Secondary metabolite fingerprinting, anti-pathogenic activity, elite chemotype selection and conservation of Curcuma caesia- an ethnomedicinally underutilized species

Abstract

Curcuma caesia Roxb. is an ethnomedicinally important, essential oil (EO) yielding aromatic plant. A total of twelve accessions of this plant rhizome were collected from six different agro-climatic zones of West Bengal, India and evaluated for their antimicrobial activities against eight disease-causing, multi-drug-resistant pathogenic strains of urinary-tract infection and respiratory-tract infection. The EO and extracts demonstrated antibacterial activity, with the highest inhibition zone of 18.00 ± 0.08 and 17.50 ± 0.14 mm against Klebsiella pneumoniae by accession 06, even where all the broad-spectrum antibiotics failed to respond. In this study, we employed high-performance thin-layer chromatography (HPTLC) to quantify curcumin, the primary secondary metabolite of C. caesia, and the highest 0.228 mg/gm of curcumin resulted from accession 06. Hence, on the basis of all aspects, accession 06 was identified as the elite chemotype among all twelve accessions. The chemical profiling of EO from accession 06 was done using gas chromatography-mass spectroscopy (GC–MS). Conceivably, about 13 medicinally significant compounds were detected. As this plant species is seasonal and has difficulties in conventional breeding due to dormancy, it must be conserved through in vitro tissue culture for a steady supply throughout the year in massive amounts for agricultural demand. A maximum number of 19.28 ± 0.37 shoots has been obtained in MS medium fortified with 6-Benzylaminopurine, Kinetin, and Naphthalene acetic acid. The genetic uniformity of the plants has been studied through Start Codon Targeted Polymorphism. Therefore, this study must help meet the need for essential phytoactive compounds through a simple, validated, and reproducible plant tissue culture protocol throughout the year.

Introduction

The medicinal plants are reservoirs of versatile groups of secondary bioactive compounds with diverse types of therapeutic significance in traditional medicine pharmacopoeias. The family Zingiberaceae is well known for its medicinal and aromatic nature and has been used as food and spice worldwide since ancient times. Genus Curcuma comprises 80 species, including Curcuma caesia, a well-known oil-yielding rhizomatous medicinal herb (Paw et al. 2021). Curcuma caesia is a native species of India and is often known as "kali-haldi," used as a cultivated plant with diverse medicinal uses (Mahanta et al. 2020). Other species of the genus, like Curcuma longa and Curcuma amada, are extensively explored, but Curcuma caesia is still unexplored in different fields of study (Borah et al. 2019). A few researchers reported that Curcuma caesia is endangered in India and Southeast Asia (Borah et al. 2019; Paw et al. 2020a, b). As per our findings, the plant is available in a few places throughout West Bengal. As a result, only 12 collections have been made due to wide unavailability. This plant's rhizomes and oil are used in the treatment of leucoderma, tumours, bronchitis, tuberculosis, spleen enlargement, hemorrhoids, epilepsy, menstrual disorder, cancer, and leprosy and are applied during snake bites and scorpion bites also (Chakraborty et al. 2019, 2023; Sharma et al. 2021; Chaturvedi et al. 2021).

The dominance of emerging deadly bacterial infections in recent years and the failure of synthetic antibiotics to combat them forced us to find an alternative way (Fair and Tor 2014). Plants are an enormous and continuous reservoir of bioactive compounds in the natural environment. Bioactive compounds in medicinal plants can synergistically affect bacterial resistance to pharmacologically necessary antibiotics (Mickymaray 2019). Previous studies have demonstrated the antimicrobial potency against some laboratory-grade non-pathogenic microbial strains by Curcuma caesia (Pandey and Gupta 2014; Kaur et al. 2018; Yadav and Kaliyaperumal 2021). However, no such work has yet been done against multi-drug-resistant strains of human pathogens. In the present study, eight (four urinary tracts infecting and four respiratory tracts infecting) multi-drug-resistant bacterial strains were tested with crude plant extract, essential oil, and pure curcumin to determine their susceptibility.

Curcumin, primarily found in various Curcuma species, is a highly potent secondary component. Its IUPAC name is (1E, 6E)-1,7-Bis(4-hydroxy-3-methoxyphenyl) hepta 1,6-diene-3,5-dione, with a molecular formula of C₂₁H₂₀O₆ and a molecular weight of 368.385 g/mol. It is also known as 1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione or diferuloylmethane. Curcumin has gained recognition for its outstanding efficacy across various applications related to human well-being. This compound exhibits antimicrobial, anti-inflammatory, anti-diabetic, hepatoprotective, neuroprotective, chemo-protective, anti-cancer, anti-allergic, and anti-dermatophytes properties. Additionally, it finds utility in addressing gastrointestinal, respiratory, and cardiovascular issues (Salehi et al. 2019a, b). This study focuses on identifying and quantifying curcumin content in different populations of C. caesia employing High-performance thin-layer chromatography (HPTLC) fingerprinting. The extensive use of HPTLC has been found in the analysis of secondary metabolites for its systematic approach to method validation, cost efficiency, and consistency in quantification (Panigrahi et al. 2017a, 2017b; Hakim et al. 2017; Gantait et al. 2020). A high-yielding chemotype with high curcumin and essential oil content and high antimicrobial efficacy was selected after screening twelve chemotypes of this plant procured from different agro-climatic zones of West Bengal. Identification and selection of elite chemotypes with high curcumin content are very much needed for the commercial exploitation of the plant. Therefore, the high-yielding chemotype can be further propagated to produce high-value compounds.

