Industrial Crops & Products 150 (2020) 112449

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Industrial Crops & Products

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Variability in chemical composition and antimicrobial activity of Tagetes T

minuta L. essential oil collected from different locations of Himalaya
Swati Waliaa,b, Srijana Mukhiac,d, Vinod Bhatte, Rakshak Kumara,c, Rakesh Kumara,b,*
a
Academy of Scientific and Innovative Research, New Delhi, India
b
Agrotechnology of Medicinal, Aromatic and Commercially Important Plants Division, CSIR-Institute of Himalayan Bioresource Technology, (Council of Scientific and
Industrial Research), Post Box No. 6, Palampur 176 061, HP, India
c
Biotechnology Division, CSIR- Institute of Himalayan Bioresource Technology, (Council of Scientific and Industrial Research), Post Box No. 6, Palampur 176 061, HP,
India
d
Department of Microbiology, Guru Nanak Dev University, Amritsar, 143005, Punjab, India
e
Natural Product Chemistry and Process Development Division, CSIR- Institute of Himalayan Bioresource Technology, (Council of Scientific and Industrial Research), Post
Box No. 6, Palampur 176 061, HP, India

A R T I C LE I N FO A B S T R A C T

Keywords: Tagetes minuta L. is an annual herb used in flavoring, perfumery, food and pharmacological industry. In the
Tagetes minuta recent past, bioactivities and therapeutic properties of T. minuta against a wide range of plant, human and animal
Essential oil pathogens remain the focus of considerable scientific studies. So in this study chemical and antimicrobial activity
Locations profiles of T. minuta grown in 16 altitudinal locations of three states in India were explored. Fresh plant material
Principal component analysis
was subjected to hydrodistillation using a Clevenger-type apparatus. The essential oil content ranged from 0.37
Antimicrobial activity
to 0.79% (v/w). Nine volatile compounds were identified by GC and GC–MS analysis. Range of major com-
pounds in sixteen locations were Z-β-ocimene (56.34−39.32%), dihydrotagetone (28.07−7.66%), E-ocimenone
(25.06−0.00%) and Z-tagetone (14.46−5.29%). Comparison of the relative concentrations of essential oil
compounds showed a significant altitudinal impact with higher Z-β-ocimene from high altitudes; while reverse
was seen in case of dihydrotagetone. Antimicrobial activity of the essential oils (EOs) was investigated against
two Gram-positive bacteria viz. Micrococcus luteus, and Staphylococcus aureus, and two gram-negative bacteria
viz. Klebsiella pneumoniae and Pseudomonas aeruginosa using agar well-diffusion and microdilution methods. The
agar well diffusion method demonstrated better activity of T. minuta EOs in gram positive bacteria as compared
to gram negative bacteria. Best activity was demonstrated against S. aureus with zone of inhibition above 9 mm.
Best potential EOs (three locations of HP and one location of Manipur) demonstrated an MIC of 25–30% (v/v).
Based on the current study, EOs of T. minuta from Indian Himalayas may be regarded as potential antibacterial
agents against S. aureus and selected EOs may have potential application as bactericidal agents.

1. Introduction
Tagetes minuta (family Asteraceae) commonly known as wild mar-
igold is commercially grown as a new multi-use crop in the respective
agro-ecological regions of various countries. It has been originated from
South America and is found in temperate grasslands and elevated re-
gions of countries like Argentina, Peru, Bolivia, Chile, and Paraguay
(Holm et al., 1997). It was initially confined to the higher elevations,
later it has been introduced in many countries like Europe, Africa,
Australia, Asia, Madagascar, India, and Hawaii (Soule, 1996). In India,
this species finds habitat in western Himalaya between altitudes of
1000–2500 m above mean sea level (amsl) in Himachal Pradesh, Uttar
Pradesh, Uttrakhand and Jammu & Kashmir (Thappa et al., 1993). It is
an annual herb with an erect woody stem approximately 1−2 m high
(Wang and Chen, 2006). Its pinnately compound leaves have number of
small, punctuate, multicellular glands in the undersurface of the leaves
which exude liquorice like aroma when ruptured. Flower heads are
numerous, yellowish green, usually in flat topped cymes. Traditionally
it has been used in various health ailments like treatment for colds,
respiratory problems, stomach disorders, antispasmodic, insecticide,
anti-septic, anti-parasitic, and sedative (Shirazi et al., 2014). T. minuta
was widely grown around the world because of its agrochemical, food,

⁎
Corresponding author at: Agrotechnology of Medicinal, Aromatic and Commercially Important Plants Division, CSIR-Institute of Himalayan Bioresource
Technology, (Council of Scientific and Industrial Research), Post Box No. 6, Palampur 176 061, HP, India.
E-mail address: rakeshkumar@ihbt.res.in (R. Kumar).

