Introduction
Medicinal plants have an important medicinal plant for
the well-being of people and beneficiation to the economy (Alrayes et al.
2018; Shhab et al. 2021). Ruta graveolens L. belongs to the
Rutaceae family. It is an odorous aromatic medicinal plant. Rutaceae family is
a perennial plant that consists of many types of secondary metabolites, such as
furanocoumarins, alkaloids, flavonoids, alkaloids, and essential oils (Diwan
and Malpathak 2007; Kengar and Paratkar 2015; Mahmoud et al. 2015). R.
graveolens known as rue has been used in traditional medicine. R.
graveolens is used for the remedy of various treatments such as eye
problems, rheumatism, dermatitis, psoriasis, vitiligo, and leucoderma pain
(Retheesh and Helen 2007; Diwan et al. 2012; Al-Ajlouni et al.
2015; Orlanda and Nascimento 2015; Hadi et al. 2019).
The
germination percentage of R. graveolens seeds was low and the
seed set also is low does not allow the production of the true-to-type plant
resulting in a great variant of secondary metabolites (Faisal and Naseem 2005;
Orlanda and Nascimento 2015; Hadi et al. 2019). In addition, R.
graveolens obtained shoots would not be genetically identical to the
parent plant and the genetic makeup may vary with the individual shoots because
this plant is a cross-pollinated plant. Due to the above problems, in vitro
propagation method is the only option for high propagation grade of genetically
uniform R. graveolens plants. Many previous studies have been
successfully propagated using in vitro propagation methods (Kunicka-Styczy
´nska and Gibka 2010; Parray et al. 2012; Reddy et al. 2015;
Shatnawi et al. 2019; González-Locarno et al. 2020). In vitro
propagation techniques may assist in the propagation and preserve endangered
and rare medicinal plants. The media compositions and plant growth regulators
play a vital role in in vitro propagation of plants. Therefore, there is
an urgent need to look for alternate means of propagation for R. graveolens which could ensure high mass-producing plants to fulfill
the demands of these plants (Atta-Alla et al. 2008; Parray et al.
2012; Shhab et al. 2021). In vitro propagated medicinal plants
could offer a ready source of even, sterile, and compatible plant material for
biochemical characterization and identification of bioactive constituents (Shhab
et al. 2021).
R. graveolens contain
pharmaceutical active compounds such as alkaloids, coumarins, volatile oils,
and flavonoids that have antimicrobial activity have antimicrobial activity.
The antimicrobial activity of R. graveolens has been studied by Reddey and
Al-Rajab (2016) and Parray et al. (2012). R. graveolens has the
capability to forbid the growth of some microorganisms and have been used in
treating sores, gum disease, and wounds. Many researchers studied bactericidal
activity using a different extract from R. graveolens. Moreover, R.
graveolens have been reported previously that crude extract is capable to
inhibit the growth of Staphylococcus aureus, Salmonella typhimurium, and
Bacillus subtilis (Reddey and Al-Rajab 2016). However, secondary
metabolites synthesized by the plants are responsible for their capability
against microorganisms (Parray et al. 2012). Therefore, the impact of
extract from different plants was studied by many researchers. Since many
secondary metabolites and their cause are sources of antibacterial agents
(Reddey and Al-Rajab 2016). Debnath (2008) indicated that plant crude extracts
were found to be the initial steps for the screening of pure compounds that
were isolated from exceptional outcomes. In addition, secondary metabolites
extracted from tissue cultures may be more easily purified because of simple
extraction procedures and the absence of significant amounts of interfering
pigments, which will minimize the cost of purifying and producing such valuable
compounds (Varma 2011). To our knowledge, the present study is the first report
to illustrate an extensive study on in vitro propagation and
antimicrobial activity of R. graveolens. Therefore, this study was
conducted to develop a simple procedure for micropropagation, also to evaluate
the antimicrobial activity of these important medicinal plants using the
different solvent procedures.
