untransformed A. bracteosa plants; ABRL1–3 = transgenic lines 1–3 of A. brac-
teosa. Each value represents mean ± SD (n = 5). * p < 0.05, and ** p < 0.01 statistically significant data.

2.5.3.
2.5.3. Antidepressant
Antidepressant Activity
Activity
The
The potential
potential antidepressant
antidepressant activity
activity of
of A.
A. bracteosa
bracteosa crude extracts was evaluated in
mice
mice by
bytail
tailsuspension
suspensiontest
testand
andresults
resultsare
aredisplayed
displayed in in
Figure 9. All
Figure transgenic
9. All lineslines
transgenic ex-
exhibited
hibited enhanced
enhanced antidepressant
antidepressant activitycompared
activity comparedtotowild-type
wild-typeandand positive
positive control
(Fluoxetine-HCl). ABRL3 demonstrated the lowest lowest immobility
immobility time
time of 38.3±
of 38.3 ± 2 s followed
by 45.7 ±±33ssininABRL1
by 45.7 ABRL1and 63.3±±4 4s in
and63.3 s inABRL2.
ABRL2.

Figure 9.
Molecules 2021, 26, x FOR PEER REVIEW
Figure In vivo
9. In vivo anti-depressant
anti-depressant activity
activity of Ajuga bracteosa
of Ajuga bracteosa crude
crude extracts.
extracts. F-HCl
F-HCl == fluoxetine
fluoxetine HCl;
11 HCl;
of 20
WT == wild
WT wild type
type untransformed A. bracteosa
untransformed A. bracteosa plants;
plants; ABRL1–3
ABRL1–3 ==transgenic
transgeniclines
lines1–3
1–3 of A. bracteosa.
of A. bracteosa.
Each value
Each represents mean ±±SD
value represents SD(n 5).* *p p< <0.05,
(n==5). and****p p< <0.01
0.05,and 0.01statistically
statisticallysignificant
significantdata.
data.

2.5.4. Anticoagulant
Anticoagulant Activity
Anticoagulant
Anticoagulant activity
activity of
of wild
wild type
type and transgenic
transgenic lines
lines of A. bracteosa is shown in
Figure
Figure 10. Transgenic
Transgenic lines
lines delayed
delayed blood
blood clotting
clotting from
from 2.46 min in negative control to
4.51 min, 5.40 min,
min, and
and 5.41
5.41 in
in ABRL1,
ABRL1, ABRL2,
ABRL2, and
and ABRL3
ABRL3 treated
treated mice,
mice, respectively.
respectively. WT
crude extracts were least effective among all plant extracts with a clotting time of 3.3 min.

In vivo
Figure 10. In vivo anti-coagulant
anti-coagulant activity of Ajuga bracteosa crude extracts. WT == wild wild type
type untrans-
untrans-
formed
formed A. bracteosa plants; ABRL1–3 = = transgenic
transgenic lines 1–3 of A. bracteosa.
bracteosa. Each value represents
mean
mean ±±SD
SD(n (n==5).
5).****p p< <0.01
0.01statistically
statisticallysignificant
significantdata.
data.

3. Discussion
3. Discussion
The plant
The plant kingdom
kingdom consists
consists of
of countless
countless species
species with
with several
several uses
uses in
in medicines.
medicines. They
They
contain valuable secondary metabolites demonstrating anti-inflammatory, anticancer,
contain valuable secondary metabolites demonstrating anti-inflammatory, anticancer, an- an-
tioxidant antidiabetic, and/or antimicrobial properties [16]. The growing demand for these
plant secondary metabolites forces the use of new green biotechnology tools to create new,
more productive in vitro transgenic plant cultures [17]. This study reports the examination
of the potential role of rolABC genes in increasing the production of secondary metabolites
Molecules 2021, 26, 4874 11 of 19

