Bio-organic and inorganic fertilizers modify leaf nutrients, essential oil properties, and antioxidant capacity in medic savory (Satureja macrantha L.)

Abstract

The present study was carried out to assess the effects of bio-organic and inorganic fertilizers on plant nutrients, essential oil composition and antioxidant capacity of Satureja macrantha L. The experiment included nine treatments viz., NPK (50:25:25 kg ha−1), Vermicompost (VC) (5 t ha–1), NPK +VC, Thiobacillus (T), T+VC, T+sulfur (S) (250 kg ha−1), T+S 500 kg ha−1, Glomus mosseae, and control (untreated plants). The results showed the highest Essential Oil (EO) content and yield were respectively observed in plants treated with the combination of VC and NPK. Total Phenol Content (TPC) in first-year plants treated with VC and second-year plants under NPK+VC were higher than other experimental plants. Total Flavonoid Content (TFC) in second-year plants under the combination of NPK was greater than other plants. N content in first year plants treated with NPK fertilizer / combination of NPK and VC was higher compared to other experimental plants. The highest P content was observed in the NPK application in first year. Gas Chromatography—Mass Spectrometry (GC/MS) analyses revealed that the main constitutes of S. macrantha EO were p- Cymene (16.30-34.64%), γ-terpinene (15.46-33.6%), and Thymol (14.82-43.09%), which had different responses to sampling time and fertilizer treatments.

Full text

Abstract
The present study was carried out to assess the effects of
bio-organic and inorganic fertilizers on plant nutrients, essential
oil composition and antioxidant capacity of Satureja macrantha
L. The experiment included nine treatments viz., NPK
(50:25:25 kg ha−1), Vermicompost (VC) (5 t ha–1), NPK +VC,
Thiobacillus (T), T+VC, T+sulfur (S) (250 kg ha−1), T+S 500 kg
ha−1,Glomus mosseae, and control (untreated plants). The
results showed the highest Essential Oil (EO) content and yield
were respectively observed in plants treated with the combina-
tion of VC and NPK. Total Phenol Content (TPC) in first-year
plants treated with VC and second-year plants under NPK+VC
were higher than other experimental plants. Total Flavonoid
Content (TFC) in second-year plants under the combination of
NPK was greater than other plants. N content in first year plants
treated with NPK fertilizer / combination of NPK and VC was
higher compared to other experimental plants. The highest P
content was observed in the NPK application in first year. Gas
Chromatography—Mass Spectrometry (GC/MS) analyses
revealed that the main constitutes of S. macrantha EO were p-
Cymene (16.30-34.64%), γ-terpinene (15.46-33.6%), and
Thymol (14.82-43.09%), which had different responses to sam-
pling time and fertilizer treatments.
Introduction
The genus satureja, belonging to Lamiaceae family, native-
ly grows in Africa, southern and southeastern Europe, the mid-
dle east, and central Asia. Iran has 13 satureja species, in which
S. macrantha L., as the most ones, grows in north-west of the
country.1,2 Phenols, flavonoids, anthocyanins, sterols, diter-
penes, and triterpenes are the main phytochemical compounds
of S. macrantha L.2,3 Essential oil of most satureja species
such as S. macrantha L. is mainly known by thymol and
Carvacrol.1-3
Fertilization is considered as the most principal factor in
plant nutrition.4 The type, amount, and application method of
fertilizer directly affect the availability of plant nutrients and
indirectly influenced physiological and biochemical mecha-
nisms in plant.4 Chemical fertilizers are extensively used to
enhance crop yield, albeit their long-term application change
soil pH, decrease beneficial soil microflora, pollute water sup-
plies, and disturb soil ecological systems.5,6 However, in recent
years, demand for organic products has increased due to their
healthy and environmental consideration especially for medici-
nal products.6Furthermore, tendency to medicinal plants culti-
vation with the application of soil amendments such as
manures, mycorrhiza, and vermicompost has been increased.5
These compounds contain nutrients for crop growth. They
improve physico-chemical attributes of the soil and increase
organic matter, cation exchange, water holding capacity, and
ultimately upgrade the quantity and the quality of medicinal
plants. 6
Literature reviews showed that inorganic and organic fertil-
izers can improve the quality and quantity of medicinal plants.