Materials and Methods
Plant materials and culture
conditions
R. graveolens plants seed were collected from five years plant from
the Al-Sareeh, Irbid, Jordan (about 600 meters above sea level, 32.3306° N
latitude and 35.8951°E Longitude). Then seeds were sterilized by using 4% NaOCl
for 10 min, then implanted in 70% ethanol with shaking for 1 minute. After
sterilization, seeds were then washed three times in deionized sterile water in
a laminar flow cabinet. The seeds were germinated firstly on agar water media,
then shoots were cultivated on Murashige and Skoog (MS) medium (1962). Agar was
added at 8 g/L agar was prior to autoclaving. The
medium pH was adjusted to 5.8. 80 mL of medium was dispensed in each 250 mL
flask. Microshoots were incubated in the growth
chamber at 24 ± 2ºC with a 16 h photoperiod and photosynthetic photon flux density
(PPFD) of 50 μmol m-2 s-1
supplied by cool white fluorescent lamps.
Elongation of microshoot
Microshoots were subculture onto80 mL MS medium (250 mL
flask) enriched with 0.05 mg/L 6-benzylaminopurine (BAP) and 30 g/L sucrose.
Effect of
thidiazuron (TDZ) or zeatin on shoot proliferation
Microshoots
length of 10 mm in length was cultivated on MS medium to enrich the various
concentrations of TDZ and zeatin. Sixteen treatments were used and each
treatment consisted of four microshoots. The culture was incubated as described
above. Six weeks later data were collected on the number of shoots, shoot
length, and leaves number.
Antimicrobial
activity
Tested plants: R. graveolens plant material (in vitro
and ex vitro) was obtained from the tissue culture laboratory of Al-Balqa
Applied University.
Fungal and bacterial growth media
The fungal
strain was cultured on (15 mL were poured to 9 cm Petri dishes) potato dextrose
agar (PDA; Himedia, India), while nutrient agar medium (NA; Fluka, Germany) was
used for bacterial strains, about 15 mL (4 mm in thickness) were poured to 9 cm
sterile Petri dishes.
Plant extractions
Plants materials (in vitro and ex vitro) (20 g) were dried
in the shade for 14 d, using in liquid nitrogen (LN), ground to a fine powder,
and then using 100 mL (methanol or ethanol) were extracted by soaking plant
material for seven days (Ndukwe et al. 2006). Then using a rotary
evaporator, the solvents were eliminated (HeidolphVV2000, Germany) under
reduced pressure at below 50ºC temperatures. According to methods, two volumes were taken from the extract (40 µL or 80 µL) were disposed of in
dimethylsulphoxide (DMSO) in (250 µg/µL) concentration and then were
evaluated opposite microbe’s activity, the crude extracts were stored at
-20ºC until used. Both (bactericide)
(oxytetracycline) and fungicide (cyclohexamine), were used as positive control
and DMSO (controls) was used as negative. For evaluated their activity to reduce bacterial and fungal growth,
methanol or ethanol extracts were dissolved in DMSO.