https://doi.org/10.1016/j.indcrop.2020.112449
Received 31 December 2019; Received in revised form 23 February 2020; Accepted 5 April 2020
Available online 18 April 2020
0926-6690/ © 2020 Elsevier B.V. All rights reserved.
S. Walia, et al. Industrial Crops & Products 150 (2020) 112449

flavoring, perfumery and pharmacological properties (Hulina, 2008). October-November) from these 16 locations.
World annual production of its essential oil was around 15 tonnes
during 2016 which is now increasing with an upward trend. Many
2.2. Essential oil isolation
producing countries of T. minuta essential oil are South Africa, India,
Zimbabwe, Egypt, France, and Argentina (Cornelius and Wycliffe,
Fresh plant material was distillated for four hours, using Clevenger
2016).
type apparatus according to the European Pharmacopeia (1975). Oil
Significance of T. minuta is due to the presence of essential oil in
distillate was dried with anhydrous sodium sulphate and stored at 4 °C
about every part of its plants excluding stem (Singh et al., 2003). T.
until gas chromatography (GC) and Gas chromatography- Mass spec-
minuta is rich in many secondary metabolite, including monoterpenes,
trometry (GC–MS) analyses. Density (gmL−1) of T. minuta essential oil
sesquiterpenes, flavonoids, and thiophenes. The major constituents are
was calculated mass/volume of oil. The essential oil content (v/w%)
Z-β-ocimene, limonene, dihydrotagetone, tagetones (E & Z) and oci-
was calculated using the formula
menones (E & Z) (Singh et al., 2016). Quality of oil is estimated by its
composition, oils with high percentage of Z-β-ocimene (40–55 %) E1
having higher value at the international market, and, however, for Essential oil content (%) = X 100
E2
biocidal activity tagetenone is essential (Cornelius and Wycliffe, 2016).
There are several biological activities in its essential oils such as anti- Where, E1: Essential oil weight, E2: Fresh sample weight
bacterial, anti-fungal, anti-viral, anti-oxidant, anti-cancerous, acar-
icidal, nematicidal, insecticidal and allelopathic activities (Gakuubi
2.3. Identification of essential oil compounds by GCMS and GC
et al., 2016a).
Besides the vast amount of information on the species morpholo-
Compounds of T. minuta essential oil were analysed by Shimadzu
gical, chemical and genetic diversity, little is known about the en-
GC–MS QP 2010 fitted with Auto injector (AOC-5000) and DB-5 fused
vironmental factors that influence this plant's qualitative and quanti-
silica capillary column (30 m ×0.25 mm i.d.; 0.25 μm film thickness)
tative essential oil profile. Variation in the percentage of essential oil
from SGE International, Ringwood, Australia. Carrier gas used was
compounds is an important conception, especially in commercially
Helium with flow rate of 1.1 mL min−1, with 240 °C injector tempera-
significant crops, as the chemistry has a direct effect on consumer
ture, 250 °C interface temperature, 50–800 amu acquisition mass range
product performance. Some authors have reported variation in the
and 70 eV ionization energy. Oven temperature programmed initially
composition of Tagetes oil secondary metabolites with different en-
for 4 min at 70 °C which was increased at rate of 4 °C min−1 to 220 °C
vironmental factors such as soil, nitrogen levels, sunlight exposure,
and maintained for 5 min (Walia et al., 2019).
elevation and fluctuations in temperature (Moghaddam and Omidbiagi,
Gas chromatography (GC) analyses were completed on a Shimadzu
2007). Tagetes essential oil from different locations of South Africa has
GC-2010 equipped with flame ionized detector (FID) and a DB-5 fused
been reported to be highly variable (Tankeu et al., 2013). The chemical
silica capillary column (30 m ×0.25 mm i.d.; 0.25 μm film thickness).
composition of essential oils from T. minuta show huge contrasts in the
Carrier gas used was nitrogen with 2 mL min−1 flow rate. Oven tem-
chemical profiles due to variation in the geographical locations
perature was programmed at 70 °C for 4 min, which increased to
(Chamorro et al., 2008; Senatore et al., 2004). Sanli and Karadogan
70–220 °C at a rate of 4 °C min−1. Temperature of injector and detector
(2017) reported that changes in altitude typically affected terpenoid
were maintained at 240 °C (Walia et al., 2019). Identification was done
biosynthesis and oxygenated monoterpenes and were higher at low
on the basis of retention indices (RI) calculated using homologous series
altitudes; while the constituents of sesquiterpenes were higher at high
of n-alkanes (C8–C24) and percentage of peak area in chromatogram.
altitudes. In particular, the effects of altitude in various medicinal and
Essential oil components were then identified by comparing their RI,
aromatic plants were only investigated superficially, while incomplete
pattern of mass fragmentation and library database of NIST-MS (Na-
information is available for T. minuta. Literature data indicated that the
tional Institute of Standards and Technology) Version 3.02 (Adams,
essential oil of T. minuta have high percentage anti microbial activity
1995; Stein, 2005).
against different gram positive and gram negative strains (Gakuubi
et al., 2016b; Shirazi et al., 2014; Muyima et al., 2004).
Based on the above findings, the objective of the present work was 2.4. Antibacterial assay
to corroborate how altitude variation could affect the chemical profiles
and antibacterial activity of T. minuta grown in different Himalayan 2.4.1. Agar well-diffusion method
locations of India. Such knowledge may be useful in selecting different The antibacterial activity of essential oils of T. minuta was evaluated
chemotypes for various uses in flavour, food and perfumery industries. by well diffusion method as described earlier by Balouiri et al. (2016)
and Valgas et al. (2007). The test bacterial organisms used were the
2. Material and methods Gram-positive bacterial strains, Micrococcus luteus MTCC 2470 and
Staphylococcus aureus MTCC 96, and Gram-negative bacteria viz. Kleb-
2.1. Sampling sites and plant material siella pneumoniae MTCC 109, Pseudomonas aeruginosa MTCC 2453. The
bacterial strains were cultured overnight in nutrient broth at 37 °C until
Improved cultivar of T. minuta HIM GOLD (IHBT.MARIGOLD.I) a suspension of 1.0 × 108 CFU mL−1 was attained. The bacterial in-
developed by CSIR-Institute of Himalayan Bioresource Technology, oculum was spread uniformly with a sterile cotton swab on Mueller
Palampur, India was cultivated in the month of June at all the 16 lo- Hinton Agar plates. A 6 mm diameter hole was aseptically punched on
cations of different altitudinal zones i.e. low, mid and high hills. the agar surface with a sterile tip, and 10 μL of the essential oils was
Standard agro practices designed by CSIR-IHBT were used for cultiva- introduced into each well. Amoxicillin (20 mcg/disc) and neomycin (20
tion of the crop at different locations. Geographic positions (latitude mcg/disc) were used as positive controls against Gram-positive and
and longitude), altitude, locations and key meteorological data (total Gram-negative bacteria respectively. The plates were incubated at 4 °C
rainfall, and average minimum and maximum temperatures) of each for 2−3 h to allow diffusion of essential oils into the agar medium,
collection site were extrapolated from the localities of meteorological followed by incubation at 37 °C for 12−24 h. The tests were performed
stations closest to each sampling site during cropping season (i.e. June in triplicate and inhibition zones were measured. The sensitivity to the
to November) and presented in Table 1. During morning hours, the different essential oils was categorized as: not sensitive (diameter
aerial parts of T. minuta plants were randomly collected from self cul- ≤8 mm); sensitive (diameter 9−14 mm); very sensitive (≥15 mm)
tivated field at same stage i.e. full bloom stage (in the month of (Ponce et al., 2003).