Factors such as deforestation, excessive utilization, and unscientific practices have contributed to the endangerment and eventual disappearance of numerous species from the environment. Many plants, especially those belonging to the Zingiberaceae family, are seasonal, thriving during specific periods of the year. C. caesia, for instance, is also a seasonal plant, displaying growth between July and November. This seasonal pattern makes it challenging to maintain a year-round supply of the plant. Moreover, the dormancy of rhizomes poses difficulties in conventional breeding efforts. Given these constraints, it is imperative to preserve this plant in an artificially controlled environment. Plant tissue culture is the most effective way to conserve plant species over the year in a small area, and time to obtain them in large amounts with stable genotypes (Gantait et al. 2014, 2015; Singh 2018; Panigrahi et al. 2018). In vitro conservation of the species of Curcuma is the most promising way to conserve the elite germplasm from extinction due to disease, natural disaster, or extensive non-scientific use.

True-to-type plant regeneration in a controlled environment is also a challenging task. Cultivation of a wild species outside its natural habitat may cause genetic alteration and significant changes in chemical profiles (Rey et al. 2020). According to the previous reports, it was established that tissue culture is a way that can be used to regenerate true-to-type plants (Haque and Ghosh 2013). DNA markers have broad applications in plant molecular genomic study (Bhat et al. 2016). Genetic diversity studies in plants are often done by mapping linked genes or quantitative trait loci (QTL) controlling important traits (Cui et al. 2015; Mahanta et al. 2023). In this study, we have employed Start Codon Targeted (SCoT) Polymorphism for genetic fidelity assay, with higher polymorphism and better marker resolvability, to study the polymorphism between both the in-vivo and ex vitro plants. This method shares the principle of using a single primer like RAPD and ISSR. This gene-targeted promising marker targets the conserved flanked region of translation initiation start codon (ATG) of plant genes (Rohela et al. 2019; Kudikala et al. 2020; Mahanta et al. 2023). Therefore, polymorphism between large numbers of plants can be validated using this method very quickly.

The present work mainly describes, the validation of intra-species variation in curcumin and essential oil content of the plants procured from different agro-climatic regions of West Bengal by HPTLC fingerprinting, selection of an elite chemotype based on curcumin content, antimicrobial activity, essential oil and crude extract content. Investigation of the antimicrobial activities of the plant extract and essential oil against four multidrug-resistant human UTIs and four RTI pathogens must help in the development of drugs against multi-drug-resistant bacterial strains. Therefore, it must be used as a future drug in pharmaceuticals. Analytical derivatization of essential oil and the presence of bioactive compounds will help to obtain bio-medicinally active compounds for industrial purposes. The enhanced in vitro propagation system for this elite chemotype must help in future agriculture to reproduce high-yielding plants in large amounts, which must be profitable in the agriculture system. Tissue culture-mediated genetically stable plant production also helps obtain the same chemotype and genotype for a long time. All the types of experiments for C. caesia conducted in the present study have been reported compactly for the first time till date. Therefore, this study must help further improve this respective plant.

Materials and methods

Plant collection and identification

A number of twelve rhizomes samples of Curcuma caesia, were collected from different agro-climatic zones of West Bengal. The plants were identified from the Central National Herbarium (CNH) of the Botanical Survey of India (BSI), Howrah, India, with Voucher no. ‘CNH/Tech. II/2022/80’ and plant specimen no. ‘RKMVC-1533’.The accessions were planted and maintained in our institute's experimental garden for future experiments (Table 1). The elite chemotype was screened from all the accessions collected, and a protocol was established for high-frequency propagation of this elite type through in vitro technique.

Table 1.