Antibacterial activity assay by the agar well diffusion method
Using sterile swap different bacterial strains were spread on Table 1: Influence of thidiazuron (TDZ) on shoot number, shoot length, and leaves number of in
vitro R. graveolens microshoots after six weeks growth periods
|
Leaves number /explant |
Length of shoot (mm) |
Number new shoots/explant |
TDZ (mg/L) |
|
4.06 ± 0.51 a |
22.19 ± 1.76 c |
1.13 ± 0.09 a |
0.0 |
|
3.38 ± 0.22 a |
10.00 ± 0.00 a |
5.38 ± 0.93 b |
0.5 |
|
4.63 ± 0.40 a |
12.50 ± 1.71 ab |
6.25 ± 0.56 b |
1.0 |
|
6.38 ± 0.34 b |
16.06 ± 1.46 b |
9.44 ± 0.87 c |
1.5 |
|
6.44 ± 0.71 b |
23.06 ± 2.53 c |
6.81 ± 0.96 b |
2.0 |
Means
followed by the same letter within the column are not significantly different
according to Duncan Multiple range test at P
≤ 0.05. Each treatment consisted of 16 replicates and each sample
contained four microshoots. Values are the means ± standard error
Table 2:
Influence of zeatin on shoot number,
shoot length, and leaves number of in vitro R. graveolens microshoots
after six weeks growth
periods
|
Leaves number /explant |
Length of shoot (mm) |
Number new shoots/explant |
Zeatin (mg/L) |
|
4.06 ± 0.51 a |
22.19 ± 1.76 a |
1.13 ± 0.09 a |
0.0 |
|
10.00 ± 1.52 a |
40.75 ± 4.53 bc |
2.19 ± 0.26 b |
0.5 |
|
21.13 ± 5.64 b |
49.56 ± 5.98 c |
3.69 ± 0.52 c |
1.0 |
|
10.19 ± 1.03 a |
44.13 ± 3.52 c |
3.56 ± 0.56 c |
1.5 |
|
9.56 ± 1.25 a |
31.75 ± 3.29 ab |
3.75 ± 0.62 c |
2.0 |
Means
followed by the same letter within the column are not significantly different
according to Duncan Multiple range test at P
≤ 0.05. Each treatment consisted of 16 replicates and each sample
contained four microshoots. Values are the means ± standard error
%20352-358_files/image002.jpg)
Fig. 1:
Formation of multiple shoots of R. graveolens after different growth
periods grew on MS media containing 1.5 mg/L TDZ. A) One-week growth period, B)
4 weeks’ growth periods, C) 6 weeks’
growth periods. Bars represent 5 mm
nutrient agar plates. A well divided into 4 quadrates on 6 mm diameter
sterile plates with a sterile cork borer composed of different R. graveolens
extracts at 40 µL or 80 µL at (250 µg/µL) concentration for
1.0 h. Then Petri dishes were cultivated at 37 ± 2°C for 24 h. Oxytetracycline
(antibiotic) was used as a positive control, which was prepared in (250 µg/µL)
and their antimicrobial activity was examined. The solvent dimethyl sulfoxide
DMSO (negative control) was added, at the end of
the cultivation growth period, the inhibition zone was scaled in mm. The
diameter of the inhibition zone was determined by measuring the microbial
compared with a standard antibiotic (Oxytetracycline). Three Petri dishes for
each treatment were used which consisted of three replicates.
Antifungal activity assay by the agar well diffusion method
A 100 µL spore suspension (1 × 108 spores/mL)
of an aliquot of each isolate was grooved on the surface in radial patterns on
media plates. Each well was made on the plates divided into 4 quadrates (6 mm diameter),
with a sterile cork borer which gains the different R. graveolens
extracts. At 250 µg/µL, 40 and 80 µL of R. graveolens aliquots extract were enriched into
wells and left for 1 h to diffuse, then the plates were incubated at 30 ± 2°C
for 48 h. Positive control (Cyclohexamine) of the antifungal was prepared in
250 µg/µL and using the same manner the antimicrobial activity was tested.
At the end of the cultivation growth
period, the inhibition zone was scaled in mm. The
solvent dimethyl sulfoxide DMSO (negative control) was added Three Petri dishes for each treatment were used which
consisted of three replicates.
Statistical
analysis
A completely
randomized design was used in this study. The results data were exposed to
ANOVA test. Duncan Multiple Range tests were used for mean separation. Data
were analyzed using SPSS programs version 16 (SPSS 2007).
Results
Impact of thidiazuron (TDZ) and kinetine
TDZ at a
concentration of 1.5 mg/L resulted in significantly the maximum number of
shoots (9.44 shoots per explants) (Fig. 1). Shoot length increased
significantly with increase TDZ concentration. The highest shoot length (23.06
mm) was produced at 2.0 mg/L. TDZ at 2.0 mg/L produced the maximum number of
leaves (6.44 leaves per explants) (Table 1).
