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. 2024 Feb 23;30(1):67–80. doi: 10.1007/s12298-024-01416-x

In vitro micropropagation and conservation of endangered medicinal plant Nepeta asterotricha Rech.f. (Lamiaceae): genetic fidelity, phytochemical and biological assessment

Mahdieh Zamani 1, Ali Sonboli 2, Mostafa Goldansaz 2, Mohammad Hossein Mirjalili 1,✉
PMCID: PMC10901756  PMID: 38435858

Abstract

An efficient in vitro protocol was introduced for the conservation of Nepeta asterotricha, a vulnerable and endangered medicinal species found in the central of Iran for the first time. Growth, phytochemical, and biological traits of in vitro regenerated plant (RP) and acclimated plant (AP) were compared to the mother plant (MP). In addition, the genetic stability of AP was assessed by using inter-simple sequence repeats (ISSR) markers. The highest number of lateral branches (4.25) was obtained from the medium with 3 mg/mL kinetin (KIN), while the highest length of lateral branches (13.25 cm) was achieved on the medium culture fortified with 3 mg/mL thidiazuron (TDZ) and 6-benzylaminopurine (BAP). The highest number of leaves (20.25) and main branch length (12.25 cm) were obtained from the medium containing 3 mg/mL TDZ. The highest number of roots (46.25) and root length (2.25 cm) was measured from the medium fortified with 1 mg/mL indole-3-butyric acid (IBA) and 0.6 mg/mL indole-3-acetic acid (IAA), respectively. RP was successfully acclimated (85%) in vivo. Molecular analysis showed that the AP was true to the type of the MP. cis-Sabinene hydrate (26.8–57.7), 1,8-cineole (6.2–24.1), 4aα,7β,7aα-nepetalactone (4.1–12.3), and terpinene-4-ol (3.2–15.0) were the major essential oils compounds. The studied samples contained rosmarinic acid (2.55–5.97 mg/g DW), cichoric acid (1.68–12.7 mg/g DW), chlorogenic acid (1.91–64.21 mg/g DW), rutin (0.59–1.09 mg/g DW), apigenin (0.52–0.72 mg/g DW), betulinic acid (0.17–2.20 mg g DW), oleanolic acid (0.84–5.37 mg/g DW) and ursolic acid (3.46–15.70 mg/g DW). Acclimated plant exhibited the highest antioxidant activity (IC50 = 196.4 μg/mL), while the methanolic extract of MP displayed the highest antibacterial activity (MIC = 8 mg/mL) against Staphylococcus aureus.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-024-01416-x.

Keywords: Acclimatization, Endemic plant, Essential oil, ISSR, Phenolic content, Triterpenoids

Introduction

The predominant portion of plant genetic resources in territories comprises medicinal and aromatic plants (MAPs), but their utilization in traditional medicine and various industries like pharmaceuticals, food, and cosmetics has led to improper harvesting and habitat destruction. Presently, over 45% of MAPs are harvested directly from their natural pastures and habitats, contributing to the accelerated decline and extinction of numerous species (Barata et al. 2016). Due to the slow and long regeneration rate of many plants in nature, over-collection, and environmental disasters, the protection of these plants may be necessary (Chen et al. 2016).

Flora of Iran encompasses about 8200 vascular plant species, with approximately 2300 primarily identified as medicinal plants (Mozaffarian 2013; Hassanpouraghdam et al. 2022). Growing commercial interest has heightened the strain on wild plant populations, particularly those serving as primary sources for MAPs. Reports indicate that a notable number of plant species face extinction annually, leading to the loss of at least one potential medicinal resource each year (Pimm et al. 1995).

Nowadays, in vitro culture techniques have proven to be a successful and valuable approach for safeguarding vulnerable plants. These techniques have been applied for micropropagation and conservation of a large variety of endangered MAPs including Jasminanthes tuyetanhiae T.B. Tran and Rodda (Nam et al. 2022), Farsetia macrantha Blatt. and Hallb. (Choudhary et al. 2020), Eryngium viviparum J.Gay (Ayuso et al. 2019) and Decalepis salicifolia Venter (Ahmad et al. 2018), Anarrhinum pubescens Loudon (Abdelsalam et al. 2021), Dyckia brevifolia Baker (Bertsouklis and Panagaki 2022), Myrmecodia tuberosa Jack. (Rittirat et al. 2022), Ceropegia lawii Hook (Bhamare et al. 2022), and Tetraclinis articulata (Vahl) Mast. (Juan-Vicedo et al. 2022).

Nepeta L. stands as one of the extensive genera within the Lamiaceae family, encompassing around 300 aromatic species, both annual and perennial. Primarily found in Turkey, Iran, and the western Himalayas (Hindu Kush), this genus includes 75 species referred to as "Pounehsa" in Persian, with 38 species (53%) being endemics in Iran (Jamzad 2013).The plants are traditionally used as anti-asthmatic, antispasmodic, diuretic, diaphoretic, and sedative agents. Further use as a topical therapy in kids with dermal eruptions was also reported (Formisano et al. 2011). Nepeta asterotricha Rech.f, is an aromatic species that is exclusively growing in the center of Iran at the heights of the Shirkouh Mountains, with an altitude of more than 2000 m, located in Yazd Province (Ezzatzadeh et al. 2014). The plant's essential oil is distinguished by various nepetalactone isomers (Masoudi et al. 2012; Ezzatzadeh et al. 2014; Goldansaz et al. 2019). Recently, the plant extracts have been acknowledged for their anti-inflammatory properties (Goldansaz et al. 2019). The plant faces a severe threat of extinction due to successive droughts, uncontrolled harvesting, and overgrazing of livestock, causing a significant decline in its populations within natural habitats. In vitro conservation through tissue culture emerges as a promising method to safeguard the genetic resources of this endangered species. Moreover, it offers the potential to utilize plant materials for producing biologically active substances through biotechnological methods.