Collection of plants from the different agro-climatic zones of West Bengal

Sl. no.	Place	District	Type of sample	GPS	Agro-ecological zone
1	Udaynarayanpur	Howrah	Rhizome	22.8172° N, 88.1047° E	Vindhyan alluvial
2	Khardaha	North 24 parganas	Rhizome	22.7003° N, 88.3753° E	Gangetic alluvial
3	Kakdwip	South 24 Parganas	Rhizome	21.8760° N, 88.1853° E	Coastal saline
4	English Bazar	Maldah	Rhizome	25.0108° N, 88.1411° E	Gangetic alluvial
5	Chirulia	Midinapore	Rhizome	21.7562° N, 87.5062° E	Coastal saline
6	Birpara	Alipurduar	Rhizome	26.7058° N, 89.1373° E	Northern hill
7	Chakdah	Nadia	Rhizome	23.0765° N, 88.5293° E	Gangetic alluvial
8	Chinsurah	Hooghly	Rhizome	22.9012° N, 88.3899° E	Vindhyan alluvial
9	Indus	Bankura	Rhizome	23.1504° N, 87.6318° E	Red laterite
10	Debrajpur	Birbhum	Rhizome	23.7941° N, 87.3746° E	Red laterite
11	Berhampur	Murshidabad	Rhizome	24.0983° N, 88.2684° E	Gangetic alluvial
12	Barmek	Kalimpong	Rhizome	27.0666° N, 88.4666° E	Teesta-Tarai alluvial

Extraction of the plant material

Extraction through Soxhlet

Mature rhizomes of C. caesia were maturely grown and collected. One hundred grams of fresh rhizomes were then subjected to drying, resulting in 84 g of dry powder using an electric grinder. The drying process occurred in open sunlight. Subsequently, 3.0 g the dried powder from each sample were weighed and underwent extraction with methanol using a Soxhlet apparatus, lasting 12 h. After extraction, the extracted metabolite is dried at 40 °C until total solvent evaporation and finally stored at 4 °C for further use. No significant increase in extract content was observed after 8 h of extraction, leading to the decision to extract all samples for a total of 12 h as per the prescribed procedure.

Clevenger extraction

A total of 300 g of freshly harvested C. caesia rhizomes were precisely weighed and underwent hydrodistillation using a Clevenger apparatus, which continued for 8 h. This process was employed to isolate the volatile aromatic essential oil from the rhizomes. Subsequently, the essential oil was collected from the apparatus's collection channel and stored at a temperature of 4 °C, awaiting further analysis. It's noteworthy that the highest yield of essential oil was achieved following 8 h of extraction. Interestingly, no significant increase in oil yield was observed even with prolonged distillation beyond the 8-h mark in the Clevenger apparatus.

High-performance thin-layer chromatography (HPTLC) fingerprinting

The samples were spotted in the forms of bands of width 3 mm with a Camag 100 µl syringe on precoated silica gel aluminium plates 60F254 (20 cm × 10 cm with 0.2 mm thickness; E. Merck, Darmstad, Germany) using a Camag Linomat V (CAMAG, Muttenz, Switzerland). The slit dimension was kept at 4 mm × 0.1 mm, and a 20 mm/s scanning speed was employed. These parameters were kept constant throughout the analysis of samples. The mobile phase consisted of toluene, chloroform and methanol in a ratio of 5: 4: 1 (v/v/v). Plates were developed in ascending order with a CAMAG twin through a glass tank pre-saturated with the mobile phase for 20 min; the length of each run was 8 cm. The TLC runs were performed under laboratory conditions (Temp: 25 ± 2 °C and % RH: 60 ± 5). The plates were then dried in the air. Densitometric analysis was performed at 425 nm with a Camag TLC scanner III operated by Win CATS software (Version 1.4.8.2031). The Rf value for curcumin was found to be 0.38. The scanning wavelength selected was 425 nm to detect the absorption maxima of the curcumin spot.

Isolation and identification of UTI and RTI bacterial pathogens

Urine samples and swab specimens collected from the diseased patients were cultured by plating the samples on MacConkey agar and Nutrient agar plates (Himedia®, India) for UTI and RTI, respectively, and grown at 37 ± 2 °C for 24 h. Respiratory specimens (e.g., sputum, pleural fluid, bronchial aspirate etc.) for RTI and urine samples for UTI were collected from the Ashok laboratory Clinical Testing Centre Private Limited, Kolkata 700,068, India (a BSL-3 laboratory and NABL accredited), maintaining rules of patient confidentiality. VITEK 2 COMPACT SYSTEM BIOMERIUEX machine was used to identify the pathogenic strains. Initial identification was made by conducting the following investigations— oxidase activity, catalase activity, Indole test, Methyl red, Citrate test, Voges-Proskauer test, and hydrogen sulfide production. Antibiotic sensitivity assays were done after the strains were identified. Four UTI and four RTI strains were screened for resistance/susceptibility against more than ten common antibiotics. The preliminary antibiotic resistance profile was established using 10 μg of standard antibiotic discs (Himedia®, India), followed by precise determination of antibiotic resistance levels for all bacterial pathogens through minimum inhibitory concentration assays.

Antimicrobial activity study

Antimicrobial activities of essential oil, crude extract of rhizome and pure curcumin were tested against urinary tract infecting (UTI) and respiratory tract infecting (RTI) human pathogenic bacterial strains. Four microorganisms from both UTI and RTI were used in the present study. The antimicrobial potentiality was investigated using the agar-well diffusion method. The study has been conducted in triplicate for each case of a total of eight pathogens.