Impact of
zeatin
3.75 shoots per
explants of were gained when MS medium containing 2.0 mg/L zeatin was used (Table 2). While, on MS medium containing 1.0
mg/L zeatin, the maximum shoot length of 49.56 mm was promoted a maximum number
of leaves per explant formed in medium containing 1.0 mg/L zeatin, the largest
callus (more than 10 mm in diameter) was recorded on 1.5 mg/L zeatin about.
Antifungal
activity
Table
3: Impact of various plant types of Ruta graveolens using ethanolic extract against different
fungal species
|
Zone of inhibition
(mm) |
Crude amount (µL) |
Fungal strain |
|||
|
Control |
Ethanol |
||||
|
Negative |
Positive |
In vitro |
Ex vitro |
||
|
0.00 ± 0.00 a |
17.33 ± 1.45 cde |
21.67
± 2.03 d |
26.33
± 1.45 cd |
40 |
P. digitatum |
|
0.00 ± 0.00 a |
22.00 ± 1.73ef |
28.00
± 1.73 e |
31.00
± 1.73 de |
80 |
|
|
0.00 ± 0.00 a |
9.33 ± 1.45 a |
17.67
± 1.76 c |
14.00
± 1.53 b |
40 |
A. niger mutant.
brown |
|
0.00 ± 0.00 a |
20.33 ± 1.45 e |
32.67
± 2.60 fg |
21.33
± 2.03 c |
80 |
|
|
0.00 ± 0.00 a |
25.33 ± 1.45 f |
0.00
± 0.00 a |
0.00
± 0.00 a |
40 |
A. niger mutant.
black |
|
0.00 ± 0.00 a |
32.00 ± 1.73 g |
12.00
± 1.15 b |
15.33
± 2.03 b |
80 |
|
|
0.00 ± 0.00 a |
11.00 ± 1.73 ab |
0.00
± 0.00 a |
11.33
± 2.03 b |
40 |
P. chrysogenum |
|
0.00 ± 0.00 a |
15.33 ± 1.45 bcd |
0.00
± 0.00 a |
21.00
± 1.73 c |
80 |
|
|
0.00 ± 0.00 a |
10.00 ± 1.73 a |
0.00
± 0.00 a |
21.00
± 2.31 c |
40 |
P. italicum |
|
0.00 ± 0.00 a |
13.67 ± 1.20 abc |
0.00
± 0.00 a |
27.00
± 1.73 d |
80 |
|
|
0.00 ± 0.00 a |
10.33 ± 0.88 a |
0.00
± 0.00 a |
10.67
± 1.76 b |
40 |
A. nidulans |
|
0.00 ± 0.00 a |
15.33 ± 1.45 bcd |
0.00
± 0.00 a |
14.00
± 1.53 b |
80 |
|
|
0.00 ± 0.00 a |
11.33 ± 1.45 ab |
30.00
± 1.53 ef |
26.67
± 2.03 d |
40 |
C. albicans |
|
0.00 ± 0.00 a |
19.33 ± 1.45 de |
36.00
± 1.73 g |
34.33
± 1.45 e |
80 |
|
Means
followed by the same letter within the column are not significantly different
according to Duncan Multiple range test at P
≤ 0.05. Each treatment consisted of three replicates and each sample
contained three Petri dishes. Values are the means ± standard error. Data
obtained after incubation 48 hours on PDA media
Table
4: Impact of various plant types of Ruta graveolens using methanolic extract against different