In recent years, there have been reports on the micropropagation of various Nepeta species, including N. nuda L. (Nedelkova et al. 2011), N. rtanjensis Diklic and Milojevic (Mišić et al. 2005), and N. camphorate Boiss. and Heldr (Darras et al. 2020). However, our literature review indicates a gap in the study of micropropagation and in vitro conservation of N. asterotricha. Thus, this study aims to establish an effective in vitro regeneration protocol for the plant, intending its application in habitat restoration and sustainable utilization. To assess the protocol's efficiency, the genetic fidelity and phytochemical stability of the regenerates were compared to the mother stock. In addition to micropropagation, the determination of phenolic acids and triterpenic acids (TAs) of the plant was carried out for the first time. This information could be interestingly applied for further conservation and commercial exploitation programs of this vulnerable aromatic plant.

Materials and methods

Plant material

The aerial parts of wild-growing N. asterotricha were collected at full flowering stage from Dehbala village (31° 34′ 53ʺ N, 54° 05′ 22ʺ E at an altitude of 2791 m) in Shirkouh Mountain located in the center of Iran (Fig. 1A). Nodal segments were also collected from the same wild sample as a mother plant (MP) (Fig. 1B), kept in wet paper and transferred to the laboratory for in vitro culture establishment. A voucher specimen (MPH-2573) has been deposited at the Herbarium of Medicinal Plants and Drugs Research Institute (MPDRI), Shahid Beheshti University, Tehran, Iran.

Fig. 1.

Fig. 1

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In vitro propagation and acclimation of Nepeta asterotricha. a Distribution map, b Wild plant that served as nodal explant, c The sterile nodal segments cultured on MS medium for further propagation, d In vitro-proliferated shoots employed as a source of explants, e In vitro-rooted plantlets, f, Well-rooted plantlets that considered for further acclimation, g Plantlets transferred to a sterile jar (750 mL) containing (200 mL) distilled water for acclimation at first week, h One-month-old acclimated plant regenerates in the greenhouse, i One-year-old acclimated plants in the ex vivo habitat field

Sterilization and culture establishment

The plant nodal segments (3 cm) were sterilized according to Bakhtiar et al. (2014). The sterile nodal segments (2 cm) were then aseptically cultured in the baby food jars with 30 mL Murashige and Skoog (1962) (MS) medium solidified with 0.8% (w/v) agar and pH was adjusted to 5.6 for further propagation (Fig. 1C). All cultures were maintained in a growth chamber at a 16/8 h photoperiod provided by cool-white fluorescent lamps at a photon flux density of 40 µmol m–2 s–1 at 25 ± 2 °C.

In vitro shoot multiplication and rooting

In vitro-proliferated shoots served as the source of explants (Fig. 1D). Various concentrations (0.5, 1, 2, and 3 mg/L) of cytokinins, i.e. 6-benzylaminopurine (BAP), kinetin (KIN) and thidiazuron (TDZ) were used for shoot multiplication. For instance, five explants were cultured in a glass jar containing 30 mL MS medium supplemented with different levels of cytokinins mentioned above. The regenerated shoots were then rooted on MS medium supplemented with 0.2, 0.6, and 1 mg/L of auxins, i.e. indole-3-acetic acid (IAA), 1- naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA) (Fig. 1E). At this experiment, each glass jar containing three proliferated shoots (3 cm) was treated as an experimental unit, with four replications for each experiment. A control, involving hormone-free MS medium culture, was implemented in both experiments.

After one month, measurements were taken for lateral branch number, main stem length, length of each lateral branch, and leaf number in each explant. Additionally, parameters such as root length, rooting percentage, and the number of roots were also assessed. The aerial parts of one-month-old in vitro-rooted plantlets were harvested, dried in the shade at ambient temperature, and then utilized for phytochemical and biological evaluations.

Acclimatization

Well-rooted regenerated plants (RP) (Fig. 1F) were carefully detached from the solidified medium and their roots were gently rinsed with sterile distilled water. Each plantlet was then relocated to a sterile jar (750 mL) containing (200 mL) of distilled water, and then covered with a plastic bag to prevent evaporation during the initial week (Fig. 1G), and kept in the chamber at 25 ± 2 °C and photoperiod 16/8 h light/dark provided by cool-white fuorescent lamps at a photon flux density of 40 µmol m−2 s −1. For the first week, the seedlings received a quarter of the MS concentration solution. After a week, pores were introduced on each plastic cover. Subsequently, in vitro regenerates were transferred to the greenhouse after three weeks. The plant regenerates were placed in the greenhouse without cover for one week and then transplanted to the plastic pot with a ratio of 3:1 cocopeat-perlite (Fig. 1H). The plants with well-developed roots were further maintained in the greenhouse under normal day length at 23 ± 2 °C during the day and 18 ± 2 °C at night, and humidity which was initially set at 70% and was decreased to 50% during the acclimatization period. The plants were nourished with a nutrient solution of one-half strength of MS medium. One-month acclimated plants (AP) were transferred to the field conditions (Fig. 1I). Irrigation was done each day and it was then reduced to once a week. The field had a semi-arid climate with an average annual temperature of 12.9 °C. The relative humidity was 40%.

Genetic fidelity evaluation

DNA isolation from one-month-old AP samples was performed using a DNA extraction kit (Parsian Bio-Tech, Iran), utilizing young leaves. The quantity and quality of isolated DNA were assessed by spectrophotometry (OD260/OD280) and agarose gel electrophoresis (1%), respectively. Polymerase chain reaction (PCR) amplification was carried out using four primers (IS5, IS7, IS23, and UBC866) for inter simple sequence repeats (ISSR) molecular markers (Table 1).

Table 1.