2
S. Walia, et al. Industrial Crops & Products 150 (2020) 112449

Table 1
Tagetes minuta sampling site characteristics.
Code Location Altitude Latitude Longitude Temperature (°C) Rainfall (mm)
(m amsl) (decimals) (decimals)
Village District State Max. Min.

L1 Yula Kinnaur Himachal Pradesh 2637 31.56 78.12 19.18 10.70 204.00
L2 Moorang Kinnaur Himachal Pradesh 2591 31.59 78.43 23.90 4.70 215.80
L3 Kotgarh Shimla Himachal Pradesh 1981 31.30 77.46 25.78 10.57 854.00
L4 Sihunta Chamba Himachal Pradesh 489 32.30 76.07 28.68 19.22 1978.00
L5 Salooni Chamba Himachal Pradesh 1829 32.72 76.04 28.73 19.10 1651.00
L6 Bharmour Chamba Himachal Pradesh 2133 32.44 76.52 22.63 14.25 1372.00
L7 Sainj Kullu Himachal Pradesh 1532 31.76 77.29 24.60 16.35 826.00
L8 Kullu Kullu Himachal Pradesh 1279 31.95 77.09 33.48 9.94 873.90
L9 Gogardhar Mandi Himachal Pradesh 1928 31.89 76.92 33.57 13.52 1396.10
L10 Rajol Kangra Himachal Pradesh 676 32.16 76.23 29.97 20.05 2081.60
L11 Jia Kangra Himachal Pradesh 1254 32.15 76.45 29.07 14.23 2147.60
L12 Thandol Kangra Himachal Pradesh 1301 32.01 76.51 29.87 20.00 2124.00
L13 Palampur Kangra Himachal Pradesh 1390 32.10 76.56 25.83 16.08 2665.50
L14 Silogi PauriGarhwal Uttrakhand 1850 29.99 78.57 19.25 11.67 1327.00
L15 Makhan Senapati Manipur 1767 24.98 93.86 21.78 13.93 1290.00
L16 Senapati Senapati Manipur 2500 25.27 94.00 27.27 19.30 1199.00

Note: amsl; above mean sea level.