For example, we can report the useful effect of Arbuscular
Mycorrhizal Fungi (AMF) on Satureja macrostema,7Sesbania
sesban,8and Leptospermum scoparium,9the functional impact
of vermicompost on Amaranthus retroflexus L.,10 Drimiopsis
maculata,11 and the helpful reaction of NPK on Cucurbita pepo
L.12 Totally, they demonstrated that the application of proper
portion for different fertilizers, separately or in combination,
can help the producers to reach the optimum products of medic-
inal plants.
To the best of our knowledge, there is no report on the com-
bination of these soil amendments on phytochemical properties
and leaf nutrients of S. macrantha L. Therefore, the aims of
present study were i) to assess the effects of NPK, vermicom-
post, thiobacillus, sulfur, and G. mosseae, on plant nutrients, ii)
to evaluate their effects on phenol, flavonoid and antioxidant
capacity, and iii) to discover their effects on essential oil content
and composition of S. macrantha L.
Correspondence: Hamid Mozafari, Department of Agronomy, Shahr-e-
Qods Branch, Islamic Azad University, Tehran, Iran
E-mail: hamidmozafari1398@gmail.com
Key words: Essential oil; Thymol; Total phenol content; Nutrients
uptake.
Conflict of Interests: The Authors declare no potential conflict of
interests.
Received for publication: 9 August 2019.
Accepted for publication: 28 November 2019.
©Copyright: the Author(s), 2020
Licensee PAGEPress, Italy
Journal of Biological Research 2020; 93:8477
doi:10.4081/jbr.2020.8477
This article is distributed under the terms of the Creative Commons
Attribution Noncommercial License (by-nc 4.0) which permits any
noncommercial use, distribution, and reproduction in any medium,
provided the original author(s) and source are credited.
[Journal of Biological Research 2020; 93:8477] [page 1]
Journal of Biological Research 2020; volume 93:8477
Bio-organic and inorganic fertilizers modify leaf nutrients, essential oil
properties, and antioxidant capacity in medic savory (
Satureja macrantha
L.)
Mitra Bakhtiari,1Hamid Mozafari,1Khalil Karimzadeh Asl,2Behzad Sani,1Mehdi Mirza2
1Department of Agronomy, Shahr-e-Qods Branch, Islamic Azad University; 2Research Institute of Forests and
Rangeland, Agricultural Research, Education and Extension Organization (AREEO) Tehran, Iran
Non-commercial use only

[page 2] [Journal of Biological Research 2020; 93:8477]
Materials and Methods
Experimental site
This work was accomplished at the experimental farm in
Alborz Research Station, Research institute of Forests and
Rangelands, Karaj (1312 m asl, 35°48´45”N, 51°01´30”E), Iran
during 2017–2018. The soil features of the experimental site con-
sisted of silt, lightly acidic (pH 6.4), high organic carbon (1.06%),
and presented a low availability of N (151.5 kg ha−1), P (15.2 kg
ha−1), and K (485.2 kg ha−1). During the study period mean maxi-
mum temperature fluctuated from 17.3°C to 28.2°C; whereas;
mean minimum temperature varied from 2.6°C to 15.4°C. The
highest precipitation occurred in March (43.2 mm) and April (39.1
mm) (Figure 1).
Fertilizers and soil amendments used
For most types of soils, the manufacturer’s suggested rate of N
is considered at a rate of 50 kg N ha−1 applying ammonium nitrate
(33 %); P was used at a rate of 25 kg P ha−1 using calcium super
phosphate (16 %); and potassium sulphate (48% K2O) was used for
K at a rate of 25 kg K ha−1. G. mosseae as a species of AMF was
applied in rhizosphere. The mycorrhizal inocula (originally isolat-
ed by the endemic AMF group of a maize farm), a combination of
sterile sand, hyphae and spores of AMF (20 spores/g inoculum)
and colonized fragments of root, were provided by Research
Institutes of Forest and Rangelands (RIFR), Iran. To produce
Vermicompost (VC), adult epigeic species of clitellate earth-
worms, Eudrilius eugineae, composted a combine of distillation
waste (plant-spent de-oiled herb) of aromatic grasses in a VC unit
during three months. This VC combination was used as the organic
fertilizer in our experiment. The VC included 1.07% N, 0.62% P
and 0.73% K.