Amplification products generated by ISSR markers among mother plant and acclimated plant of Nepeta asterotricha

No Primer Sequence (5ʹ → 3ʹ) Total number of bands
1 IS5 GACACACACACACACACC 3
2 IS7 ACGACGACGACGACGG 4
3 IS23 CTCCTCCTCCTCRC 5
5 UBC866 CTCCTCCTCCTCCTCCTC 4
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The ISSR primer amplification took place in a 10 μL reaction volume, containing 3 μL of DNA template (at a concentration of 10 ng/μL), 0.64 μL of primer, 1.5 μL of sterile distilled water, and 4.8 μL Taq DNA Polymerase Master Mix RED (Ampliqon, Denmark). The PCR program included an initial denaturation of 4 min at 94 °C, followed by 35 cycles of 1 min at 94 °C, 60 s annealing at 52 °C 128 with 60 s extension at 72 °C, and a final extension of 10 min at 72 °C.

Phytochemical analysis

Isolation and analysis of the essential oil

Powdered aerial parts of MP, RP, and AP (10 g) were hydro-distilled (3 h) by using a Clevenger-type apparatus by the British Pharmacopeia (1998). The essential oils were then analyzed by gas chromatography-flame ionization detection (GC-FID) and GC-mass spectrometry (GC–MS) as described previously (Raeisi et al. 2015). The essential oil composition was determined according to Ebrahimi et al. (2008), calculating their retention indices under temperature conditions for n-alkanes (C6–C24) and the oil on a DB-5 column under identical chromatographic settings. Individual compounds were identified by comparing their mass spectra with those in the internal reference mass spectra library or with authentic compounds. In addition, confirmation was achieved by comparing their retention indices with authentic compounds or with those reported in the literature (Adams 2007). The relative area percentages obtained by FID were utilized for quantification without correction factors (Ebrahimi et al. 2008).

Extraction and measurement of total phenol and total flavonoid contents

Dried and powdered materials (100 mg) of MP, in vitro RP, and AP were extracted in 10 mL methanol incubated for 40 min in an ultrasonic bath, and subsequently centrifuged at 2000 rpm for 5 min. The extracts were then stored in a freezer at -20 °C for subsequent analysis. Total phenolic content (TPC) was determined using the the Folin-Ciocalteu method as milligram gallic acid equivalent per gram of plant dry weight (mg GAE/g DW) according to Kamtekar et al. (2014). Similarly, total flavonoid content (TFC) was assessed using a solution of 4% sodium hydroxide (NaOH), 5% sodium nitrite (NaNO2), and 10% aluminum chloride (AlCl3) in distilled water as mg rutin equivalent per gram of plant dry weight (mg RUE/g DW) according to Selseleh et al. (2020).

HPLC–PDA analysis

The dried methanolic extracts, dissolved in 10 mL high-performance liquid chromatography (HPLC) grade methanol were used for the quantification of phenolic acids, following the methodology outlined in a previous study (Ghaderi et al. 2019). The analysis was conducted using HPLC-photodiode array (PDA) (Waters 2695 separations module), equipped with a C18 column (250 mm × 4.6 mm, 5 μm, Waters) and a UV detection (waters 2487).

Extraction and analysis of TAs were also performed according to previously established procedures (Srivastava and Chaturvedi 2010; Mirjalili et al. 2016).

Antioxidant and antibacterial activity

Antioxidant activity of the studied samples was assessed using methanolic extracts by 1,1- diphenyl-2-picrylhydrazyl (DPPH) method, following the established protocol (Kamtekar et al. 2014). To evaluate antibacterial activity, the samples were tested against both Gram-positive (Staphylococcus aureus ATCC 25923) and Gram-negative (Escherichia coli ATCC 25922) bacteria using the broth microdilution method, adhering to the guidelines recommended by CLSI as recommended by CLSI (Clinical and Laboratory Standards Institute, 2012) with minor modifications based on the procedure outlined by Selseleh et al. (2020).

Statistical analysis

A factorial experiment was conducted within a completely randomized design (CRD), with four replications for the current study. The data were analyzed using statistical analysis system (SAS) software (version 9.4). The means were also compared using the Duncan multi-domain test at the probability level of 0.05. Cluster analysis of molecular data based on matrix similarity coefficients and our unweighted pair group method with arithmetic mean (UPGMA) method using numerical taxonomy and multivariate analysis system (NTSYS-pc) software (version 2.02).

Results

Shoot multiplication and root induction

The results revealed that the measured parameters of branching (lateral shoot number and length, main shoot length and leaf number) and rooting (rooting percentage, root number, and length) were significantly affected by the type and concentration of plant growth regulators (PGRs). The highest number of lateral branches (25.4) was observed in the explants cultured on the medium containing 3 mg/L KIN, while the highest length of the lateral branches (13.25 cm) was obtained in the treatment of 3 mg/L TDZ and 3 mg/L BAP. The medium containing 3 mg/L BAP resulted in the highest length of the main stem (12.25 cm). The highest leaf number per plantlet (20.25) was obtained on the culture medium containing 3 mg/L TDZ (Table 2). Conversely, the lowest values for side branch number (2.01), side branch length (6.87 cm), and main shoot length (5.01 cm) were recorded in the treatment of TDZ 0.5 mg/L. The lowest leaf number (10.01) was also measured in the hormonal treatment of 0.5 mg/L KIN.

Table 2.

Effect of cytokinin type and concentration on shoot multiplication of Nepeta asterotricha