2.4.2. Microdilution method different altitudinal locations are summarized in Table 2.

The minimum inhibitory concentration (MIC) values for the strain
showing sensitivity to the essential oils in a well diffusion test were
determined following the microdilution method as described previously
by Tadtong et al. (2009). The bacterial inoculum was prepared by ad-
justing the final concentration to McFarland no. 0.5 standard turbidity.
The essential oils were prepared in 10% DMSO to make a stock con-
centration (v/v) of 90%, 80%, 70%, 60% and 50%. Briefly, in a 96-well
plate, 95 μL of nutrient broth was added followed by the addition of
5 μL of the prepared inoculum. To this, 100 μL of the stock solutions
were added to make a final concentration (v/v) of 45%, 40%, 35%,
30%, and 25%. 200 μL of broth without inoculum and essential oils
served as a negative control. Amoxicillin (1 mg mL−1) was used as a
positive control. The plate was incubated at 37 °C for 24 h and bacterial
growth was checked by reading absorbance at 600 nm.
2.5. Statistical analysis
Statistical analysis was performed according to standard analysis of
variance (ANOVA) using SYSTAT-12 software (Chicago, IL, USA).
Variation among treatments was compared using least significant dif-
ference (LSD) at 5% level of probability. Multivariate principal com-
ponent analysis was performed using PAST3 software to evaluate the
effect of treatments on essential oil compounds.
3. Results and discussion
3.1. Essential oil content and composition
The fresh samples of T. minuta on hydrodistillation yielded yellow
oil with licorice-like aroma. Essential oil content varied from 0.37 to
0.79% in different altitudinal locations, a range that is compliant with
earlier reports (Singh et al., 1992). Significantly higher essential oil
content was obtained in L1 (Yula 2637 m amsl) from higher altitudinal
location. Overall, analysis of essential oil identifies 9 different com-
pounds whose GCMS profile is shown in Fig. 1. The GC-FID analysis of
essential oil identified six predominant compounds: Z-β-ocimene, di-
hydrotagetone, E-tagetone, Z-tagetone, Z-ocimenone and E-ocimenone
which is consistent with earlier studies (Babu and Kaul, 2007;
Moghaddam and Omidbiagi, 2007). The total area percentages of T.
minuta components were 89.37–96.21%. The results showed that the
essential oil of T. minuta collected from different altitudinal locations
presented one major chemical group of terpenes: acyclic monoterpenes,
ranging between 83.58 and 92.63%. Detailed data on the distribution in
Essential oil analysis showed that the major compound was Z-β-
ocimene (56.34%), followed by dihydrotagetone (28.07%), E-ocime-
none (25.06%), Z-tagetone (14.46%), Z-ocimenone (10.23%) and E-
tagetone (2.39%) represented in Table 2. These findings are consistent
with the major volatile fraction found in previous reports on T. minuta,
however quantitative difference was observed when compared (Babu
and Kaul, 2007; Senatore et al., 2004; Pazcel et al., 2018). Studies of
Tankeu et al. (2013) and Rathore et al. (2018) also reported that major
compounds in T. minuta collected from South Africa and India are Z-β-
ocimene and dihydrotagetone. The percentage of Z-β-ocimene was
significantly higher in location L1 (Yula 2637 m amsl) which remained
statistically at par with L2 (Moorang 2591 m amsl), while lowest in L4
(Sihunta 489 m amsl). Z-β-ocimene showed very interesting results i.e.
decrease down with decreasing altitude. Similar results by Singh et al.
(2003); Thappa et al. (1993) and Bansal et al. (1999) reporting lower
concentration of Z-β-ocimene in Lucknow (13.3%) than essential oil
from Jammu (35.32%) and Himachal Pradesh (45.00%). These results
corroborates our findings concluding that difference in essential oil
composition in different locations may be ascribed to adaptation to
particular habitats, along with plants age and genetic variation.
Dihydrotagetone was found significantly higher in L10 (Rajol 676 m
amsl) and significantly lower in L2 (Moorang 2591 m amsl). It was clear
that dihydrotagetone decreases with increase in altitude (Table 2). Si-
milar result was observed by Ramaroson-Raonizafinimanana et al.
(2009) while reporting considerably lower concentration of dihy-
drotagetone in Madagascar than essential oil from India, Turkey,
Rwanda and France. The next predominant compounds E-ocimenone,
Z-ocimenone, E-tagetone and Z-tagetone were found significantly
higher in L7 (Sainjh 1532 m amsl), L6 (Bharmour 2133 m amsl), L4
(Sihunta 489 m amsl) and L15 (Makhan 1767 m amsl), respectively,
while sabinene, limonene and trans-caryophyllene were significantly
higher in L14 (Silogi 1850 m amsl). According to previous reports, the
chemical composition of essential oils from T. minuta show huge con-
trasts in the chemical profiles due to variation in the geographical lo-
cations (Chamorro et al., 2008; Senatore et al., 2004). Similarly, dif-
ferences in the relative quantities of essential oil constituents in
Lavender and Zanthoxylum armatum populations are attributed to var-
iations in geographical location (Skoula et al., 1996; Bhatt et al., 2017).
Melito et al. (2013, 2016) and Homer et al. (2000) explored the in-
fluence of altitude level and climatic conditions on the secondary me-
tabolites profiles in Helichrysum italicum and Melaleuca alternifolia.
These studies support our finding that qualitative and quantitative
disparity in chemical components of T. minuta essential oil in different