Experimental details
The experiment consisted of nine treatments viz., NPK
(50:25:25 kg ha−1), VC (5 t ha–1), NPK +VC, Thiobacillus (T),
T+VC, T+sulfur (S) (250 kg ha−1), T+S 500 kg ha−1, G. mosseae,
and control (untreated plants). The experiment was fulfilled in ran-
domized complete block design (RCBD) with three replications.
Seeds of S. macrantha L. were sown in the culture trays prepared
with a cocopeat perlite mixture (1:1 volume). Two-month
seedlings were transplanted in the farm at a spacing of 50 cm (row
to row) × 50 cm (plant to plant) during march 2017 and 2018.
Inorganic fertilizer (N, P, and K) were applied utilizing urea (N
50%). Irrigation was fulfilled weekly during non-rainy season.
Determination of N, P and K content
Dried powder (100 mg) of leaves was precisely transferred to
a digestion tube and 2 cm3of AR grade concentrated sulphuric acid
was added. It was heated on a temperature controlled assembly at
80 °C for 2 h. On heating, the contents of the tube turned black. It
was cooled at room temperature for 15 min and then 0.5 cm3of
H2O2 (30%) was added drop by drop. The addition of H2O2fol-
lowed by gentle heating was repeated until the contents of the tube
being changed colorless. The aliquot (peroxide-digested material)
was used to estimate percentage content of N, P and K in the shoot
on dry weight basis.
Estimation of N content
Dried powder (0.2 g) of aerial parts was used based on diges-
tion method. After preparing the digested solution, we determined
the concentrations of N content by Kjeldahl.13 For this purpose, 0.2
g of dried plant was combined with 20 ml of sulfuric acid and a
Kjeldahl tablet. The mixture was then placed in a digestive tube in
the oven at 400 ° C until the sample color would change to a light
green. The resulting solutions were cooled at room temperature
and diluted by adding sulfuric acid (0.01 N) until the color would
turn to red. The volume of acid consumed was recorded and N con-
tent calculated as follows: N%= A*N*1.4/W; where N is normality
of the used acid, A is the volume of used acid, and W is weight of
leaf sample.
Estimation of P content
For P measurement, a 5 cm3aliquot was taken in a10 cm3grad-
uated test tube. To it, 1 cm3molybdic acid (2.5%) was added care-
fully, followed by addition of 0.4 cm3of 1-amino-2-naphthol-4-
sulphonic acid. When the color turned blue, the volume was made
up to 10 cm3with double distilled water (DDW). The O.D. of the
solution was recorded at 620 nm using the spectrophotometer. The
P content was estimated using a standard graph prepared by graded
dilutions of monopotassium phosphate.14
Estimation of K content
K content in the leaves was determined flame photometrically
in the peroxide-digested material with the help of emission spectra
using specific filters. In the flame-photometer, the solution (perox-
ide digested material) was discharged through an atomizer in the
form of a fine mist into a chamber, where it was drawn into a
flame. Combustion of the elements produced the light of a partic-
ular wavelength [(λ max for K= 767 nm (violet)]. The light pro-
duced was conducted through the appropriate filters to impinge
upon a photoelectric cell that activated a galvanometer, resulting in
the digital reading of the respective samples.15
Essential oil extraction
In order to identify the EO content, during peak flowering sea-
son, 100 g of dried aerial parts from each treatment were hydrodis-
tilled in the Clevenger type apparatus for 3 hr. The EO content was
measured and reported as v/w percentage. The EO yield (kg ha−1)
was measured with multiplying the EO content with the plant yield
of the experimental treatments. Anhydrous sodium sulfate was
used to dry EO samples, and finally the samples were stored at 4°C
to further analysis of Gas Chromatography (GC) and Gas
Chromatography—Mass Spectrometry (GC-MS).
Article
Figure 1. Mean temperature and rainfall in the case study.