fungal species
|
Zone of inhibition
(mm) |
Crude amount (µL) |
Fungal strain |
|||
|
Control |
Methanol |
||||
|
Negative |
Positive |
In vitro |
Ex vitro |
||
|
0.00 ± 0.00 a |
17.33 ± 1.45 cde |
26.67
± 2.03 def |
23.00
± 1.15 d |
40 |
P. digitatum |
|
0.00 ± 0.00 a |
22.00 ± 1.73ef |
33.33
± 2.03 g |
28.00
± 1.73 e |
80 |
|
|
0.00 ± 0.00 a |
9.33 ± 1.45 a |
25.00
± 2.31 de |
11.67
± 1.76 ab |
40 |
A. niger mutant.
Brown |
|
0.00 ± 0.00 a |
20.33 ± 1.45 e |
29.67
± 1.76 efg |
15.00
± 1.73 b |
80 |
|
|
0.00 ± 0.00 a |
25.33 ± 1.45 f |
11.67
± 1.76 bc |
16.33
± 1.45 bc |
40 |
A. niger mutant.
Black |
|
0.00 ± 0.00 a |
32.00 ± 1.73 g |
16.00
± 1.73 c |
22.00
± 1.73 d |
80 |
|
|
0.00 ± 0.00 a |
11.00 ± 1.73 ab |
0.00
± 0.00 a |
13.00
± 1.73 ab |
40 |
P. chrysogenum |
|
0.00 ± 0.00 a |
15.33 ± 1.45 bcd |
0.00
± 0.00 a |
20.33
± 1.45 cd |
80 |
|
|
0.00 ± 0.00 a |
10.00 ± 1.73 a |
23.33
± 2.03 d |
12.33
± 1.45 ab |
40 |
P. italicum |
|
0.00 ± 0.00 a |
13.67 ± 1.20 abc |
29.33
± 2.03 efg |
20.33
± 2.03 cd |
80 |
|
|
0.00 ± 0.00 a |
10.33 ± 0.88 a |
7.33
± 0.88 b |
8.33
± 0.88 a |
40 |
A. nidulans |
|
0.00 ± 0.00 a |
15.33 ± 1.45 bcd |
13.33
± 1.45 c |
12.00
± 1.73 ab |
80 |
|
|
0.00 ± 0.00 a |
11.33 ± 1.45 ab |
31.00
± 1.73 fg |
29.00
± 2.31 e |
40 |
C. albicans |
|
0.00 ± 0.00 a |
19.33 ± 1.45 de |
39.00
± 1.73 h |
36.33
± 2.03 f |
80 |
|
Means
followed by the same letter within the column are not significantly different
according to Duncan Multiple range test at P
≤ 0.05. Each treatment consisted of three replicates and each sample
contained three Petri dishes. Values are the means ± standard error. Data
obtained after incubation 48 hours on PDA media
R. graveolens crude in
vitro and ex vitro extract of invariant solvent shown to be very
efficacious against bacteria and fungi. The methanolic and ethanolic extracts
were effective against bacteria and fungi. However, the activities against some
microbes were quite similar to a positive control (Tables 3–6). The in vitro
ethanolic leaf crude extract was not identifying to be effective for some fungi
including Penicillium chrysogenum,
P. italicum, and Aspergillus
nidulans, On the other hand, methanolic extract was not effective
against P. chrysogenum. Maximum
inhibition with ethanolic extract was found against Candida albicans, P. digitatum and P. italicum. While methanolic leaf crude extract was found to
be active against C. albicans, P. digitatum, and
P. italicum. The activity of the ethanolic and methanolic extract
was similar to the antifungal agent tested (Tables 3 and 4). Both ex vitro and
in vitro extract showed to be similar in antifungal properties.
Antibacterial
activity
Crude ex
vitro and in vitro extract was very effective against some bacterial
species. In vitro leaf extract showed a higher inhibition zone against
the A. niger
mutant. brown compared with
ex vitro plants. The antibacterial activities of methanolic
extract and ethanolic leaf crude extracts compared with an antibiotic used were
represented in (Tables 5 and 6). The results of the antibacterial activity showed that ex vitro and in vitro extracts
were more efficacious apposite to
Gram-positive and Gram-negative bacteria. On the contrary, Gram-negative
bacteria were more resistant. In vitro and ex vitro methanolic and ethanolic crude leaf
extracts showed similar results (Tables 5 and 6).