Agar cup method

The agar well-diffusion method was used to determine the antibacterial potency of the crude extracts, essential oil extracted from rhizomes and pure curcumin, following the methodology described by Deans and Ritchie (1987) with minor modifications. The plant extract was dissolved in DMSO (dimethyl sulfoxide) at 100 mg/ml and applied at 50 mg/cup, whereas essential oil at 3.0 μl/cup and curcumin at 0.1 mg/cup was applied on microbial strains. Muller Hinton agar (MHA) plates (Himedia®, India) were used to determine the zone of inhibition. The plates were punched with a cork borer of 6.0 mm diameter, and different concentrations of the samples (crude plant extract, EO and curcumin) were poured into the agar cups. After incubation of the plates at 37 °C for 24 h, zones of inhibition were recorded.

Elite chemotype selection

The process of elite chemotype selection was executed by evaluating essential oil, crude extract, and curcumin content, primarily based on their remarkable antimicrobial capabilities. This study strongly emphasises identifying and choosing chemotypes with high yields, while disregarding other ecological variations. These selected chemotypes hold immense potential for cultivation and utilization across various domains, including pharmaceuticals, agriculture, and industry. The goal is to harness their unique properties to develop valuable products, contributing to advancements in medicine, agriculture, and various industrial applications, ultimately benefiting society through enhanced and sustainable resource utilization.

GC–MS analysis of essential oil from an elite type

1 μl of the oil sample, extracted from the plant rhizome, was diluted with 999 μl of n-hexane, and 1 μl of this diluted solution was injected in GC–MS with a split ratio of 1:10 in a TG-5MS column (30 m × 0.25 mm, 0.25 μm, Thermo Fisher Scientific). The inlet temperature was maintained at 260 ºC throughout the time. The initial oven temperature was set at 50 ºC for 2 min, which was increased to 60 ºC at a rate of 2 ºC/min. The temperature was held at 60 ºC for 2 min followed by an increase to 210 ºC at a rate of 3 ºC/min and held for 2 min at this stage. Finally, the temperature was reached at 270 ºC at a rate of 10 ºC/min with a final holding time of 7 min (Total run time 74.6 min). The carrier gas flow (helium) was kept at 1 ml/min throughout the analysis. For MS ion source temperature and MS transfer, line temperature was kept at 200 ºC and 280 ºC, respectively. The electron impact was set at 70 eV, and a 40–600 amu scan range was maintained. The compounds were identified using the NIST 20 library by comparing the mass spectra with the database (National Institute of Standards and Technology, Gaithersburg, USA).

In vitro conservation of elite plant

Following the standardized protocol of Haque and Ghosh (2018), the in vitro establishment of elite chemotypes of C. caesia was done. The rhizomes of elite type were planted in a mixture of soil and organic manure (2:1 ratio V/V) and maintained in a pot. During May–June, axillary buds (6–8 mm) sprouted and were used as explant to introduce in the tissue culture system. The buds were thoroughly washed under running tap water for 10 min, then 25 gm/l Bavistin® was used to wash for 15 min and finally with liquid detergent (30 ml/l Tween-20) for 3 min. The buds were subsequently surface-disinfected with a freshly prepared 1.5 gm/l aqueous solution of mercuric chloride (HgCl₂) for 10 min, and then sterile distilled water was used to wash the explant thrice to remove traces of HgCl₂. Furthermore, the explants implanted in MS medium (Murashige and Skoog 1962) were fortified with 1.5 mg/l 6-benzyl amino purine (BAP) and 1.0 mg/l α-naphthalene acetic (NAA) following the result obtained by Ghosh et al (2013). Tubes were then kept under a 16 h/8 h photoperiod at 25 ˚C in the tissue culture laboratory.

Shoot and root multiplication

The infection-free explants are implanted in the MS medium supplemented with different cytokinins including 6-benzylaminopurine (BAP), Kinetin (KIN), and Thidiazuron (TDZ). The cytokinins are provided in the medium with varied concentrations. The four different concentrations of BAP, KIN and TDZ (1.0, 2.0, 3.0, and 4.0 mg/l) and their combinations are used for shoot multiplication upon multiplication and response. MS medium without PGRs is used as a control. The measurements of shoot length and the rate of multiplication were observed every 10-day interval for up to 60 days. The complete data of the percentage of shoots and length of the shoot were recorded 60 days after the inoculation. The shoots are implanted in the MS medium supplemented with three different auxins indole butyric acid (IBA), indole-3-acetic acid (IAA) and α-naphthalene acetic acid (NAA) for the rooting experiment. Different auxin concentrations are also used to study the optimum number of root regenerations. Varied concentrations of IBA, NAA and IAA (0.5, 1.0, and 1.5 mg/l) have been used to study the better result of root induced from the multiplied shoot. Furthermore, the effect of auxin (NAA or IAA at 0.5, 1.0, and 1.5 mg/l) in combination with optimum shoot multiplication causing PGRs combination (3.0 + 1.0 + 0.5 mg/l, 3.0 + 1.0 + 1.0 mg/l BAP + KIN + NAA and BAP + KIN + IAA) has been done to get better results.