3
S. Walia, et al.
Fig. 1. GCMS chromatogram of analysed essential oil of T. minuta.
geographical locations showed that along with genetic variation, en-
vironmental factors also plays an important role in the variation of
these compounds.
3.2. Correlation between altitude and essential oil compounds
Correlation between EO profiles and the altitude revealed nine
compounds correlated to quote (Table S1). Among them, three com-
pounds (Z-β-ocimene, Z-ocimenone and trans-caryophyllene) resulted
positive correlation, with Z-β-ocimene showing highly positively cor-
relation at 1% level. However, six of them showed a negative correla-
tion with altitude (sabinene, limonene, dihydrotagetone, E-ocimenone,
Z-tagetone, and E-tagetone). The positive correlation between major
essential oil compound and altitude revealed that altitude might be
responsible for increased percentage of Z-β-ocimene around 37.00%
(R2 = 0.3700) in T. minuta aerial parts, while dihydrotagetone reported
negative correlation of around 10.60% (R2 = 0.1060) (Fig. S1). These
results showed differences in volatiles of plant from different sampling
sites which confirm the influence of the environmental conditions on
secondary metabolites production. Similar trend was observed by
Melito et al. (2015) who reported that Helichrysum italicum EO from
Sardinia presents an increasing level of monoterpenes compounds ac-
cording to the altitude, whereas the sesquiterpenes fraction showed an
opposite trend.
3.3. Principal component analysis (PCA)
To determine the variation between composition and treatments,
principal component analysis (PCA) was applied to nine compounds of
T. minuta essential oil from different altitudinal locations. The PCA plot
(Fig. 2A) showed that the first two Principal Components (PC-1 and PC-
2) represented total variance of 62.66%. PC-1 explaining 37.73% of
total variation, accounting positive contribution of sabinene, limonene,
Z-β-ocimene, dihydrotagetone and trans-caryophyllene rich samples,
and negative contribution of Z & E tagetone and Z & E ocimenone
(Fig. 3A). PC-2 explaining 24.93% of total variance, clearly distin-
guishes sabinene, limonene, E-tagetone, Z & E ocimenone and trans-
4
Industrial Crops & Products 150 (2020) 112449
caryophyllene samples having positive contribution, however, Z-β-oci-
mene, dihydrotagetone and Z-tagetone with negative contribution
(Fig. 3B).
The two-dimensional PCA score plot identified five clusters of dif-
ferent locations based on the chemical composition of their essential
oils (Fig. 2B). The cluster I included six locations (L3, L4, L5, L6, L7 &
L12) reporting significantly higher E-tagetone (2.39%), Z-ocimenone
(10.23%) and E-ocimenone (25.06%) in location L4, L6 and L7, re-
spectively. Cluster II (L1 & L2) from Kinnaur (H.P.) explained sig-
nificantly higher concentration of Z-β-ocimene (56.34% & 54.14%) and
lower concentration of dihydrotagetone (11.11% & 7.66%). Cluster III
(L8, L9, L10, L11 & L13) reported significantly higher dihydrotagetone
in L10. The cluster IV included two locations from Manipur (L15 & L16)
reporting higher Z-β-ocimene (51.41% & 52.43%) and dihydrotagetone
(24.26% & 21.06%). Cluster V (L14) from Uttrakhand explained sig-
nificantly higher concentration of sabinene (0.47%), limonene (4.89%)
and trans- caryophyllene (0.52%) as compared to other locations
(Table 3). Variations in the comparative concentrations of these com-
pounds suggest their adaptive ecological importance under diverse
environmental surroundings.
3.4. Hierarchical cluster analysis (HCA)
To categorize the studied species by their compound individuality,
HCA was performed according to the essential oil compositions of 16
locations; unweighted pair-group method (square Euclidean distance)
was used to construct a dendrogram (Fig. 4). By setting an arbitrary cut-
off criterion at a distance of 3.5 (50% of the maximum distance), the
altitudinal locations are separated from each other. HCA generated
dendrogram indicates the existence of five main groups that were si-
milar to the PCA groups, demonstrating the chemical similarities be-
tween altitudinal locations. Group A is formed by the essential oil ob-
tained from L14 from Uttrakhand; L1 and L2 from Kinnaur (H.P.)
formed group B; group C (L8, L9, L10, L11 and L13) are formed of
locations from Kullu, Mandi and Kangra regions of Himachal Pradesh;
L3, L4, L5, L6, L7 and L12 from Shimla, Chamba, Sainjh (Kullu) and
Thandol (Kangra) of Himachal Pradesh formed group D; while those
 S. Walia, et al.