Non-commercial use only

Gas Chromatography (GC) analysis
Thermo-UFM ultrafast gas chromatograph equipment with a
ph-5 fused silica column (10m length × 0.1 mm id., film thickness
0.4 µm) was used to anlyze EOs. Oven temperature was main-
tained at 60 º C for 5 min and then programmed to 285 º C at a rate
of 5 º C min–1; Flame Ionization Detector (FID) and injector tem-
perature were 290 º C and 280 º C, respectively; helium was
applied as carrier gas with an inlet pressure of 0.5 kg cm-2.
Gas Chromatography—Mass Spectrometry (GC-MS)
GC-MS analyses were accomplished by Varian 3400 GC-MS
system equipment with AOC-5000 auto injector and DB-5 fused
silica capillary column (30 m × 0.25 mm i.d.; film thicknesses
0.25 µm). Temperature was programmed from 60°C to 250°C with
3 °C min–1; Injector and interface temperature were 260 °C and
270 °C, respectively; acquisition mass range of 40–340 amu; ion-
ization voltage of 70 eV; the carrier gas was helium at a velocity of
45 cm sec–1.
Component identification
Homologous series of n-alkanes (C7–C25) determined the
retention index for all volatile constituents. According to Adams,
the components of oil were identified by matching their Retention
Indices (RI) and mass spectra. EO components were identified by
GC/MS spectroscopy.
Determination of Total Phenolic Content (TPC)
Folin–Ciocalteu reagent was selected to measure TPC spec-
trophotometrically.16 100 µl of the MeOH solution of the precisely
measured weight of investigated plant 1–10 (2.54, 2.58, 2.25, 4.03,
4.80, 2.13, 4.62, 1.47, 1.58, 15.05 mg/mL respectively) were
mixed with 0.75 mL of Folin–Ciocalteu reagent and allowed to
stay at 22º C for 5 min. The mixture was supplied with 0.75 ml of
NaHCO3. Absorbance was measured at 725 nm by UV–vis spec-
trophotometer (Varian Cary 50) after 90 min at 22 ºC. Standard
curve was calibrated by Gallic acid (0–100 mg/; r > 0.99). The
results were represented as mg Gallic acid /g Dry weight.
Determination of Total Flavonoid Content (TFC)
The method with aluminum chloride was applied to measure
the total flavonoid content.17 Briefly, the mixture containing 0.5
mL of sample and 300 μL of NaNO2(1:20 w/v) was vortexed for
10 s and left to stand at 24 º C for 5 min. After that, the reaction
mixture was changed by 300 μL of AlCl3(1:10 w/v), 2 mL of
NaOH (1 M) and 1.9 mL of distilled water, and then vortexed for
10 s. The absorbance was determined at 510 nm. Quercetin con-
centrations ranging from 0 to 1200 μg/mL were prepared and lin-
ear fit was used for calibration of the standard curve.
Radical scavenging activity
Free radical scavenging activities of the extracts was measured
using a DPPH radical described by Brand-Williams et al.: 0.1 ml
of the extract solution was mixed with 1.0 ml of DPPH solution
and 4 ml of methanol. 18 The absorbance wavelength was 517 nm
performed by UV–vis spectrophotometer (Varian Cary 50). The
scavenging effect was determined as follow:
DPPH scavenging% =
[1/(A 517 nm, sample – A 517 nm, control)] × 100. (1) (Eq. 1)
Statistical analysis
The data (n= 3) were subjected to one-way analysis of vari-
ance (ANOVA) and using the SAS software package for Windows
(SAS, version 9.3, SAS Institute, Cary, NC). Duncan’s multiple
range tests showed the comparison of mean values. The data were
statistically investigated at 5% probability level.
Results
Macronutrients (NPK)
The interaction of fertilizer and sampling time was signifi-
cant on NPK (P≤0.05). N in first year plants treated with NPK
fertilizer / combination of NPK and VC was higher than that in
other plants (Figure 2). The highest P content was observed in the
NPK application in first year as 0.28±0.029%. In contrast, the
lowest P was in the untreated plants in both first and second year
(Figure 3). K in plants with application of NPK fertilizer in first
[Journal of Biological Research 2020; 93:8477] [page 3]
Article
Figure 2. Nitrogen (N) content under fertilizer and time. Means
followed by the same letter are not significantly different
(P<0.05) as determined by Duncan’s multiple range test.