Cytokinin Level (mg/L) LSN (cm) LSL (cm) MSL (cm) NL
BAP 0.5 2.25 ± 0.50fg 9.50 ± 5.80bcd 6.05 ± 4.10ed 15.50 ± 1.91cde
1 2.75 ± 0.50def 11.28 ± 2.49abc 7.62 ± 1.79bcd 12.50 ± 1.01g
2 2.25 ± 0.50fg 10.12 ± 3.77abcd 7.91 ± 2.55bcd 11.50 ± 1.91gh
3 3.25 ± 0.50bcd 13.25 ± 3.97a 9.95 ± 0.57ab 13.50 ± 3.01efg
KIN 0.5 2.50 ± 0.57efg 8.01 ± 1.41dc 6.42 ± 1.12ed 10.01 ± 1.63h
1 3.50 ± 0.57bc 8.80 ± 0.85bcd 7.70 ± 1.11bcd 11.50 ± 1.01gh
2 3.75 ± 0.50ab 8.50 ± 0.81bcd 7.67 ± 0.78bcd 13.01 ± 1.15fg
3 4.25 ± 0.50a 11.37 ± 0.85abc 9.12 ± 0.85bc 16.01 ± 0.10cd
TDZ 0.5 2.01 ± 0.10g 6.87 ± 0.69d 5.01 ± 1.41e 15.01 ± 0.81def
1 2.50 ± 0.57efg 8.87 ± 0.85bcd 6.87 ± 0.85cde 17.50 ± 1.01bc
2 3.01 ± 0.10cde 11.07 ± 0.94abc 9.01 ± 0.91bc 19.25 ± 0.95ab
3 3.02 ± 0.11cde 13.25 ± 1.70a 12.25 ± 89a 20.25 ± 1.25a
Control 0 2.13 ± 0.13fg 12.01 ± 0.85ab 9.80 ± 1.55ab 14.87 ± 1.75def
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Data shown are mean ± standard deviation (n = 4)

Different letters in columns of each character indicating statistically differences mean at p < 0.05 by Duncan's multiple range test

LSN, lateral shoots number; LSL, Lateral shoot length; MSL, Main shoot length; NL, Number of leaves

The highest rooting percentage (100%) was achieved in hormonal treatments of 1 mg/L of NAA, IBA, and IAA, while the lowest rooting percentage (49.49%) was observed from the free-hormonal MS medium (control). The highest root length (2.75 cm) was attained from the medium supplemented with 0.6 mg/L IAA. The hormonal treatment of 1 mg/L IBA resulted in the highest number of roots (Table 3). Conversely, the lowest values for root length (1.25 cm) and number of roots (13.50) were recorded in the treatments involving 1 mg/L IBA and 0.2 mg/L NAA, respectively.

Table 3.

Effect of auxin type concentration on root induction and elongation of Nepeta asterotricha

Auxin Level Rooting (%) Root length (cm) Number of roots
NAA 0.2 83.33 ± 15.24a 2.25 ± 0.28bc 13.50 ± 2.51g
0.6 95.00 ± 5.77a 2.25 ± 0.28bc 24.01 ± 4.32d
1 100.00a 2.37 ± 0.25abc 31.50 ± 1.01c
IBA 0.2 96.25 ± 4.78a 2.25 ± 0.50bc 22.50 ± 2.38ed
0.6 100.00a 1.75 ± 0.50d 34.75 ± 4.11bc
1 100.00a 1.25 ± 0.28e 46.25 ± 2.50a
IAA 0.2 49.50 ± 15.05b 2.25 ± 0.28bc 24.75 ± 4.50d
0.6 97.50 ± 5.01a 2.75 ± 0.28a 18.25 ± 1.25ef
1 100.00a 2.50 ± 0.10ab 39.05 ± 1.15b
Control 0 49.43 ± 18.83b 1.97 ± 0.32dc 15.43 ± 3.84fg
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Data shown are mean ± standard deviation (n = 4)

Different letters in columns of each character indicating statistically differences mean at p < 0.05 by Duncan's multiple range test

The acclimation process of RP from in vitro conditions to the ex-situ habitat was successfully executed. According to the developed acclimation protocol, 85% of well-rooted RP were effectively acclimated under field conditions. The appearance of AP exhibited normalcy without any morphological abnormalities.

Genetic fidelity

Genetic homogeneity and stability of in vitro regenerates derived from nodal segments of N. asterotricha MP during micropropagation conditions were assessed using ISSR markers. Initially, four ISSR primers were tested (Table 1), all of which generated informative and reproducible bands (3–5 bands per primer), ranging in size from nearly 200 to 800 bp. A total of 16 scorable bands, averaging 4 per primer, were produced, and these bands were found to be monomorphic across all the analyzed in vitro regenerates originating from nodal segments of MP (Fig. 2 and Supplementary Fig. 1). Notably, the primers IS23 and IS5 were produced the highest and lowest number of bands, respectively. No polymorphism was detected in the analysis, indicating that the RP is similar to the MP.

Fig. 2.

Fig. 2

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ISSR marker analysis generated by the primers IS-7 a IS-23, b UBC866, c Lane L corresponds to 3 kb DNA ladder; Lane M, DNA from mother plant; Lanes 1–4, DNA from randomly selected acclimated plantlets in the greenhouse

Essential oils content and composition

The order of essential oil content (w/w %) in the studied plant samples was MP (0.58%) > RP (0.41%) > AP (0.21%). The chemical composition of essential oils (EOs) is detailed in Table 4. A total of twenty-seven compounds were identified in MP, which constitutes 97.0% of the total essential oil content, which mainly includes twelve monoterpene hydrocarbons (8.8%), twelve oxygenated monoterpenes (86.9%) and three sesquiterpene hydrocarbons (2.1%). cis-Sabinene hydrate (35.0%), 1,8-cineole (24.1%), linalool (12.3%), and 4aα,7β,7aα-nepetalactone (5.3%) were found as main constituents.

Table 4.