R. graveolens in vitro and ex vitro leaf crude
ethanolic extract was more effective against Gram-positive bacteria tested.
Ethanolic and methanolic in vitro leaf extracts showed a varying degree
of inhibition against bacteria. Maximum inhibition was found in S. aureus, followed by Micrococcus latus and then B. cereus. The results of the current study
show that the solvent used plays an important role in the antimicrobial efficacious. Moreover, the ex vitro and in vitro leaf
extracts showed similar antibacterial properties.
Discussion
Table
5: Impact of various plant types of Ruta graveolens using methanolic extract against different
fungal species
|
Zone of inhibition
(mm) |
Crude amount (µL) |
Strain |
|||
|
Control |
methanol |
||||
|
Negative |
Positive |
In vitro |
Ex vitro |
||
|
0.00 ± 0.00 a |
34.67 ± 0.88 d |
30.33 ± 1.45 d |
20.33
± 1.45 d |
40 |
S. aureus |
|
0.00 ± 0.00 a |
41.00 ± 1.15 e |
33.00 ± 2.08 d |
22.67
± 1.45 d |
80 |
|
|
0.00 ± 0.00 a |
30.67 ± 1.20 c |
25.67 ± 1.20 c |
20.67
± 1.20 d |
40 |
B. cereus |
|
0.00 ± 0.00 a |
36.33 ± 1.20 d |
30.67 ± 1.20 d |
22.00
± 1.73 d |
80 |
|
|
0.00 ± 0.00 a |
35.67 ± 0.88 d |
30.33 ± 1.45 d |
15.33
± 1.20 c |
40 |
Micrococcus latus |
|
0.00 ± 0.00 a |
43.67 ± 1.20 e |
33.33 ± 1.03 d |
22.33
± 1.76 d |
80 |
|
|
0.00 ± 0.00 a |
10.67 ± 1.20 a |
0.00 ± 0.00 a |
0.00
± 0.00 a |
40 |
Salmonella typhimurium |
|
0.00 ± 0.00 a |
12.67 ± 1.20 a |
0.00 ± 0.00 a |
0.00
± 0.00 a |
80 |
|
|
0.00 ± 0.00 a |
13.33 ± 1.20 a |
0.00 ± 0.00 a |
0.00
± 0.00 a |
40 |
Pseudomonas aeruginosa |
|
0.00 ± 0.00 a |
24.00 ± 1.53 b |
0.00 ± 0.00 a |
0.00
± 0.00 a |
80 |
|
|
0.00 ± 0.00 a |
24.33 ± 1.20 b |
12.00 ± 2.08 b |
11.67
± 1.76 b |
40 |
Escherichia coli |
|
0.00 ± 0.00 a |
50.33 ± 0.88 f |
1 4.00 ± 1.15 b |
15.33
± 0.88 c |
80 |
|
Means
followed by the same letter within the column are not significantly different
according to Duncan Multiple range test at P
≤ 0.05. Each treatment consisted of three replicates and each sample
contained three Petri dishes. Values are the means ± standard error. Data
obtained after incubation 24 hours on NA media
Table
6: Impact of various plant types of Ruta graveolens using ethanolic extract against different
bacterial species
|
Zone of inhibition
(mm) |
Crude amount (µL) |
Strain |
|||
|
Control |
Ethanol |
||||
|
Negative |
Positive |
In vitro |
Ex vitro |
||
|
0.00 ± 0.00 a |
34.67 ± 0.88 d |
30.33 ± 1.76 c |
19.33
± 2.03 c |
40 |
S. aureus |
|
0.00 ± 0.00 a |
41.00 ± 1.15 e |
34.00 ± 1.53 d |
21.67
± 1.76 c |
80 |
|
|
0.00 ± 0.00 a |
30.67 ± 1.20 c |
28.33 ± 1.45 c |
20.33
± 0.88 c |
40 |
B. cereus |
|
0.00 ± 0.00 a |