tioxidant antidiabetic, and/or antimicrobial properties [16]. The growing demand for these
plant secondary metabolites forces the use of new green biotechnology tools to create new,
more productive in vitro transgenic plant cultures [17]. This study reports the examination
of the potential role of rolABC genes in increasing the production of secondary metabolites
and compares the pharmaceutical efficacy of transgenic regenerants and in vitro grown
untransformed A. bracteosa plants.
Minerals are requisite for disease resistance and normal physiology of the human body;
however, high concentrations could be a health risk. Major elements including sodium,
potassium, calcium, and magnesium are involved in protein synthesis, nerve transmission,
bone development, muscle contraction, and enzyme activation [18]. Here we found that all
studied elements were below the maximum permissible limit as recommended by WHO
and European pharmacopeia [19]. Among 10 detected elements, potassium was most
abundant in all samples. However, the Na/K ratio was below 1 which is required to main-
tain normal blood pressure [20]. Only minute amounts of 6 trace elements were detected.
These trace elements have well reported anti-inflammatory, antianemia, antidiabetic, and
anticancer properties [21].
Next, phytochemical analysis was carried out to investigate the effects of transfor-
mation on secondary metabolite production. Qualitative assays screened several medic-
inally active molecules in A. bracteosa based on their polarities. Considerable amounts
of alkaloids, phenolics, flavonoids, and glycosides were present in all extracts. These
phytoconstituents possess antimicrobial, antioxidant, anti-inflammatory, and anticancer
properties [22]. Saponins and tannins used as astringents, hepaprotective, cardioprotective,
and anticancer agents [23] were present in moderate quantities. The presence of terpenoids
in transgenic plants can impart antioxidant, antibacterial, antiviral, chemoprotective, and
neuroprotective characters [24,25] to these transgenics.
The antioxidant activity of phenolics and flavonoids primarily depends on the pres-
ence of hydroxyl groups [26]. In a previous study on A. bracteosa, the highest amount of phe-
nolic content was found in crude extracts prepared in more polar solvents (10.75 ± 0.70 µg
GAE/mg DW of methanolic extracts) than the non-polar solvent systems (0.33 ± 0.0 µg
GAE/mg DW demonstrated by n-hexane extract). A similar pattern of results was obtained
for flavonoid content [10]. The current study demonstrated significantly higher values of
TPC and TFC in transformed regenerants as compared to untransformed plants. Our obser-
vations are in accordance with the studies reporting enhanced TPC and TFC of Artemisia
dubia, A. annua, and Lactuca sativa plants transformed with rolABC genes [27–29].
RP-HPLC delineated a marked surge in polyphenols of transformed plants than their
untransformed counterparts. These differences support our previous findings indicating
that rolABC genes enhanced the expression of biosynthetic pathway genes in transformed
regenerants of A. bracteosa [15]. We found that ABRL3 presented the highest expression of β-
hydroxy β-methylglutaryl-CoA reductase (HMGR), farnesyl diphosphate synthase (FDS), 4-
hydroxy-3-methyl-but-2-enyl pyrophosphate synthase (HDS), and phenylalanine ammonia-
lyase (PAL) genes owing to the significant phenolic content and antioxidant activity of
this line. Higher expression of the metabolic pathway genes can easily be correlated to
the higher production of phytoecdysteroids and hence the polyphenols contents. These
polyphenols are reported to prevent aging and age-related disorders such as cancer, cardiac
malfunctions, diabetes, neurodegenerative problems, and inflammatory disorders [3]. Their
protective mechanisms involve suppression of oxidative stress, maintenance of nuclear
factor kappa B (NF-κB) and interleukins (IL-1β), activation of gluconeogenesis, inhibition
of angiogenesis, and cyclooxygenases (COX-1 and COX-2) [30]. Our results show that six
polyphenol markers, namely, plumbagin, thymoquinone, catechin, emodin, gentisic acid,
and luteolin were below the detection limit in all crude extracts. However, some earlier
studies [10] reported catechin in ethyl acetate and aqueous fractions of A. bracteosa. These
findings are likely to be related to the diverse nature of metabolites and polarity in different
solvent systems [31].
Molecules 2021, 26, 4874 12 of 19