Essential oil composition of Nepeta asterotricha samples studied

No Compounds CRI LRI MP (%) RP (%) AP (%)
1 α-Thujene 926 930 0.4 0.6 0.7
2 α-Pinene 935 939 0.6 0.6 0.5
3 Sabinene 975 975 1.2 1.2 1.7
4 β-Pinene 983 979 1.8 1.2 0.8
5 Myrcene 990 990 0.6 0.2 0.5
6 α-Terpinene 1021 1017 0.7 1.7 0.4
7 ρ-Cymene 1030 1024 – 5.1 0.9
8 Limonene 1033 1029 0.6 – –
9 1,8-Cineole 1037 1031 24.1 13.0 6.2
10 (E)-β-Ocimene 1048 1050 0.5 – 0.2
11 γ-Terpinene 1062 1059 1.6 4.0 2.0
12 cis-Sabinene hydrate 1081 1070 35.0 26.8 57.7
13 Terpinolene 1091 1088 0.4 0.4 0.1
14 Linalool 1108 1096 12.3 1.8 1.6
15 trans-Sabinene hydrate 1111 1098 0.7 3.2 1.8
16 p-Menth-2-en-1-ol 1132 1120 – 0.2 0.2
17 Citronellal 1153 1157 – – 0.2
18 Lavandulol 1169 1169 0.2 – –
19 δ-Terpineol 1179 1166 1.2 0.5 0.4
20 Terpinen-4-ol 1189 1177 3.2 15.0 3.4
21 α-terpineol 1205 1188 3.7 2.3 1.7
22 Neral 1248 1238 0.1 0.9 0.6
23 Geranial 1264 1267 0.2 1.2 0.5
24 4aα,7α,7aα-Nepetalactone 1374 1360 0.4 – 0.2
25 4aα,7α,7aβ-Nepetalactone 1407 1387 – 0.9 2.3
26 4aα,7β,7aα-Nepetalactone 1413 1392 5.3 12.3 4.1
27 (E)-Caryophyllene 1424 1419 0.6 0.4 1.9
28 trans-α-Bergamotene 1434 1434 – – 0.2
29 (E)-β-Farnesene 1453 1456 0.1 0.1 0.2
30 (Z)-α-Bisabolene 1503 1507 1.3 1.2 5.1
31 Caryophyllene oxide 1589 1583 – 0.5 –
Monoterpene hydrocarbons 8.8 15.6 8.2
Oxygenated monoterpenes 86.9 78.6 81.4
Sesquiterpene hydrocarbons 2.1 2.5 7.5
Total identified 97.9 96.8 97.3
Essential oil content (%w/w) % 0.5 % 0.4 % 0.2
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CRI, calculated retention index (RI), LRI, literature RI, retention indices determined in the present work relative to n-alkanes C6-C24 on DB-5 column; MP, mother plant; RP, regenerated plant; AP, acclimated plant. The major essential oils compounds are presented bold

A total of twenty-six compounds were characterized in RP, representing 96.8% of the total oil, mainly including of eleven monoterpenes hydrocarbons (15.6%), eleven oxygenated monoterpenes (78.6%), and four sesquiterpene hydrocarbons (2.5%). The main components were cis-sabinene hydrate (26.8%), terpinen-4-ol (15.0%), 1,8-cineole (13.0%), 4aα,7β,7aα-nepetalactone (12.3%) and p-cymene (5.1%). Twenty-nine components representing 97.3% of the total oil were determined in AP, which consisted mainly of thirteen monoterpene hydrocarbons (8.2%), twelve oxygenated monoterpenes (81.4%), and four sesquiterpene hydrocarbons (7.5%). cis-Sabinene hydrate (57.7%), 1,8-cineole (6.2%), (Z)-α-bisabolene (5.1%), and 4aα,7β,7aα-nepetalactone (4.1%) were the main constituents. Remarkably, the content of this phenolic monoterpene in the oil of RP, was significantly higher than the oil of AP and MP (Table 4).

Phytochemical characteristics

The phenolic content in the methanolic extracts of the plant was determined using HPLC–PDA. All studied samples exhibited the presence of chlorogenic acid (1.91–64.25 mg/g DW), chicoric acid (1.68–12.70 mg/g DW), rutin (0.59–1.38 mg/g DW), rosmarinic acid (2.55–5.97 mg/g DW), and apigenin (0.51–0.70 mg/g DW) (Table 5). Rutin was not detected in RP. The highest level of chlorogenic acid (64.25 ± 0.33 mg/g DW), chicoric acid (12.7 ± 0.06 mg/g DW), and rosmarinic acid, (5.94 ± 0.04 mg/g DW) was found in AP, while the highest level of rutin (1.38 ± 0.28 mg/g DW) and apigenin (0.70 ± 0.01 mg/g DW) were observed in MP (Table 5).

Table 5.

Phenolic and triterpene acids content of mother plant (MP), regenerated plant (RP), and acclimated plant (AP) of Nepeta asterotricha

Compound Content (mg/g DW ± SD)
MP RP AP
Chlorogenic acid 20.54 ± 0.29b 1.91 ± 0.04c 64.25 ± 0.33a
Chicoric acid 6.39 ± 0.17b 1.68 ± 0.09c 12.7 ± 0.06a
Rutin 1.38 ± 0.28a – 0.59 ± 0.07b
Rosmarinic acid 2.55 ± 0.15b 2.81 ± 0.07b 5.97 ± 0.04a
Apigenin 0.70 ± 0.01a 0.51 ± 0.01b 0.52 ± 0.01b
Betulinic acid 2.20 ± 0.01a 0.37 ± 0.02b 0.17 ± 0.01c
Oleanolic acid 5.37 ± 0.02a 1.64 ± 0.01b 0.84 ± 0.01c
Ursolic acid 15.70 ± 0.03a 5.78 ± 0.02b 3.46 ± 0.01c
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Data shown are mean ± standard deviation (n = 3)

Different letters in columns of each character indicating statistically differences mean at p < 0.05 by Duncan's multiple range test

HPLC analysis revealed the presence of TAs in the studied plant samples. This is the first report on the quantification of these metabolites in the plant. The highest levels of BA (2.20 ± 0.01 mg/g DW), OA (5.37 ± 0.02 mg/g DW), and UA (15.70 ± 0.03 mg/g DW) were found in MP. Regenerated plants was also contained (0.37 ± 0.02 mg/g DW) BA, (1.64 ± 0.01 mg/g DW) OA, and (5.78 ± 0.02 mg/g DW) UA. The lowest levels of BA (0.17 ± 0.01 mg/g DW), OA (0.84 ± 0.01 mg/g DW), and UA (3.46 ± 0.01 mg/g DW) were observed in AP. As can be ascertained, UA is the main TAs in the three samples.