Figure 3. Phosphorous (P) content under fertilizer and time.
Means followed by the same letter are not significantly different
(P<0.05) as determined by Duncan’s multiple range test.
Non-commercial use only

[page 4] [Journal of Biological Research 2020; 93:8477]
and second year, as 4.4±.16% and 4.1±.15% respectively, was
greater than other treatments (Figure 4).
Essential Oil (EO) content and yield
Both EO content and yield were affected by fertilizer and time
(P≤0.05). The highest EO content (2.025±0.1%) and EOY
(55.04±3.7 kg/ha) was respectively observed in plants under the
combination of VC and NPK (Table 1). In contrast, the lowest EO
content and yield was recorded in untreated plants (0.72±0.06%,
7.26±0.9 kg/ha), and plants inoculated with G. mosseae
(0.81±0.06%, 10.71±1.6 kg/ha) (Table 1). Both EO content and
yield in second year (1.37± 0.4 %, 27.4±7 kg/ha) was greater than
those in first year (1.09±0.4 %, 23.2±8 kg/ha) (Table 2).
Total Phenolic Content (TPC), Total Flavonoid Content
(TFC), and DPPH-scavenging activity
The TPC, TFC, and DPPH-scavenging activity were influ-
enced by the interaction of fertilizer and time (P≤0.05). TPC in
first-year plants treated with VC, and second-year plants treated
with NPK+VC, as well as second-year plants with TB + sulfur 500
were higher than other plants (Figure 5). TFC in second-year
plants treated with combination of NPK and VC (15.2 ±1.7 mg
quercetin/gr DW) and second-year plants with VC (14. 8 ±0.5 mg
quercetin/gr DW) were greater than other experimental plants
(Figure 6). DPPH-scavenging activity in second-year plants under
TB + S 500 (86.33±3 %) was higher compared with other plants
(Figure 7).
Essential oil composition
The GC/MS analysis showed 24 compounds of S. macrantha
L. EO, which most of these components were monoterpene (Tables
Article
Figure 4. Potassium (K) content under fertilizer and time. Means
followed by the same letter are not significantly different
(P<0.05) as determined by Duncan’s multiple range test.
Figure 5. Total phenolic content (TFC) under fertilizer and time.
Means followed by the same letter are not significantly different
(P<0.05) as determined by Duncan’s multiple range test.
Figure 6. Total flavonoid content (TFC) under fertilizer and time.
Means followed by the same letter are not significantly different
(P<0.05) as determined by Duncan’s multiple range test.
Figure 7. DPPH-scavenging activity under fertilizer and time.
Means followed by the same letter are not significantly different
(P<0.05) as determined by Duncan’s multiple range test.
Non-commercial use only

3 and 4). p-Cymene, γ-terpinene, and Thymol were the main con-
stitutes of S. macrantha EO (Tables 3 and 4). p-Cymene was dif-
ferent during two year and fertilizer treatments. This in plants
treated with the combination of VC and NPK (30.08%) was higher
than other treatments in first year. In contrast, its high content was
changed to the combination of TB and S250 (29.95%) in second
year. In first year, γ-terpinene in EOs of plants under the combina-
tion of VC and NPK was higher compared to other treatments.
However, its maximum content in second year was observed in
plants inoculated with G. Mosseae (30%). Thymol- acetate was
fluctuated from the untreated plants (24.91%) to the combination
of TB and S500 (43.09 %) in first year. This trend in second year
varied from control (14.82%) to application of NPK (37.57%).
Discussion
NPK fertilizer in combination with VC increased the N, K, and
P contents. A 92% increase of N content in aerial parts was
observed in second-year plants under combination of NPK fertiliz-
er and VC compared to untreated plants. In addition, we found a
77% increase of K content in first-year plants under NPK fertilizer
relative to untreated plants. This increase in first year for P content
was recorded 2.5 fold in NPK-treated plants compared to untreated
plants. NPK fertilizer increase the availability in rhizosphere and
[Journal of Biological Research 2020; 93:8477] [page 5]
A
Table 1. Essential oil (EO) content and yield of S. macrantha L.
under different fertilizer treatments.