All extracts were evaluated for TAC, TRP, and antioxidant activity through DPPH,
hydroxyl radical scavenging, and iron-chelating power assays. Transgenic lines fared
better for reducing power and antioxidant activity than untransformed plants. A similar
pattern of results was found in Artemisia annua [32] and lettuce [33] plants transformed with
different rol genes. Previously, Kayani et al. (2016) [8] also determined the phytochemical
content and antioxidant activity of naturally growing A. bracteosa plant extracts in different
solvent systems. They observed that the aerial and root extracts of A. bracteosa prepared
in methanol and chloroform represented the best TPC, TFC, TAC, TRP, and free radical
scavenging among all solvents. Moreover, among 15 different extracts of A. bracteosa, the
maximum total reducing power (23.90 ± 0.70 µg AAE/mg DW) and total antioxidant
capacity (11.30 ± 0.80 µg AAE/mg DW) were exhibited by methanol extract with superla-
tive percent extract recovery (17.50 ± 0.80% w/w) [10]. Similarly, Hafeez et al. (2017) [34]
reported potential DPPH radical scavenging activity of A. bracteosa root extracts along with
antibacterial and antidiabetic activity in both polar and non-polar solvents.
To find the main components responsible for the antioxidant activity of the crude
extracts we developed a correlation with total phenolic content and total flavonoid content.
We observed a significant positive correlation between antioxidant activities and plant
phenolics. Whereas moderate correlation with TFC suggests that reported antioxidant
properties are attributed to higher phenolics than flavonoids content [10].
Despite the low bioavailability of dietary polyphenolics and phytoecdysteroids, most
of their biometabolites display higher biological activity both in vitro and in vivo. These
metabolites account for the health benefits of their parent phytochemicals [5,35]. Consider-
ing this, we tried to demonstrate the in vivo pharmacological potential of A. bracteosa.
Hot plate assay is a simple and sensitive method to assess anti-nociceptive drugs to
relieve pain [36]. The results of analgesic activity showed that transgenic plant extracts
protected mice from visceral pain and produced comparable effects to standard drug
aspirin. Nonsteroidal anti-inflammatory drugs (NSAIDs) can be used to treat inflammation
associated with pain. However, their overuse can lead to serious side effects. Therefore,
herbal products are used as an alternative to antagonize inflammatory agents [36]. Blood
brain barrier (BBB) is the key target for the therapeutic delivery of central nervous system
(CNS) drugs. Inflammation and neurodegeneration are often associated in neurological
disorder [37]. Different studies suggest that polyphenols, alkaloids, terpenoids, steroids,
and lignans can attenuate the inflamed and damaged BBB thus acting as curative agent
against neurovegetative disorders [38,39] Previously, ferulic acid was physiologically
detected in the cerebrospinal fluid (CSF) of mice and rat [40,41]. Interestingly, a recent
study demonstrated the presence of non-endogenous caffeic acid in the human CSF. These
studies confirm that plant phenolics can cross the BBB in humans and reach the neurons
and microglia [42]. However, the exact mechanisms by which they may permeate the BBB
are not fully understood. Additionally, little is known about their further metabolism in the
brain [43]. Different studies suggest that with anolides and phenolics present in A. bracteosa
inhibit the activity of prostaglandins and COX [44,45]. Terpenoids, phytoecdysteroids,
and phenolics attribute to anti-depressant and anticoagulant properties of transformed
A. bracteosa plant extracts. Thus, this plant can be a potent agent to treat neurodegenerative
and thrombolytic disorders [8,27]. Our findings are in agreement with former results [33]
verifying that rol genes increased the pharmaceutical value of methanolic extracts of
transformed lettuce in rats.