Total phenol content, total flavonoid content, and antioxidant activity

There was no significant difference in TPC (Table 6), however, the highest TPC was measured in MP (49.94 mg GAE/g DW). However, the TFC exhibited significant difference among the three samples studied. The highest TFC was obtained in AP (89.183 mg RUE/g DW). Furthermore, the antioxidant activity was considerably different in the three sample extracts. The highest level of antioxidant activity was observed in AP (IC50 = 196.42 μg/mL).

Table 6.

Total phenol content (TPC), total flavonoid content (TFC), antioxidant activity, and antibacterial property of Nepeta asterotricha samples studied

No Sample TPC (mg GAE/g DW ± SD) TFC (mg RUE/g DW ± SD) IC50 (μg/mL ± SD) Antibacterial activity MIC (μg/mL)
Escherichia coli ATCC 25922 Staphylococcus aureus ATCC 25923
1 MP 49.94 ± 4.90a 69.19 ± 21.32b 312.73 ± 18.01b 80 80
2 RP 39.62 ± 6.27a 53.08 ± 6.16b 825.24 ± 35.69a 160 160
3 AP 46.16 ± 2.25a 183.89 ± 2.31a 196.42 ± 0.6c 80 160
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Data shown are mean ± standard deviation (n = 3)

Different letters in columns of each character indicating statistically differences mean at p < 0.05 by Duncan's multiple range test

MP, mother plant; RP, regenerated plant; AP, acclimated plant; GAE, gallic acid equivalent; RUE, rutin equivalent

Antibacterial activity

The antibacterial activity of the methanolic extracts from the studied samples was evaluated against two bacterial strains by determining the lowest inhibitory concentration (MIC) as outlined in Table 6. The MIC of MP extracts against both bacteria was 80 μg/mL, while RP extracts exhibited a MIC of 160 μg/mL against both studied bacteria. Furthermore, AP extract against E. coli and S. aureus was obtained as 80 μg/ml and 160 μg/mL, respectively. Our findings indicate that MP and AP extracts exhibited the highest antibacterial activity against the tested bacteria.

Discussion

It can be concluded that high concentrations of cytokinins have a positive effect on the length of each lateral branch, the average length of the main stem, and the leaf number. Previous research on N. nuda highlights that optimal shoot length is achieved with 0.4 and 0.7 mg/L BAP, while the highest shoot number is observed with 0.8 mg/L BAP (Nedelkova et al. 2011). Similarly, in N. nuda subsp. albiflora, a study indicated that the highest shoot number per explant, and highest shoot length were attained with 0.1 mg/L KIN (Erdağ et al. 2018). The result also indicates the highest number of roots, indicating that the root length decreased with the increase in the number of roots. The obtained results showed that the use of auxins had a positive effect on the percentage of rooting, length, and number of roots.

Molecular analysis demonstrated that the conditions used in the direct micropropagation of the plant did not change the genetic nature of the regenerates. Consequently, the developed method proves effective for mass propagation and in vitro cloning of N. asterotricha. The genetic structure of the plants could be affected by variable culture conditions such as PGRs, so the genetic fidelity identification of in vitro-derived plant materials could be a remarkably cost-effective and time-saving step for breeding or conservation programs (Bairu et al. 2011; Aremu et al. 2013). Our results align with previous findings that confirm the genetic stability of various plant species under in vitro conditions. The genetic stability of several plant species adapted to in vitro conditions has been previously proven which is in agreement with our obtained results. For instance, a study on the homogeneity of regenerated seedlings from native Thymus persicus plant, using random amplified polymorphic DNA (RAPD) primers, indicated a high level of genetic uniformity in the regenerated samples (Bakhtiar et al. 2014).

In another investigation, the genetic stability of in vitro seedlings of Decalepis salicifolia with the MP was examined using RAPD and ISSR primers, revealing a high genetic stability of the in vitro plants (Ahmad et al. 2018). Similarly, the genetic fidelity of micropropagated Sapindus trifoliatus Linn regenerated plants was evaluated using RAPD primer, resulting in monomorphic patterns identical to those of the mother plants (Asthana et al. 2011). The genetic fidelity of the regenerated Solanum erianthum D. Don was also evaluated using RAPD molecular markers that showed a high number of identical monomorphic bands (Sarkar and Banerjee 2020). Behera et al. (2018) have verified the genetic stability of the Paederia foetida L. regenerates using ISSR markers. Consequently, molecular markers such as RAPD and ISSR stand out as affordable and widely used tools for studying the genetic stability of in vitro culture-raised plantlets (Khurana-Kaul et al. 2012; Razaq et al. 2013).

Various factors such as altitude, temperature, nutrition, and PGRs are known to impact the accumulation of essential oil in certain members of the family Lamiaceae (Yavari et al. 2010; Sharafzadeh and Zare 2011). The reduction in essential oil content observed in the AP in this study might be attributed to ex-situ environmental conditions, including altitude, longitude, temperature, and light intensity. Additionally, the essential oil content of in vitro RP could be influenced by additives in the culture medium and PGRs. Manan et al. (2016) highlighted that the micropropagated plants of Ocimum basilicum L. exhibited essential oil production, possibly due to the presence glandular trichrome on their surfaces.

These results have also been confirmed by Morone-Fortunato and Avato (2008) that showed PGRs can affect glandular trichomes density, especially on the upper leaf epidermis, which led to increase in the essential oil accumulation of RP of Origanum vulgare L. Treatment with gibberellic acid and a type of diterpenoid (calliterpenone), was shown to positively impact the density and total number of GTs, potentially enhancing essential oil accumulation in Mentha arvensis L. (Bose et al. 2013). The effects of PGRs on essential oil content and composition have also been studied in Micromeria pulegium (Rochel) Benth. (Stojičić et al. 2016). Enhanced essential oil content in the MP may be attributed to the reproductive phase and its habitat climatic conditions. Variability in the essential oil content has been reported in other Nepeta species i.e. N. heliotropifolia Lam and N. congesta subsp. cryptantha (Akdeniz et al. 2020).