Acclimatization and field transfer

After the development of sufficient shoots and roots, healthy plantlets were carefully washed thoroughly after being taken out from the culture vials. Earthen pots containing Sterile 50 gm Soilrite™ (Keltech Pvt. Ltd. Bangalore, India) were used for hardening the plantlets and transferring them to a playhouse. Sufficient moisture (90–99%) was maintained by spraying the plants with water and covering the plants in the earthen pots using transparent plastic. After 4 weeks, the plantlets were uncovered and transferred to the shade net house for proper growth.

Polymorphism and reproducibility study through SCoT primers

Genetic stability of in vivo (wild plant) and ex vitro (tissue culture derived plant) plants was studied by amplifying their genomic DNA using 15 primers based on a conserved region flanking ATG, the translational start codon. The SCoT primers with their melting temperature are listed in the table (Table 2). Himedia, India, synthesized all the SCoT primers. All 15 primers were initially screened in both types of plants for the ability to produce reproducible polymorphic patterns. The 50 μl of PCR amplification reaction consisted of 40 ng of genomic DNA, 1 unit of Taq DNA polymerase, 0.5 μM of primer, 10 mM of dNTPs, and 1X Taq buffer. Thermal Cycler (MJ MiniTM, Bio-Rad) was used to carry out the PCR reaction by the standardized method as follows: denaturation- initially for 3 min at 94 °C followed by 35 cycles with 45 s at 94 °C, annealing at primer-specific annealing temperature, the extension of 90 s at 72 °C, and a final extension at 72 °C for 5 min; it was preserved at 4 °C until agarose (1.2%) gel electrophoresis was done with 1X TAE buffer.

Table 2.

Genetic fidelity assessment of in vitro regenerants and the mother plant of Curcuma caesia using SCoT primers, their sequences with number and size range of amplicons

SCoT primers
Primer	Sequence	Tm (○C)	Number of amplified bands per primer	Total number of bands	Size of amplicon
SCoT-1	CAACAATGGCTACCACCA	52.6	6	54	400–2000
SCoT-2	CAACAATGGCTACCACCC	53.6	4	36	300–1900
SCoT-3	CAACAATGGCTACCACCG	53.9	4	36	500–1700
SCoT-4	CAACAATGGCTACCACCT	52.3	4	36	500–1700
SCoT-5	CAACAATGGCTACCACGA	52.6	5	45	300–1500
SCoT-6	CAACAATGGCTACCACGC	54.4	3	27	500–2200
SCoT-7	CAACAATGGCTACCACGG	53.9	5	45	300–2500
SCoT-8	CAACAATGGCTACCACGT	52.9	3	27	200–500
SCoT-9	CAACAATGGCTACCAGCA	52.9	7	63	300–1000
SCoT-10	CAACAATGGCTACCAGCC	53.9	4	36	400–1500
SCoT-11	AAGCAATGGCTACCACCA	54.4	7	63	300–2000
SCoT-12	ACGACATGGCGACCAACG	58.4	7	63	200–2500
SCoT-13	ACGACATGGCGACCATCG	58.0	4	36	300–1500
SCoT-14	ACGACATGGCGACCACGC	61.3	5	45	300–1500
SCoT-15	ACGACATGGCGACCGCGA	62.6	4	36	400–1000

Statistical analysis

The shoot multiplication and root induction experiments were conducted in triplicate, each replicate consisting of a minimum of 30 explants. The experiments were arranged in a completely randomized design to ensure unbiased results. The response percentage was determined for each of the three replicates individually, and then the mean of these values was calculated. Statistical analysis was carried out using one-way analysis of variance (ANOVA) in SPSS software (IBM® SPSS, version 21.0, Chicago, Illinois). Subsequently, Tukey’s test was employed at a significance level of P ≤ 0.05 within the same software to discern significant differences between means. Tukey’s test was chosen due to its ability to generate confidence intervals, accommodate the number of comparisons made, and facilitate the comparison of multiple pairs of variables to identify correlations. Prior to ANOVA, Arcsin transformation was applied to the percentage data to stabilize variances. The results were then presented in a table, with the transformed data converted back to the original scale for clarity.

Results and discussion

Extraction of plant material

Plant secondary metabolites are the treasure trove of modern medicine. Plants contain diverse compounds with significant biological roles, mainly as chemical messengers and defensive compounds (Alamgir and Alamgir 2017; Aguirre-Becerra et al. 2021). There are compounds found in plant extracts, such as phenolics, flavonoids, terpenoids, phenolic acids, tannins, stilbenes, and lignans (Oluwole et al. 2022). Isolation of those compounds from the respective plants is also a challenging task. Different researchers have developed different techniques to isolate these compounds in a substantial amount. Soxhlet-derived plant secondary metabolite extraction utilizing different solvents is an efficient method of extraction, as elaborated by many researchers (Akolade et al. 2019; Arenaz 2021). Essential oil extraction from plants has been done through the hydrodistillation method and is one of the most popular ways of extraction (Mollaei et al. 2019; Drinić et al. 2020; Katekar et al. 2022).