4. Materials and Methods

4.1. Source of Plant
The plant material was collected from the premises of Quaid-i-Azam University, Is-
lamabad, Pakistan. These plants were identified by Prof. Dr. Rizwana Aleem Qureshi (Tax-
onomist), Department of Plant Sciences, Quaid-i-Azam University, Islamabad. A voucher
specimen number (HPM-460) was deposited in the herbarium of Quaid-i-Azam Univer-
sity. Fresh green plants were surface sterilized, and tissue cultured on Murashige and
Molecules 2021, 26, 4874 13 of 19

Skoog (MS) medium. Transgenic hairy roots were generated through rolABC containing
A. rhizogenes mediated transformation. Intact plants were regenerated from hairy roots
by following the previously optimized method in our lab [46]. Polymerase chain reaction
(PCR) was performed for the confirmation of transformation and stable integration of rol
genes in transformed roots and regenerants [15,46]. Untransformed tissue cultured plants
and transgenic regenerants of A. bracteosa plants were used in this study.

4.2. Elemental Analysis

For the digestion of leaf samples, an earlier reported method [47] was employed with
heated at 550 ◦ C for 4 h and then cooled at room temperature. Then Nitric acid (6 M;
10 mL) was added for acid digestion. After filtration, the solution was diluted up to a
25 mL mark with deionized water. Reference standards of 12 micro and macro elements
(Sigma-Aldrich, Burlington, MA, USA) were used to quantify the essential elements and
to find the possible accumulation of hazardous heavy metals in A. bracteosa. Estimation
of all elements was carried out on a Fast Sequential Atomic Absorption Spectrometer
(Varian 240AA FS, Melbourne, Australia). The operating parameters for working elements
were optimized according to the manufacturer’s recommendations.slight modifications.
Powdered leaf samples were weighed (100 mg each) and heated in an oven at 110 ◦ C in a
china dish for the removal of moisture. The dried samples were.

4.3. Crude Extracts Preparation for Biological Activities

Aerial parts of three independent transgenic lines (ABRL1, 2, and 3) and in vitro grown
untransformed A. bracteosa plants (WT) were shade dried after rinsing with water. Leaves
were ground and 1 g powdered material was allowed to set in a mixture of methanol:
chloroform (1:1; 5 mL). After 1 h the mixture was sonicated for 10 min followed by 20 min
of shaking and the process was repeated three times. Finally, the plant extracts were
filtered, and pooled filtrates were dried, weighed, and stored at room temperature for
further analysis.

4.4. Phytochemical Profiling

4.4.1. Qualitative Assays
Crude extracts of A. bracteosa were evaluated for the presence of major families of
secondary metabolites such as alkaloids, glycosides, flavonoids, phenols, tannins, saponins,
terpenoids, coumarins, betacyanin (β-cyanin), anthocyanin, and sterols using standard
quality procedures based on coloring reaction and/or precipitation [48].

4.4.2. Quantitative Assays

Estimation of Total Phenolics and Flavonoids Content
The total phenolic content (TPC) was calculated by Folin–Ciocalteu (FC) assay using
gallic acid as standard [49]. Briefly, 20 mg of each dried extract was dissolved in 1 mL of
DMSO. Gallic acid (1 mg, Merck, Kenilworth, NJ, USA) was dissolved in 1 mL of DMSO
and further diluted. In this experiment, 5 µL of plant extract was mixed with 98 µL of
10 times diluted FC reagent in 96 well plate (Thermo Scientific, Waltham, MA, USA). After
5 min, 98 µL of 6% sodium carbonate was added and incubated at 25 ◦ C for 90 min. Finally,
the absorbance was measured at 630 nm with a microtiter plate reader (Elx 800, BioTek,
Winooski, VT, USA). TPC was expressed as gallic acid equivalents (µg GAE/mg dry weight
of plant extract). Total flavonoid content (TFC) was determined by the already optimized
colorimetric method [27] with slight modifications. 5 µL of each extract (20 mg/mL) was
mixed with equal volumes of potassium acetate (1 M) and 10% aluminum chloride (20 µL
each). Then, 155 µL of distilled water was subsequently added and the mixture was left at
37 ◦ C for half an hour. After incubation, absorbance was determined at 405 nm and TFC
was expressed as quercetin equivalents (µg QE/mg extract).
Molecules 2021, 26, 4874 14 of 19