36.33 ± 1.20 d |
30.00 ± 1.73 c |
20.33
± 1.45 c |
80 |
|
|
0.00 ± 0.00 a |
35.67 ± 0.88 d |
12.00 ± 1.73 b |
28.33
± 1.45 d |
40 |
Micrococcus latus |
|
0.00 ± 0.00 a |
43.67 ± 1.20 e |
14.33 ± 1.45 b |
34.67
± 1.20 e |
80 |
|
|
0.00 ± 0.00 a |
10.67 ± 1.20 a |
0.00 ± 0.00 a |
0.00
± 0.00 a |
40 |
S. typhimurium |
|
0.00 ± 0.00 a |
12.67 ± 1.20 a |
0.00 ± 0.00 a |
0.00
± 0.00 a |
80 |
|
|
0.00 ± 0.00 a |
13.33 ± 1.20 a |
0.00 ± 0.00 a |
0.00
± 0.00 a |
40 |
P. aeruginosa |
|
0.00 ± 0.00 a |
24.00 ± 1.53 b |
0.00 ± 0.00 a |
0.00
± 0.00 a |
80 |
|
|
0.00 ± 0.00 a |
24.33 ± 1.20 b |
0.00 ± 0.00 a |
0.00
± 0.00 a |
40 |
E. coli |
|
0.00 ± 0.00 a |
50.33 ± 0.88 f |
12.67 ± 1.20 b |
9.00
± 1.15 b |
80 |
|
Means
followed by the same letter within the column are not significantly different
according to Duncan Multiple range test at P
≤ 0.05. Each treatment consisted of three replicates and each sample
contained three Petri dishes. Values are the means ± standard error. Data
obtained after incubation 24 hours on NA media
In vitro R. graveolens plantlets were
established successfully, with only a very low contamination percentage (data
not shown). The sterilization procedure used in this study gave satisfactory
results. Micropropagation has been used to promote conservation and maintenance
of free disease-free plants under controlled environmental conditions (Luan et
al. 2006; Alrayes et al. 2018). Huettema and
Precee (1993) reported that low concentrations (<1 µM) increased axillary proliferation compared to other cytokines.
on the other hand, TDZ inhibits the length of the shoot. Moreover, TDZ can
enhance adventitious shoots, somatic embryos and the formation of callus. The adding of different concentrations of TDZ or zeatin
to the media of R. graveolens enhance multiplication (Tables 1 and 2). In the current study TDZ promotes multiple shoots (Table
1).
Medium
containing 1.5 mg/L TDZ produced
maximum number of shoots (9.4 shoots per explants). However, this
study showed that TDZ at low concentrations had efficiency in the production of
adventitious buds (Murthy et al. 1998). This is opposite to the previous
finding in Artemisea herba-alb and Stevia rebaudiana
Shatnawi et al. 2011a, b. On
the other hand, TDZ increased the axillary bud formation and decreased the
length of the newly developed shoot because it released apical dominance. Reddy et al. (2015) reported that shoot
sprouting percentage, shoot number, and length were increased with increasing
TDZ concentration. Therefore, the
TDZ effect depends on exposure duration, explant, and plant (Huettema and Preece 1993; Reddy et al. 2015).