Table 2
Composition of T. minuta essential oils collected from different altitudinal regions.
Treatments Essential oil Essential oil Sabinene Limonene Z-β-ocimene Dihydrotagetone E- tagetone Z-tagetone Z-ocimenone E-ocimenone Trans Acyclic Cyclic Bicyclic Total
density (g content (v/w Caryophyllene monoterpenes monoterpenes monoterpenes
mL−1) %)

Litt. R.I. – 976 1031 1040 1054 1146 1153 1231 1239 1418 Grouped components
Exp. R.I. – 971 1029 1032 1052 1146 1153 1233 1241 1393
L1 0.85 0.79 0.25 3.44 56.34 11.11 0.94 5.29 5.39 12.10 0.37 91.16 3.43 0.62 95.22
L2 0.98 0.78 0.19 2.10 54.14 7.66 1.76 8.74 6.65 12.85 0.11 91.79 2.10 0.30 94.20
L3 0.95 0.62 0.25 2.37 41.77 9.99 1.23 10.61 7.82 20.03 0.48 91.44 2.36 0.73 94.54
L4 0.88 0.71 0.37 4.44 39.94 15.09 2.39 11.94 6.05 15.17 0.31 90.58 4.44 0.68 95.70
L5 0.85 0.56 0.28 3.13 41.01 17.17 2.39 10.35 6.98 14.63 0.27 92.52 3.13 0.55 96.21
L6 0.95 0.58 0.21 2.44 39.32 10.11 1.96 8.60 10.23 22.37 0.26 92.59 2.44 0.47 95.51

5
L7 0.91 0.72 0.16 1.37 41.09 9.33 1.24 9.61 5.65 25.06 0.37 91.46 1.97 0.53 93.88
L8 0.62 0.68 0.22 2.29 49.81 11.84 0.58 7.05 3.40 17.59 0.52 90.76 1.97 0.74 93.30
L9 0.77 0.67 0.22 1.90 46.94 14.55 0.66 10.09 3.65 16.67 0.25 92.63 2.45 0.46 94.93
L10 0.89 0.37 0.20 2.65 41.30 28.07 0.88 7.90 2.65 10.90 0.15 91.65 1.80 0.35 94.70
L11 0.87 0.60 0.15 2.75 40.81 18.60 1.10 10.69 2.91 14.75 0.23 88.85 2.75 0.38 91.99
L12 0.89 0.71 0.26 3.02 47.56 10.89 1.77 9.82 6.45 14.68 0.45 91.16 3.01 0.71 94.89
L13 0.94 0.68 0.25 2.66 44.54 20.23 1.29 8.82 5.15 11.72 0.30 91.75 2.65 0.54 94.95
L14 0.83 0.62 0.47 4.89 48.45 24.89 0.36 5.43 0.24 4.21 0.78 83.58 4.88 1.25 91.72
L15 0.65 0.69 0.17 1.55 51.41 24.26 0.80 14.46 0.15 0.57 0.14 91.65 1.55 0.31 93.51
L16 0.72 0.72 0.19 4.65 52.43 21.06 1.27 9.37 0.00 0.00 0.40 84.12 4.65 0.59 89.37
SEm ± 0.01 0.03 0.01 0.07 0.92 0.81 0.13 0.35 0.57 1.00 0.03 0.709 0.16 0.02 0.71
LSD 0.02 0.08 0.03 0.21 2.64 2.32 0.37 1.01 1.63 2.88 0.07 2.053 0.46 0.08 2.05