Quantification of Polyphenols by RP-HPLC

For RP-HPLC analysis, polyphenols were extracted from aerial parts of wild plant and
transgenic lines according to the previously reported procedure [50]. HPLC-DAD system
(Agilent Technology, Ratingen, Germany) was attached to an analytical column (Sorbex RXC-
8) with the dimensions, 5 µm, 4.6 by 250 mm, for separation of polyphenols. This separation
was achieved using two mobile phases; water:methanol:acetonitrile:acetic acid (85:10:5:1)
were present in mobile phase A while mobile phase B included methanol:acetonitrile:acetic
acid (60:40:1). The process was operated by regulating the gradient program as follows;
(t (min), % B); (0, 50), (20, 50), (25, 100), and (40, 100) with 1 mL/min flow rate at 350 bars.
Then, 20 µL of each crude extract and standard was injected. Identification and quantifica-
tion of polyphenols were carried out by comparison of the peaks of each metabolite with
particular retention time in the standards.

4.5. In Vitro Assays

4.5.1. Total Antioxidant Capacity Assay
Total antioxidant capacity (TAC) assay of plant extracts was evaluated by the method
of Phatak and Hendre [51] with some modifications. An aliquot of 10 µL of samples was
mixed with 190 µL of molybdenum (IV) solution (4 mM ammonium molybdate, 0.6 M
sulphuric acid, and 28 mm sodium phosphate) and incubated at 95 ◦ C. After 90 min,
absorbance was measured at 630 nm and TAC was reported as ascorbic acid equivalent
(µg AAE/mg DW).

4.5.2. Total Reducing Power (TRP)

Reducing power activity was estimated according to the method of Wintola and Afo-
layan [52] with some modifications. In this assay, 125 µL of all extracts and standards were
thoroughly mixed with phosphate buffer (0.2 M; pH 6.6) and 1% potassium ferricyanide
(313 µL each). After 20 min of incubation at 50 ◦ C, 313 µL of trichloroacetic acid (10%;
TCA) was added. This mixture was centrifuged at 3000 rpm for 10 min. the upper layer
(100 µL) was mixed with an equal volume of distilled water and 20µL of ferric chloride (1%).
Absorbance was measured at 630 nm and results were recorded as ascorbic acid equivalent.

4.5.3. Antioxidant Assays

Diphenyl-2-Picryl-Hydrazyl (DPPH) Radical Scavenging Activity
Antiradical activities of potent antioxidants from crude extracts were detected by the
previously reported DPPH method [33]. Ascorbic acid (1 mg, Merck, Darmstadt, Germany)
dissolved in 1 mL of DMSO was used as a positive control. The assay was performed by
mixing 150 µL of freshly prepared DPPH solution (0.1 mM) with 50 µL of different dilutions
of extracts. The resultant mixture was incubated in dark at 37 ◦ C for 1 h. Decolorization
of DPPH indicated the antioxidant activity of test samples. Consequently, a reduction
in absorbance was recorded at 515 nm. The radical scavenging activity of DPPH was
calculated by the following Formula (1):

Absorbance of control − Absorbance of sample

 
Percent radical scavenging = × 100 (1)
Absorbance of control

Hydroxyl Ion (OH) Scavenging Assay

The OH scavenging potential of extracts was determined by classical thiobarbituric
acid (TBA) and deoxyribose-based method [53]. For this assay, a 2.8 mM solution of 2-
deoxyribose (w/v) was prepared in phosphate buffer (50 Mm; pH 7.4). Then, 125 µL of
2-deoxyribose (2.8 mM), 25 µL EDTA (0.1 M), 25 µL ferric chloride (100 M), 25 µL of 0.2 M
H2 O2 , and 25 µL of ascorbic acid (0.3 M) were mixed with 100 µL of plant extract. The
whole mixture was kept at 37 ◦ C for 1 h. Afterward, 250 µL of 2.8% TCA and 1.25 mL
of 1% TBA prepared in sodium hydroxide (50 mM) were added. The resulting solution
was heated for 15 min in a water bath and then placed for cooling. Then absorbance
Molecules 2021, 26, 4874 15 of 19

was measured at 540 nm and OH radical scavenging activity was calculated through the
following Formula (2):