Treatment EO content (%) EO yield (kg/ha)
Control 0.72±0.06 e 7.26±0.9 e
NPK 1.33±0.13 c 30.78±2.2 c
Vermicompost 1.43±0.1 bc 31.89±2.8 c
NPK + Vermicompost 2.025±0.1 a 55.04±3.7 a
Vermicompost +
Thiobacillus 1.52±0.53 b 37.31±2.3 b
Thiobacillus 1.01±0.07 d 16.06±4 d
Thiobacillus + S250 1.09±0.11 d 18.61±3.2 d
Thiobacillus + S500 1.165±0.1 d 20.19±4.1 d
G. mosseae 0.81±0.06 e 10.71±1.6 e
Table 2. Essential oil (EO) content and yield of S. macrantha L.
during 2017 and 2018.
Time EO content (%) EO yield (kg/ha)
2017 1.09±0.4 b 23.2±8 b
2018 1.37± 0.4 a 27.4±7 a
Table 3. Percentage of essential oil (EO) composition in S. macrantha L. under different fertilizer treatments in 2017.
No Compound RI Control NPK VC NPK+VC TB+VC TB TB+S250 TB+S500 GM
(%) (%) (%) (%) (%) (%) (%) (%) (%)
1 α-thujene 934.61 1.53 0.85 2.22 1.17 0.86 1.23 1.28 0.92 0.59
2 α-pinene 946.09 2.17 0.7 0.75 1.5 1.07 1.9 1.95 0.75 0.7
3 Camphene 962.66 0.18 0.11 0.21 0.16 0.1 0.14 0.19 0.12 1.87
4 Sabinene 976.79 2.49 1.89 1.01 2.46 2.2 2.1 2.2 1.73 0.52
5 β-pinene 1007.94 1.29 0.41 0.54 0.97 0.77 1.28 1.17 0.41 0.13
6 3-octanone 1033.65 0.18 0.17 0.18 0.18 0.14 0.17 0.21 0.17 2.03
7 Myrcene 1043.62 2.67 2.65 2.35 2.53 2.43 2.59 3.21 2.55 1.88
8 α-terpinene 1046.08 2.32 0.33 0.55 1.18 1.55 0.98 0.95 0.15 2.04
9 p-cymene 1053.41 27.54 17.47 25.4 30.08 22.72 26.53 16.4 16.3 17.74
10 β-phellandrene 1055.84 0.27 0.23 0.26 1.67 1.94 1.37 0.27 0.25 0.11
11 (Z)-β-ocimene 1060.65 2.62 0.38 1.11 0.19 0.14 0.18 1.19 0.21 0.13
12 (E)-β-ocimene 1063.04 0.12 0.12 0.1 0.24 0.25 0.24 0.17 0.12 0.29
13 γ-terpinene 1084.13 28.2 28.93 24.4 30.39 27.51 29.92 33.6 28.23 25.69
14 cis-sabinene hydrata 1100 0.26 0.23 0.18 0.16 0.15 0.13 0.18 0.25 0.23
15 Terpinene-4-ol 1219.85 0.3 0.15 0.52 0.13 0.18 0.2 0.16 0.16 0.19
16 Carvacrol 1322.63 0.2 0.21 0.28 0.54 0.15 0.14 0.95 0.17 0.96
17 Thymol-acetate 1330.04 24.91 40.48 34.23 22.06 34.13 27.25 31.86 43.09 39.56
18 (E)-βcaryophyllene 1476.54 0.43 0.51 0.16 1.37 1.2 0.2 1.41 0.62 1.63
19 Aromadendrene 1497.09 0.12 0.28 0.55 0.19 0.13 1.08 0.2 0.47 0.21
20 Germacrene d 1520.15 0.74 1.28 0.32 0.16 0.12 0.14 0.18 1.1 0.18
21 Bicyclogermacrene 1534.32 0.56 0.22 0.62 0.97 0.73 0.14 0.91 0.14 1.01
22 β-bisabolene 1542.75 0.22 0.14 0.17 0.2 0.17 0.77 0.27 0.12 0.25
23 ɵ-cadinene 1548.33 0.4 0.72 0.29 0.42 0.38 0.16 0.49 0.57 0.41
24 Spathulenol 1556.66 0.19 0.26 0.78 0.16 0.1 0.32 0.18 0.18 0.18
Total (%) 99.91 98.72 97.18 99.08 99.12 99.16 99.58 98.78 98.53
VC, Vermicomost; TB, thiobacillus; S, sulfur; GM, glomus mosseae.