The primary components of the essential oil from the plant have been previously documented, including 1,8-cineole (26.1%), terpinen-4-ol (14.8%), 4aα, 7α, 7β-nepetalactone (8.6%), and cis-sabinene hydrate (8.5%) (Ezzatzadeh et al. 2014). In another study, the major constituents of the stem and flower essential oils of N. asterotricha were terpinen-4-ol (22.8% and 27.7%), respectively (Masoudi et al. 2012). The other notable compounds in the essential oils of the stem and flower of the plant were γ-terpinene (14.1% and 8.1%) and 1,8-cineole (6.7% and 14.6%), respectively. Consequently, the essential oil of N. asterotricha can be characterized by three main compounds: cis-sabinene hydrate, 1,8-cineole, and 4aα,7α,7β-nepetalactone.

This variation has been widely described in the Lamiaceae family. For example, in vitro Salvia stenophylla Burch. ex Benth plantlets produced β-caryophyllene and cis-α-bisabolene in comparison with field plants (Musarurwa et al. 2010). Variation in the terpene profile of Mentha arvensis field plants, in vitro plants, callus, and acclimatized plantlets has also been reported (Phatak and Heble. 2002). Qualitative differences in the essential oil composition of in vitro- and in vivo-derived plants of Salvia przewalskii Maxim have also been reported by Skała et al. (2007). Those differences can be due to the in vitro conditions, particularly the existence of PGRs (Makowczyńska et al. 2016). Furthermore, the chemical variability among the studied EOs may be attributed to the environmental factors related to the plant habitats and in vitro culture conditions.

Environmental factors often exert a distinct influence on the biosynthesis levels and quality of specialized metabolites (SMs) in plants (Yang et al. 2018). The findings of this study indicate that RP and AP of N. asterotricha maintain the capacity to produce major essential oil components like MP. However, the chemical profile and relative content of the essential oil compounds vary among the three conditions. Consequently, N. asterotricha presents an appealing prospect for cloning valuable genotypes. Additionally, in vitro culture techniques of the plant can be interestingly used for future commercial and sustainable production of precious bioactive compounds such as 1,8-cineole. Moreover, the essential oil quality is assured and this protocol can be also applied to propagate selected chemotypes for industrial purposes.

Quantitative variations in phenolic compounds among the studied samples may stem from distinctions between in vivo and in vitro growth conditions. The majority of rosmarinic acid in the flower extract (0.397 ± 0.01 g/100 g DW) of N. humilis Bentham has also been reported (Gökbulut and Yilmaz 2020). They have also been found chlorogenic acid, luteolin, and apigenin in the plant flower extract. Similarly, chlorogenic, rosmarinic, and quinic acids were identified as the major phenolic compounds in the methanolic extract of N. nuda subsp. Lydiae (Aras et al. 2016).

Isolation of phenolic compounds including rosmarinic acid, caffeic acid, apigenin, and apigenin7-O-B- glucopyranoside has been reported from the aerial parts of N. curviflora (Rabee et al. 2020). Another investigation identified rosmarinic acid and sinapic acid in N. cataria L. with concentrations ranging from 0.63 to 2.41 mg/g DW and 0.35 to 0.74 mg/g DW, respectively (Mihaylova et al. 2013).

As it could be ascertained, rosmarinic acid is a predominant phenolic compound in most Nepeta species. This compound has significant biological properties such as antibacterial, antiviral, anti-inflammatory, antioxidant, and anti-proliferative activity (Yoshida et al. 2005). All these beyond biological activities make rosmarinic acid as a valuable metabolite for the food pharmaceutical, and cosmetic industries (Khojasteh et al. 2020). Notably, our results demonstrate that N. asterotricha is capable of producing high levels of rosmarinic acid and chlorogenic acid which may be interesting as a productive and promising source of natural antioxidants.

According to the obtained results, the highest content of phenolic acids was observed in AP, therefore, several environmental factors such as; high temperature, direct sunlight, stress of plant transfer, etc. have increased the biosynthesis of phenolic compounds in AP.

Environmental factors such as soil composition, temperature, rainfall, and humidity can influence the concentrations of phenolic compounds in medicinal plants (Kouki and Manetas 2002; Avila-Peña et al. 2007). The higher level of phenolic compounds in the AP sample can be considered for the sustainable production of these valuable SMs without disrupting the plants’ natural habitat.

The higher concentration of UA in the MP may be attributed to the optimal conditions of the plant habitat for the biosynthesis of this compound. The priority of the wild-growing MP over in vitro RP for the production of TAs has also been reported in T. persicus (Bakhtiar et al. 2014). They have also concluded that the higher content of TAs in the MP could be attributed to climatic fluctuations.

Biological factors of TAs containing anti-inflammatory, antioxidant, anti-fungal, anti-HIV, and immunomodulatory activities have been recognized so far (Gbaguidi et al. 2005; Novotny et al. 2003). Based on their substantial anticancer and biological activity mentioned above, demand for these valuable compounds has gradually increased.

Plants belonging to the Lamiaceae family are recognized as abundant sources of free TAs, along with various other compounds (Mirjalili et al. 2016). Phytochemical investigations have shown that the Lamiaceae family is identified by having a high content of UA. Salvia L. species such as Salvia fruticosa, S. officinalis, and S. virgate are known to produce significant quantities of OA and UA, constituting up to 1% of their total DW (Martin et al. 2009; Haas et al. 2014). Triterpenic acids are not exclusive to the Lamiaceae family, as evidenced by their presence in various plant families. For instance,Silphium leaves contain 17.03 mg/g DW of OA, while Silphium trifoliatum L. and S. integrifolium inflorescences contain OA ranging in the range of 22.05–17.95 mg/g DW, respectively. Calendula officinalis flowers contained 20.53 mg/g DW of OA. The content of OA in Panax quinquefolium roots has been reported as 3.15 mg/g DW. Ursolic acid has also been reported as a plentiful TAs in S. integrifolium and S. trifoliatum leaves at the level of 14.98 mg/g DW (Kowalski. 2007). Based on the other study, OA was abundant in Ortosiphon stamineus Benth., Crataegus monogyna, Lagerstroemia speciose L., and Arctostaphylos uva-ursi (L.) Spreng leaves (7.77, 4.16, 2.10, and 1.03 mg/g DW), (Caligiani et al. 2013).