Absorbance of control − Absorbance of sample

 
Percent OH radical scavenging = × 100 (2)
Absorbance of control
In this study, gallic acid was used as positive control while DMSO was a negative control.

Chelating Power Assay

The Ferrous ion (Fe2+ ) chelating ability of plant extracts was investigated by the
reported method of Dastmalchi et al. [54]. In this method, 100 µL of plant samples were
added to 50 µL ferrous chloride (2 mM) and incubated for 5 min. Then 200 µL ferrozine
(5 mM) was added to initiate ferrous-ferrozine complex formation reaction. The mixture
was re-incubated for 10 min then absorbance was measured at 540 nm. The ability of plant
extracts to inhibit complex formation was measured by the following Formula (3):

Absorbance of control − Absorbance of sample

 
Percent iron chelating power = × 100 (3)
Absorbance of control

4.6. In Vivo Biological Activities

4.6.1. Test Animals
For the experiment, healthy male BALB/c albino mice (4–6 weeks; 25–30 g) were
procured from the NIH, Islamabad, Pakistan. Test mice were maintained under con-
trolled environmental conditions (23.0 ± 2.0 ◦ C temperature; 60–70% of relative humidity;
12 h light/dark cycle) in the Primate facility Quaid-i-Azam University (QAU), Islam-
abad, Pakistan. All the mice were fed with a pellet diet and had free access to water ad
libitum. All the experiments were approved by the Animal Ethical Committee, QAU
(BEC-FBS-QAU2019-157).

4.6.2. Experimental Design

Plant extracts and standard compounds were prepared in normal saline (0.9% NaCl)
and administered orally according to mice body weight. In these experiments, 30 male
mice were randomly divided into 6 groups as follows:
Normal control group: 200 mg/kg normal saline.
Positive control group: 10 mg/kg standard drugs.
Group-1: 200 mg/kg crude extract of a wild-type plant (WT).
Group-2: 200 mg/kg crude extract of ABRL1.
Group-3: 200 mg/kg crude extract of ABRL2.
Group-4: 200 mg/kg crude extract of ABRL3.

4.6.3. Acute Toxicity Test

Before performing in vivo experiments, animals were tested for toxic effects of A. bracteosa
plant extracts. The oral administration of plant crude extracts (up to 1 g/kg) proved to
be non-toxic and did not produce mortality for the next 24 h. Guidelines 425 of the
Organization for Economic Corporation and Development (OECD) were strictly followed
during the study.

4.6.4. Analgesic Activity (Hot-Plate Method)

Analgesic activity was determined by a previously optimized method [55] with slight
modifications. Before any dosage, mice were placed on a hot plate (set at a temperature of
55 ◦ C) to determine initial reaction time based on stimulation of pain by heat for a cut-off
time of 30 s. After 1 h of dosage, a licking or jumping reaction was observed on a hot plate
at different time intervals of 1, 2, and 3 h. The standard drug, aspirin was used as positive
control while normal saline was a negative control.
Molecules 2021, 26, 4874 16 of 19

4.6.5. Anti-Inflammatory Activity

The anti-inflammatory activity of A. bracteosa crude extracts was assessed through
the carrageenan-induced hind paw edema model [56]. Diclofenac potassium was used as
a positive control. Then, 1 mL/kg of freshly prepared λ-carrageenan (1% w/v in normal
saline) was injected into mice paw. After 30 min of edema induction, an oral dose of
crude extracts and controls was administered. Control reading of paw volume was taken
immediately before and after injecting carrageenan into the sub plantar region by using
Plethysmometer (UGO Basile 7140, Varese, Italy). Change in paw volume was measured for
up to 3 h. Anti-inflammatory activity was calculated as percent edema inhibition through
the following Formula (4):