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[page 6] [Journal of Biological Research 2020; 93:8477]
plant can uptake these essential elements properly. VC can
increase the availability of essential elements for plant particularly
the amounts of N and P. The increase of N, P, and K uptake by
leaves of plants have been reported under VC,19 NPK,20 and com-
bination of VC and NPK.21
Inorganic fertilizer, VC, and combination of TB and sulfur
improved the antioxidant activity of S. macrantha. Phytochemical
properties of humid substance in VC improve the antioxidant
capacity of plants.22 Phenolic acids are an important group of sec-
ondary metabolites, which found in medicinal plants, and they
show strong antioxidant activities due to their carboxyl groups and
hydroxyl. 23 Hydroxycinnamic acids and hydroxybenzoic acids are
two main groups of phenolic acids. Flavonoids via demolishment
and detoxification of free radicals have strong impacts on cell biol-
ogy.23 In this regard, VC can play an important role in use of
organic manufacturing systems to improve the flavonoids biosyn-
thesis in plants. An increase of phenol and flavonoid content in
Pumpkin under NPK fertilizer were recorded by Oloyede et al..12
Some reports have shown the positive influence of VC in increas-
ing the synthesis of flavonoids in plants. The amounts of phenolic
compounds are strongly correlated with antioxidant power of the
plant.24 The application of humic substances enhances the plant
ability to vanish free radicals by increasing phenolic compounds.22
This elucidates the role of these fertilizers in the compounds
biosynthesis to motivate high production of shikimic acid, causing
more production of phenolic compounds such as flavonoids.4 How
to use the elements is economically and ecologically equals to the
amounts of elements.4To note a few, C: N ratio in plants encour-
ages the production of carbon-containing metabolites such as phe-
nolic acids. The C: N ratio in organic fertilizers can induce stronger
antioxidant activity in plants by increasing their phenolic com-
pounds. VC due to the presence of humic substances has a signifi-
cant role in plant health and viability. These substances increase
the plant resistance to stressful conditions via enhancing the
biosynthesis of phenolics.25 Outbreak of many chronic diseases
such as cardiovascular disease, cancer, and liver disorders can be
prevented and curved by plant antioxidants. Therefore, the boost of
antioxidant capacity in plants is a principal strategy in preventing
human maladies.26 Soil calcium carbonate determines the applica-
tion of sulfur and TB for plants. The previous works have shown
different responses of plants to applications of sulfur and TB on
soil.27,28 TB increased the oxidation of sulfur, which cusses the
reduction of soil pH. Therefore, nutrients availability in soil
increased, which results in augmentation of plant growth.28
EO quality and quantity was influenced by soil amendments
and time. Similar to our work,3reported that the main EO compo-
nents of S. macrantha L. were p-Cymene, γ-terpinene, and
Thymol. EO yield increase by application of soil amendments par-
ticularly the integrated NPK and VC. The similar trends were
observed during previous works, where significantly greater EO
Article
Table 4. Percentage of essential oil (EO) composition in S. macrantha L. under different fertilizer treatments in 2018.