RI, retention indices; SEm ± , standard error mean; LSD, least significant difference.
Industrial Crops & Products 150 (2020) 112449
S. Walia, et al. Industrial Crops & Products 150 (2020) 112449

Fig. 2. Relationship between essential oil components and sampled locations in the space of first two principal components. (A) PCA distribution of essential oil
components, (B) individual clusters of different locations based on essential oil components.

from Manipur (L15 and L16) formed group E. Group A was the most
distant group and was clearly distinguished from the other groups in the
analysis. The variation in essential oil compounds between different
populations of T. minuta tends to reflect the effect of environment on
composition, affected by variations in the habitat and microclimatic
conditions which are similar with the studies of Hajdari et al. (2015).
3.5. Antibacterial activity of T. minuta essential oils
The antibacterial plate assay demonstrated a strong activity of T.
minuta essential oils against Gram-positive bacteria as compared to the
Gram-negative group (Table 4). After 12−24 h of incubation, none of
the tested strains were very sensitive to the essential oils. Only S. aureus

6
S. Walia, et al.
Fig. 3. Loading plot of principal component analysis (A) loading plot PC-1 (B) loading plot PC-2.
MTCC 96 was sensitive to the essential oil from locations L3 (Kotgarh),
L9 (Gogardhar), L11 (Jia), and L15 (Makhan), with zones of inhibition
above 9 mm (Fig. 5). All of the oils exhibited considerable inhibition of
M. luteus MTCC 2470, but did not fall under the sensitive category.
Among all the oil extracts, L4 (Sihunta), L6 (Bharmour), L12 (Thandol),
and L16 (Senapati) showed the least activities of ≤ 3 mm against M.
luteus MTCC 2470, while in the case of S. aureus MTCC 96, L16 (Se-
napati) showed the least inhibition of 3.33 mm. None of the Gram-ne-
gative bacteria were sensitive to any of the oil extracts. The maximum
7
Industrial Crops & Products 150 (2020) 112449
inhibition zone produced against K. pneumoniae MTCC 109 was 4 mm
by the extract L3 (Kotgarh). Some of the oils i.e. L12 (Thandol), L7
(Sainjh), L16 (Senapati), and L13 (Palampur) did not produce any in-
hibition zone against this particular strain. In the case of P. aeruginosa
MTCC 2453, most of the oil extracts did not inhibit the strain, while
very little inhibition was shown by L10 (Rajol), L1 (Yula), L7 (Sainjh),
and L16 (Senapati). Our results showed some of the essential oils from
location L3 (Kotgarh), L9 (Gogardhar), L11 (Jia), and L15 (Makhan) as
potential antibacterial agents against S. aureus MTCC 96 (Fig. 5). MIC
S. Walia, et al. Industrial Crops & Products 150 (2020) 112449

Table 3
Clusters variability in major essential oil constituents (%) of T. minuta as affected by different altitudinal locations.
Components Cluster I Cluster II Cluster III Cluster IV Cluster V

Sabinene 0.16−0.37 0.19−0.25 0.15−0.25 0.17−0.19 0.47

Limonene 1.37−4.44 2.10−3.44 1.90−2.75 1.55−4.65 4.89
Z- β-ocimene 39.32−47.56 54.14−56.34 40.81−49.81 51.41−52.43 48.45
Dihydrotagetone 9.99−17.17 7.66−11.11 11.84−28.07 21.06−24.89 24.89
E- tagetone 1.23−2.39 0.94−1.76 0.58−1.29 0.80−1.27 0.36
Z-tagetone 8.60−11.94 5.29−8.74 7.05−10.69 14.46−9.37 5.43
Z-ocimenone 5.65−10.23 5.39−6.65 2.91−5.15 0.15 0.24
E-ocimenone 14.63−25.06 12.10−12.85 10.90−17.59 0.57 4.21
Trans Caryophyllene 0.26−0.48 0.11−0.37 0.15−0.52 0.14−0.40 0.78

Fig. 4. Dendrogram based on the unweighted pair-group method (square Euclidean distance) of the studied T. minuta.