Triterpenes have also been isolated from different Nepeta species of which UA was a common TAs found in these species (Formisano et al. 2011). Terpenoids are important types of natural products in N. tenuifolia Benth (Shan et al. 2021). Ursolic acid, a complex molecule with 10 chiral centers, has yet to be chemically synthesized in the laboratory, making plant materials the current focus for its extraction due to their potential. So, the plant materials are currently considered for the extraction of this valuable compound (Szakiel et al. 2012; Rubashvili et al. 2020). Our findings proposed that N. asterotricha can be interestingly considered as a good potential plant source for the commercial production of TAs especially UA through biotechnological approaches like cell suspension culture.

Phenolic and polyphenolic compounds are abundantly found in food products produced from plant sources, which have extensive antioxidant activity (Van Acker et al. 1996). The observed presence of these compounds in the analyzed extracts proposes their significant role in plants.

It is likely that AP increases the synthesis of phenolic and flavonoid compounds due to the environmental conditions such as direct sunlight, stress created during adaptation, high field temperature, etc. and thus increases the amount of antioxidant activity. The rise in phenolic and flavonoid compounds correlate directly with the augmentation of antioxidant activity.

Flavonoids particularly those with hydroxyl group, play a crucial role in inhibiting the negative effects of free radicals in most plants. (Das and Pereira 1990). The action of flavonoids involves scavenging or chelating processes (Cook and Samman 1996). Recognized for their potent free radical-scavenging abilities, plant phenolics serve as natural antioxidants. Therefore, the identification of these compounds in the plant materials herbs is thus a significant finding of this study. In the previous report, the methanolic extract of N. asterotricha was documented to contain 54.60 mg GAE/g DW and 101.34 mg RUE/g DW of total phenol and total flavonoids, respectively (Mirjalili et al. 2021).

In another study, N. pogonosperma Jamzad and Assadi exhibited a TPC of 35.20 mg GAE/g DW, with an antioxidant activity (IC50) of 195.87 μg/mL (Khalighi-Sigaroodi et al. 2013). The total phenolic content of N. melissifolia Lam was reported as 31.60 mg GAE/g DW (Proestos et al. 2013). In another report, methanolic extract of N. juncea Benth leaves, flowers, and roots contained 69.54, 45.61, and 21.33 mg GAE/g DW, respectively. The TFC of the studied samples were also 9.62, 26.42, and 47.31 mg quercetin equivalent/g DW, respectively (Sharifi-Rad et al. 2020). Bošnjak-Neumüller et al. (2017) reported the TPC and TFC of N. rtanjensis Diklic and Milojevic as 62.73 ± 1.80 mg GAE/g and 83.30 ± 1.40 mg RUE/g of extract that showed antioxidant activity (IC50 = 112.59 ± 0.95 μg/mL). These findings collectively suggest that Nepeta species are rich in phenolic and flavonoid compounds, showcasing significant antioxidant activity.

Antibacterial activities have also been observed in various Nepeta species. For instance, methanolic extract of N. juncea against S. aureus and E. coli demonstrated MIC values of 25 μg/ml and 50 μg/mL, respectively (Sharifi-Rad et al. 2020). Another study reported the antibacterial activity (MIC) of the methanolic extract of N. nervosa, N. rtanjensis, and N. sibirica against S. aureus has been reported as 50, 50, and 100 μg/mL. The value of MIC of the plant species against E. coli was also 50, 100, and 100 µg/mL, respectively (Nestorović et al. 2010).

Conclusions

A successful protocol for in vitro shoot multiplication and rooting of N. asterotricha involved culturing nodal segments on MS culture medium with 3 mg/mL KIN and 1 mg/L of each of the three auxins, maintaining a 16/8 h photoperiod. The acclimation of plants ex vitro was achieved, and molecular analysis verified the true to type nature of the acclimated plants. Phytochemical analysis of the RPs have also shown that they can produce bioactive compounds similar to the MP. Furthermore, the metabolic potential of N. asterotricha in the biosynthesis and production of TAs especially UA was confirmed for the first time. Hence, the developed technique and in vitro plant materials can be exploited in the conservation, domestication, breeding programs and commercial production of valuable plant SMs by biotechnological methods as well.

Supplementary Information

Below is the link to the electronic supplementary material.

12298_2024_1416_MOESM1_ESM.tif (1MB, tif)

Fig. S1. ISSR marker analysis generated by the primer IS-5, Lane L corresponds to 3 kb DNA ladder; Lane M, DNA from mother plant; Lanes 1–4, DNA from randomly selected acclimated plantlets in the greenhouse (TIF 1063 KB)

Acknowledgements

The authors thank the Shahid Beheshti University Research Council for financial support of this project. We also wish to thank Dr. Hassan Esmaeili and Dr. Hamid

Ahadi for their kind help in molecular and phytochemical analyses, respectively.

Author contributions

MZ contributed to the conception of the study, in vitro cultures establishment, data extraction, statistical analysis, and writing of the manuscript. AS helped in plant identification, statistical analysis, and editing the manuscript. SMG contributed in plant materials collection, formal analysis, and revising the manuscript. MHM supervised the whole experiments and wrote the manuscript. All authors read and approved the final manuscript.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

12298_2024_1416_MOESM1_ESM.tif (1MB, tif)

Fig. S1. ISSR marker analysis generated by the primer IS-5, Lane L corresponds to 3 kb DNA ladder; Lane M, DNA from mother plant; Lanes 1–4, DNA from randomly selected acclimated plantlets in the greenhouse (TIF 1063 KB)

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