Soxhlet extraction

The polarity of the solvent is the prime criterion for separating the compounds from the plant materials. Among different solvents (polar, mid-polar, non-polar) used for the extraction of curcumin from plant materials, methanol has been found to be the best in terms of the yield of the extract. Secondary metabolites have been found to extract more with methanol than other solvents previously (Zhang et al. 2020; Ng et al. 2020). Hence, methanol was used for the soxhlet extraction of the dried rhizome of the plant for 12 h (Chakraborty et al. 2022b, a). The resulting content of extracted material from accession 06 is 29.66% is the highest (v/v) following, 21.06%, 20.00%, 22.14%, 20.53%, 21.87%, 23.65%, 21.03%, 20.54%, 19.87%, 19.14% and 24.08% from accession 01–05 and 07–12, respectively. Accession 06 was found to have the most significant amount of secondary metabolite-producing plant compared to other accessions.

Clevenger extraction

The primary method for extracting essential oil from plant material is hydrodistillation. This study used 300 g of fresh rhizomes for oil extraction, with an extraction duration of 8 h. It was observed that prolonging the extraction beyond 8 h in the Clevenger apparatus did not result in a higher overall yield. Conversely, shorter extraction times yielded lower quantities of oil. Consequently, all subsequent essential oil extractions from different accessions were also conducted for 8 h. The highest recorded yield of essential oil (0.65% v/w, based on the fresh weight of rhizomes) was from C. caesia (Accession 06), surpassing the yields from accessions 01–05 and 07–12, indicating that Accession 06 had the highest production of essential oil among all evaluated accessions.

HPTLC fingerprinting

As discussed earlier, curcumin is the most significant bioactive phytocompound used for different purposes. C. caesia is a plant species under the genus Curcuma enriched with curcumin. Previous curcumin extraction and quantification literature are mainly based on C. longa (Chanda and Ramachandra 2019; Sontsa-Donhoung et al. 2022; Singh et al. 2022). No such proper quantification and extraction have been established yet on C. caesia. The present study mainly focuses on quantifying curcumin from this plant to provide an alternative resource. Eco-geography plays a significant role in plant's secondary metabolite production (Thakur et al. 2020; Verma et al. 2021). Contents of different pharma compounds from a single plant could be different when the plant is from different origins (Tungmunnithum et al. 2018). Screening and identifying the best chemotype is the primary criterion for production at the industrial level. In the present study, we have collected plant samples from different agro-geological regions and estimated their curcumin content through HPTLC fingerprinting (Fig. 1). According to the result, 0.228 mg/gm of curcumin was found from accession 06 followed by 0.209, 0.194, 0.217, 0.202, 0.206, 0.210, 0.202, 0.198, 0.191, 0.188, and 0.212 for accession 01–05 and 07–12 with an Rf value of 0.38. The result is very conclusive on the basis of curcumin accumulation in the plant. Therefore, it can be used as an alternative resource for curcumin in natural habitats.

Fig. 1.

A HPTLC chromatogram of twelve accessions (01–12) and standard curcumin (13–15). B The 3D spectrum of curcumin with Rf values. C The absorbance of curcumin from samples and standards

Identification of pathogens and study of antibiotic resistance

The VITEK 2 COMPACT SYSTEM BIOMERIUEX machine-based identification of pathogens was made in the present study. Initially, the pathogen was identified based on the following biochemical assays: oxidase activity, catalase activity, Indole test, Methyl red, Citrate test, Voges-Proskauer test, and hydrogen sulfide production. Antibiotic sensitivity tests with 10 commercially available antibiotics showed that all of the pathogenic isolates of respiratory and urinary tract infections were resistant to multiple antibiotics (Table 3).

Table 3.

Antimicrobial resistance of eight pathogenic bacteria against 15 well-known antibiotics

Pathogen	Bacterial strain		Antibiotics
			Ciprofloxacin	Amoxicillin	Erythromycin	Aztreonam	Teicoplanin	Clindamycin	Vancomycin	Rifampicin	Tetracycline	Ertapenum	Ampicillin	Tiacarcillin	Piperacillin	Cefalotin	Cefoxitin
	Strain type	Strain name
Isolate no. 12467	RTI	Escherichia coli	R	S	S	R	R	R	R	S	R	R	R	S	I	−	R
Isolate no. 12498	RTI	Escherichia coli	R	S	R	R	R	I	R	I	R	R	R	−	S	R	R
Isolate no. 13443	RTI	Klebsiella pneumoniae	S	S	S	R	R	R	R	I	R	R	I	R	S	R	R
Isolate no. 13321	RTI	Klebsiella pneumoniae	R	S	R	I	R	R	R	S	R	R	I	I	R	R	I
Isolate no. 34256	UTI	Escherichia coli	I	I	R	−	S	R	R	I	R	R	R	I	I	−	S
Isolate no. 34521	UTI	Pseudomonas aeruginosa	I	R	R	S	R	R	S	R	R	R	I	R	R	S	S
Isolate no. 42640	UTI	Staphylococcus saprophyticus	S	R	R	R	R	−	S	R	R	−	R	R	S	R	S
Isolate no. 42238	UTI	Klebsiella pneumoniae	S	I	R	I	R	R	I	R	R	S	R	I	R	R	R