Antimicrobial
activity
Nowadays there is a high
demand for discovering new substances from plants to use against many
microorganisms, which can affect human health. R. graveolens ex vitro
leaf showed effectiveness against both bacteria and fungi. R. graveolens
in vitro and ex vitro leaf extracts of R. graveolens prepared
in ethanol and methanol showed high activity against microorganisms tested
(Tables 3-6). However, both ethanol and methanol extracts were found to be
positive against gram-positive bacteria and gram-negative bacteria. The methanolic extract
showed the high effective opposite to the bacteria
study (S. aureus, B. cereus, Micrococcus latus, and S. typhimurium
the activity was quite similar to antibiotic tested. Using in vitro ethanolic
plants extract maximum inhibition was found in Micrococcus
latus, followed B. cereus,
and S. aureus (Table 6). While using in vitro ethanol
plant extract with fungi, the highest zone of inhibition was found in C. albicans followed P. digitatum, P. italicum and A. niger mutant. brown. The activity of the in vitro extract using
methanol with fungi was found to be C.
albicans, P. digitatum,
A. niger
mutant. black, and P. italicum.
In this study,
the methanolic extract of R. graveolens had an antimicrobial effect in
accordance with the other investigations realized on different kinds of
explants (Ojala et al. 2000; Oliva et al. 2003). The
methanolic and ethanolic extract shows effectiveness against fungi tested in
this study where the activity was similar to then antifungal tested. R.
graveolens leaves would be useful in developing antimicrobial substances. R.
graveolens extract might have the mode of action on DNA strands that cause
cell death. Preethi et al. (2008) indicated that R. graveolens
at higher concentrations acted as a pro-oxidant rather than an antioxidant,
which influences mitochondrial absorbency transition pore (Kushnareva and
Sokolove 2000; Preethi et al. 2006). This plant has strong
antispasmodic properties. R. graveolens plants accumulate linear
furanocoumarins (psoralens) and acridone or furoquinolone alkaloids. The
acridone alkaloids were detected in all organs particularly in endodermal and
vascular tissue (Kushnareva and
Sokolove 2000; Preethi et al. 2006).
In vitro extracts show high antimicrobial activity against tested
bacterial species; it may be due to the presence of a high concentration of
toxic compounds as result in HPLC analysis (Al-Ajlouni et al. 2015). It
has been reported it may contain flavonoids
rutin, alkaloids quinolone, furoquinolone, acridone, (psoralens), essential
oils like 2-nananone, 2- undecyl acetate, graveoline, coumarins like
furocoumarin pyranocoumarin and (Sinshemoke et al. 2000; Preethi et al. 2006). This may be attributed to the
cause of its high antimicrobial activity because it contains different
secondary metabolites. In conclusion, this study builds up good evidence that R.
graveolens may possibly be used as natural medical utilization for microbes and would help
for the development of a new alternative medicine system that has no side
effects.
Conclusion
The present study has resulted in the founding of a
consistent and reproducible protocol of R. graveolens which could
be used for mass multiplication as well as antibacterial and antifungal activity against both some bacteria and fungi. In
vitro, R. graveolens plantlets were
established successfully. Moreover, TDZ at 1.5 mg/L resulted in significantly
the maximum number of shoots (9.44 shoots per explants). The crude leaf extract
from in vitro and ex vitro R. graveolens dissolved in different solvent solutions showed diverse
activity against both some bacteria and fungi. Moreover, this study builds up
worthy indication that R. graveolens may possibly be used as normal
medical utilization for microbes and would help for the expansion of a new
alternative medicine system that has no side effects.
Acknowledgments
The authors would like to express their gratitude for
the Jordanian Ministry of Higher Education for their financial support (grant
number Z.B 2/27/2008).
Author Contributions
MA, MS, and WS planned the experiments, and sample
collection. SA, MM and SAb interpreted the results, MA, MS, MM and WS made the
write original, editing, and statistically analyzed the data, and made
illustrations. All authors commented on the manuscript, reviewed drafts of the
paper, and approved the final draft.
Conflict of Interest
All authors declare no conflict of interest
Data Availability
Data presented in this study will be available on a fair
request to the corresponding author
Ethics Approval
Not applicable in this manuscript
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