No Compound RI Control NPK VC NPK+VC TB+VC TB TB+S250 TB+S500 GM
(%) (%) (%) (%) (%) (%) (%) (%) (%)
1 α-thujene 934.61 1.88 1.59 1.35 1.28 1.29 1.58 1.92 1.35 1.63
2 α-pinene 946.09 1.94 1.72 0.76 2.01 2.05 3.98 2.72 2.44 1.58
3 Camphene 962.66 1.04 0.2 0.24 0.24 0.16 0.21 0.28 0.23 0.18
4 Sabinene 976.79 2.12 1.17 1.94 1.93 2.17 0.33 2.39 2.13 2.12
5 β-pinene 1007.94 2.16 0.94 0.27 1.31 1.29 2.28 1.47 1.46 0.79
6 3-octanone 1033.65 2.25 0.19 0.18 0.16 0.16 2.56 0.16 0.16 0.19
7 Myrcene 1043.62 1.29 2.63 2.35 1.24 2.31 0.23 2.46 2.26 2.85
8 α-terpinene 1046.08 2.31 0.8 0.97 1.15 0.57 1.96 1.25 1.09 0.67
9 p-cymene 1053.41 16.34 20.74 26.63 26.42 24.63 33.64 29.95 23.06 22.66
10 β-phellandrene 1055.84 2.35 0.27 0.26 0.23 0.24 3.03 0.36 0.26 0.29
11 (Z)-β-ocimene 1060.65 2.36 0.97 1.11 1.44 0.68 3.77 1.49 1.42 0.88
12 (E)-β-ocimene 1063.04 2.38 0.16 0.1 0.09 0.2 0.16 0.19 0.17 0.18
13 γ-terpinene 1084.13 15.46 27.15 25.57 26.65 24.58 23.69 26.06 23.08 30
14 cis-sabinene hydrata 1100 2.54 0.27 0.18 0.23 0.15 0.22 0.25 0.25 0.2
15 Terpinene-4-ol 1219.85 3.01 0.34 0.52 0.44 0.47 0.44 0.35 0.13 0.38
16 Carvacrol 1322.63 3.13 0.22 0.27 0.28 0.35 0.36 0.21 0.47 0.28
17 Thymol- acetate 1330.04 14.82 37.57 34.22 29.57 35.59 16.72 24.51 36.49 32.49
18 (E)-βcaryophyllene 1476.54 3.91 0.17 0.16 0.55 0.23 0.54 0.22 0.49 0.74
19 Aromadendrene 1497.09 4.11 0.73 0.54 1.29 0.77 0.69 0.75 0.32 0.64
20 Germacrene d 1520.15 4.16 0.51 0.32 0.89 0.15 1.04 0.44 1.03 0.27
21 Bicyclogermacrene 1534.32 4.45 0.4 0.62 0.36 0.61 0.28 0.37 0.71 0.66
22 β-bisabolene 1542.75 4.48 0.28 0.18 0.67 0.32 0.94 0.22 0.58 0.3
23 ɵ-cadinene 1548.33 - - 0.29 0.41 0.62 0.3 - 0.41 -
24 Spathulenol 1556.66 - 0.81 0.26 0.31 1.03
Total (%) 98.49 99.02 99.84 99.1 99.9 99.98 98.02 99.99 99.98
VC, Vermicomost; TB, thiobacillus; S, sulfur; GM, glomus mosseae.
Non-commercial use only

yield observed in satureja plants supplied with VC, sulfur, AMF
such as Glomus fasciculatum, G. intraradices and G. mosseae, and
plant growth promoters like Pseudomonas fluorescens and
Bacillus subtilis.10,29 The EO quality was changed by application
of the soil amendments and time. For example, in the first year, a
10% increase of p-cymene amount was observed using the combi-
nation of NPK and VC in respect to untreated plants. However, this
increase in second year was 61%. The change of EO composition
amount can be due to the physiology of plant, the biochemical
pathways of producing the corresponded compounds, and the
responses of plants to time and site.30
Conclusions
In the study, organic, Bio-organic and inorganic fertilizers
improved plant nutrients, essential oil composition and antioxidant
capacity of S. macrantha L. We found the improvement of nutrients
uptake, antioxidant potential, and EO properties under most soil
amendments. However, the VC in combination with NPK fertilizer
gave the best results for increasing the EO quality and quantity as
well as the antioxidant capacity of this species. Hence, we can rec-
ommend the 5 t ha–1 VC in combination with 50:25:25 kg ha−1 NPK
for improving the quality and quantity of S. macrantha products.
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