R Resistant, S Sensitive, I Intermediate, ‘ − ’ Not tested, UTI Urinary tract infection, RTI Respiratory tract infection

Antimicrobial activity study

Different researchers have repeatedly reported on the antimicrobial activity of different secondary plant metabolites and essential oils (Mangalagiri et al. 2021; Chebbac et al. 2021; Chakraborty et al. 2022a-b). Essential oil from the plants of the genus Curcuma, other than this species, has been reported previously for its antimicrobial activity (Mishra et al. 2018; Jena et al. 2020; Sam et al. 2020). Only a limited number of reports currently exist that investigate the antimicrobial effectiveness of Curcuma caesia against specific laboratory-grade non-pathogenic microbial strains (Pandey and Gupta 2014; Kaur et al. 2018; Paw et al. 2020a, b; Yadav and Kaliyaperumal 2021). While previous reports indicate the effectiveness of Curcuma caesia in combating bacterial strains, there is currently no documented evidence of its efficacy against pathogenic multi-drug resistant bacterial strains to the best of our knowledge. In this present study, we have examined the antimicrobial activity of plant crude extract and essential oil against previously described human pathogens that cause deadly human diseases. In cases where conventional antibiotics proved ineffective, both the test extract and essential oil demonstrated notable antimicrobial efficacy against these challenging pathogens. The evaluation of antimicrobial activity encompassed diverse methods, including the agar cup technique and determination of minimum inhibitory and cidal concentrations. Remarkably, this study represents a pioneering effort against multidrug-resistant pathogenic strains, an area previously unexplored. It firmly substantiates the documented antimicrobial potential of this plant's essential oil, aligning with prior research findings. Furthermore, the crude plant extract from C. caesia exhibited substantial antimicrobial activity against both non-pathogenic and pathogenic strains, corroborating previous observations regarding related species. To our knowledge, such compelling antimicrobial activity had not been reported previously for C. caesia.

Agar cup assay

Agar cup assay was performed in the Muller Hinton agar medium as described in the method section following punching the plate. The concentration of the crude extract was tested at a concentration of 50 mg/cup, while essential oil was used at a concentration of 3.0 μl/cup. Maximum inhibition zones were found against the ‘Isolate no. 13321’ (Klebsiella pneumoniae) among the RTI pathogens by crude plant extract and essential oil. The zones of inhibition were 18.00 ± 0.08 mm and 17.70 ± 0.14 mm for essential oil and crude extract, respectively. The lowest inhibition was found against Isolate no. 12467, with zones of inhibition of 12.50 ± 0.21 mm and 11.03 ± 0.04 mm, respectively. In the case of UTI pathogens, the highest inhibition was recorded against Isolate no. 42238, with zones of inhibition of 16.53 ± 0.04 mm and 15.66 ± 0.04 mm, respectively. The minimum inhibition against UTI pathogen was recorded against Isolate no. 34521, with zones of inhibition of 8.46 ± 0.12 mm and 7.33 ± 0.16 mm, respectively, for essential oil and crude extract. Curcumin, the primary active metabolite of this plant, was also examined against the pathogen. The result was very promising but not unexpected (Table 4) on the basis of the results obtained by previous researchers (Zorofchian et al. 2014). Hence, the results are very encouraging that the secondary metabolites and essential oil extracted from this plant also effectively kill human pathogens.

Table 4.

Antimicrobial activity of essential oil, plant crude extract of Curcuma caesia and curcumin