Edema volume in control − Edema volume in test

 
Percent edema inhibition = × 100 (4)
Edema volume in control

4.6.6. Anti-Depressant Activity by Tail Suspension Test (TST)

Tail suspension test is a simple and inexpensive method designed by Steru et al. [57]
to assess anti-depressant activity in mice. Briefly, after pretreatment with positive drug and
crude extracts, each mouse was suspended by the tail for 6 min, using an adhesive tape.
The immobility score (in seconds) was recorded during the final 4 min of the experiment.
Data collected in TST were expressed as arithmetic means of immobility time for each
experimental group.

4.6.7. Anticoagulant Assay

A capillary tube was used to analyze the anticoagulation activity of the crude extracts
of A. bracteosa [36]. Briefly, after 1 h of oral dosage, the mice’s tail was pricked with a sterile
needle, and blood was collected in a capillary tube. These tubes were placed at 37 ◦ C for
10 s. Then a small part of the tube was broken at intervals till fibrin threads appear between
two parts of the tube. The time interval between the appearance of blood on mice’s tails
and thread formation was the blood clotting time.

4.7. Statistical Analysis

All the experiments were performed in triplicates. One-way analysis of variance
(ANOVA) was used to find variability between data by Statistix 8.1 (Analytical Software,
Tallahassee, FI, USA). IC50 values were calculated through GraphPad Prism Software
Version 7.0 (GraphPad Prism® Software, Inc., San Diego, CA, USA) by plotting transformed
inhibitors vs. log values of inhibition. The correlation between IC50 values of antioxidant
assays and TPC and TFC was found by GraphPad 7. Here, p > 0.05 was considered
significant, and data were represented as the mean of 3 values ± SD (Standard Deviation).

5. Conclusions
In conclusion, present findings suggest that transgenic A. bracteosa plants are a rich
source of essential elements and polyphenols. Overall, all transgenic lines showed signifi-
cant antioxidant, analgesic, anti-inflammatory, antidepressant, and anticoagulant activities
as compared to the untransformed wild-type plant extracts. We could see that rol genes
influenced positively the production of active secondary metabolites and this could be
linked to the enhanced activities exhibited by the transgenic plant extracts. This study
provides more rationalized scientific reasons for the folklore use of this plant. However,
further investigations are needed to understand the underlying protective mechanisms.

Author Contributions: Conceptualization, S.R., W.K.K. and B.M.; data curation, S.R.; formal analysis,
S.R., N.A. and M.K.O.; funding acquisition, M.K.O.; investigation, S.R.; methodology, S.R.; project
administration, B.M.; software, A.K.; supervision, B.M.; validation, I.A.A.; visualization, R.M. and
S.A.A., writing—original draft, S.R. and W.K.K.; writing—review and editing, Y.A.A. and B.M. All
authors have read and agreed to the published version of the manuscript.
Molecules 2021, 26, 4874 17 of 19

Funding: This study was funded by the Deanship of Scientific Research at King Saud University
through research grant No (RG-1439-63).
Institutional Review Board Statement: The study was conducted according to the guidelines 425 of
the Organization for Economic Corporation and Development (OECD) and approved by the Animal
Ethical Committee, Quaid i Azam University, Islamabad, Pakistan (BEC-FBS-QAU2019-157).
Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets used and/or analyzed during the current study are
available from the corresponding author on reasonable request.
Acknowledgments: The authors extend their appreciation to the King Saud University for funding
this work through research grant No (RG-1439-63). We are thankful to Ihsan-ul-Haq, Assistant
Professor, Department of Pharmacy, Quaid i Azam University, Islamabad, Pakistan for providing
polyphenol standards and extending the HPLC facility.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of plant material are available from the authors.

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