Table 4
Inhibition zones of T. minuta essential oils against four pathogenic strains.
Treatments Diameter of inhibition zone (mm), 10 μL essential oil

GRAM POSITIVE GRAM NEGATIVE

Micrococus luteus Staphylococcus Klebsiella Pseudomonas aeruginosa

MTCC 2470 aureus pneumoniae MTCC109 MTCC 2453
MTCC 96

L1 4.66 ± 1.50 5.00 ± 0.00 2.66 ± 0.50 0.50 ± 0.00

L2 6.33 ± 0.50 8.00 ± 1.00 2.33 ± 0.50 –
L3 6.66 ± 1.15 11.33 ± 1.15 4.00 ± 0.00 –
L4 3.66 ± 0.50 7.00 ± 0.00 1.33 ± 0.50 –
L5 6.33 ± 0.50 7.66 ± 0.50 2.66 ± 0.50 –
L6 6.33 ± 0.50 8.33 ± 1.50 3.00 ± 0.00 –
L7 3.00 ± 0.00 4.66 ± 0.50 – 1.00 ± 0.00
L8 4.33 ± 0.50 7.66 ± 0.50 1.66 ± 0.50 –
L9 4.33 ± 0.50 11.00 ± 2.00 2.66 ± 0.50 –
L10 6.00 ± 0.00 7.33 ± 1.15 2.33 ± 0.50 0.50 ± 0.00
L11 8.00 ± 1.00 11.00 ± 1.00 3.66 ± 0.50 –
L12 3.66 ± 0.50 5.66 ± 1.15 – –
L13 6.00 ± 0.00 6.00 ± 1.00 – –
L14 4.00 ± 0.00 7.33 ± 1.50 1.33 ± 0.50 –
L15 3.66 ± 1.15 10.66 ± 0.50 3.00 ± 0.00 –
L16 3.00 ± 1.00 3.33 ± 0.50 – 0.66 ± 0.20

Data represent means ± standard deviation of triplicate samples.

8
S. Walia, et al.
Fig. 5. Inhibition zones of T. minuta essential oils against pathogenic strains (Staphylococcus aureus, Klebsiella pneumoniae and Micrococcus luteus).
values were determined for these selected oils, and our result demon-
strated the MIC of L3 (Kotgarh) at 25% (v/v), L9 (Gogardhar) at 30%
(v/v), L11 (Jia) at 25% (v/v), and L15 (Makhan) at 25% (v/v). Similar
to our findings, reports on the antibacterial activities of the essential
oils of T. minuta are available that shows Gram-positive bacteria to be
more susceptible to these extracts than Gram-negative bacteria
(Gakuubi et al., 2016b; Shirazi et al., 2014; Muyima et al., 2004).
9
Industrial Crops & Products 150 (2020) 112449
According to Trombetta et al. (2005) Gram-positive bacteria are usually
more susceptible to the antibacterial activity of lipophilic essential oils
because of the lipophilic nature of the Gram-positive cell membrane, as
compared to the hydrophilic character of the cell membrane component
of Gram-negative bacteria. The essential oil is thought to act upon
bacteria by penetration into the lipid portion of plasma membrane,
thereby interrupting and causing leakage of the intracellular
S. Walia, et al.
components (Trombetta et al., 2005).
4. Conclusion
The growing interest in the food, taste, perfumery and pharma-
cology industries contributes to the investigation of environmental
conditions that have affected the aromatic plants qualitative as well as
quantitative composition and yield. This research explored the impact
of environmental conditions on chemical profiles of T. minuta plants
collected from different regions of India. The obtained results strongly
suggested that altitudinal variation affected the chemical composition
of this species. The variations in essential oil composition shown in this
study provide meaningful information for diversified phytotherapy and
for improved utilization in food, flavour and fragrance industries.
However, with the present data, the increase in essential oil compo-
nents could not be determined conclusively from north to east of the
province; a physiological response to altitudinal changes could be seen
resulting from selective pressures. Nevertheless, there appears to be a
tendency in these regions to generate more Z-β-ocimene and less di-
hydrotagetone with increase in altitude, which is likely to have an
adaptive benefit. Investigation of the antibacterial activity of the es-
sential oils of T. minuta revealed that some of these oils had a good
antibacterial activity against Staphylococcus aureus MTCC 96 and de-
monstrates their potential application as a bactericidal agent. Such
knowledge may be useful in selecting particular chemotypes with uses
in pharmaceutical, flavour, food and perfumery industries.
Author credit statement
Swati Walia- Experiment execution, Data recording, oil analysis,
identification of compounds and paper writing
Srijana Mukhia-Microbial analysis and paper writing
Vinod Bhatt-Verification of Identified compounds of essential oil
and paper editing
Rakshak Kumar-Verification of Microbial analysis results and paper
editing
Rakesh Kumar-Overall guidance, planning, execution and paper
editing
Declaration of Competing Interest
None.
Acknowledgments
The authors are grateful to the Director, CSIR-IHBT, Palampur for
necessary facilities during the course of study. We also thank Mr. Shiv
Kumar, Senior Technical Officer for providing technical support during
the work. Council of Scientific and Industrial Research, New Delhi
under CSIR-Aroma Mission (HCP 0007) is also acknowledged. This is
IHBT Publication No. 4534.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.indcrop.2020.112449.
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