Concentration			RTI strains (Zone of inhibition in mm)				UTI strains (Zone of inhibition in mm)
			Isolate no. 12467	Isolate no. 12498	Isolate no. 13443	Isolate no. 13321	Isolate no. 34256	Isolate no. 34521	Isolate no. 42640	Isolate no. 42238
Essential oil (µl/cup)	3.0	Accession 01	8.70 ± 0.35	10.76 ± 0.28	13.93 ± 0.20	15.23 ± 0.09	8.03 ± 0.04	7.66 ± 0.16	12.30 ± 0.16	14.33 ± 0.20
	3.0	Accession 02	8.03 ± 0.09	9.13 ± 0.12	12.53 ± 0.04	13.83 ± 0.04	7.90 ± 0.08	7.13 ± 0.09	11.93 ± 0.16	13.06 ± 0.09
	3.0	Accession 03	10.56 ± 0.09	13.26 ± 0.24	15.20 ± 0.16	17.56 ± 0.26	10.30 ± 0.08	8.13 ± 0.04	12.73 ± 0.04	16.03 ± 0.04
	3.0	Accession 04	11.20 ± 0.16	12.93 ± 0.12	14.46 ± 0.09	17.03 ± 0.04	10.10 ± 0.14	8.06 ± 0.09	12.20 ± 0.21	15.33 ± 0.04
	3.0	Accession 05	11.93 ± 0.09	12.50 ± 0.08	15.30 ± 0.21	17.26 ± 0.09	10.30 ± 0.21	8.33 ± 0.04	12.23 ± 0.04	16.16 ± 0.12
	3.0	Accession 06	12.50 ± 0.21	13.90 ± 0.08	15.86 ± 0.12	18.00 ± 0.08	10.66 ± 0.04	8.46 ± 0.12	12.96 ± 0.04	16.53 ± 0.04
	3.0	Accession 07	9.96 ± 0.12	9.70 ± 0.08	13.77 ± 0.05	16.70 ± 0.08	8.47 ± 0.05	7.67 ± 0.09	10.23 ± 0.12	14.77 ± 0.12
	3.0	Accession 08	9.43 ± 0.17	12.47 ± 0.05	14.20 ± 0.16	16.90 ± 0.08	9.73 ± 0.17	7.03 ± 0.12	11.33 ± 0.12	14.97 ± 0.09
	3.0	Accession 09	8.96 ± 0.12	9.23 ± 0.09	12.97 ± 0.17	15.97 ± 0.05	9.90 ± 0.08	8.27 ± 0.12	10.70 ± 0.08	13.67 ± 0.12
	3.0	Accession 10	8.53 ± 0.12	9.63 ± 0.05	14.80 ± 0.09	16.60 ± 0.08	8.83 ± 0.05	6.77 ± 0.05	11.10 ± 0.14	13.07 ± 0.09
	3.0	Accession 11	8.73 ± 0.09	9.30 ± 0.08	12.27 ± 0.05	14.57 ± 0.12	7.97 ± 0.05	6.13 ± 0.17	10.50 ± 0.08	11.83 ± 0.12
	3.0	Accession 12	11.43 ± 0.05	10.23 ± 0.17	15.23 ± 0.17	17.60 ± 0.16	10.13 ± 0.12	8.13 ± 0.09	12.67 ± 0.12	15.47 ± 0.12
Crude extract (µg/cup)	50	Accession 01	7.96 ± 0.04	10.06 ± 0.04	11.46 ± 0.12	13.93 ± 0.04	0	0	11.06 ± 0.09	12.50 ± 0.21
	50	Accession 02	8.13 ± 0.04	9.96 ± 0.16	11.43 ± 0.30	13.66 ± 0.04	6.66 ± 0.09	0	9.06 ± 0.09	12.73 ± 0.04
	50	Accession 03	9.86 ± 0.04	12.96 ± 0.04	14.26 ± 0.24	17.30 ± 0.08	0	6.96 ± 0.04	11.96 ± 0.16	14.90 ± 0.14
	50	Accession 04	10.1 ± 0.14	12.46 ± 0.16	13.83 ± 0.04	16.80 ± 0.08	0	0	11.66 ± 0.04	13.13 ± 0.09
	50	Accession 05	10.83 ± 0.12	13.03 ± 0.04	14.53 ± 0.04	17.16 ± 0.04	7.06 ± 0.04	7.16 ± 0.04	12.06 ± 0.04	14.10 ± 0.08
	50	Accession 06	11.03 ± 0.04	13.3 ± 0.08	14.90 ± 0.08	17.70 ± 0.14	7.33 ± 0.16	7.53 ± 0.16	12.53 ± 0.12	15.66 ± 0.04
	50	Accession 07	9.80 ± 0.22	11.93 ± 0.12	12.03 ± 0.17	14.63 ± 0.12	6.90 ± 0.08	7.17 ± 0.12	11.23 ± 0.17	15.57 ± 0.12
	50	Accession 08	8.33 ± 0.05	12.23 ± 0.12	12.77 ± 0.09	15.93 ± 0.12	6.47 ± 0.12	6.83 ± 0.05	11.73 ± 0.05	15.27 ± 0.12
	50	Accession 09	9.33 ± 0.12	11.70 ± 0.16	13.33 ± 0.05	16.17 ± 0.12	6.13 ± 0.17	7.30 ± 0.08	11.87 ± 0.12	14.17 ± 0.17
	50	Accession 10	7.97 ± 0.09	11.90 ± 0.08	13.37 ± 0.17	14.87 ± 0.05	7.13 ± 0.17	6.50 ± 0.14	10.17 ± 0.12	14.17 ± 0.12
	50	Accession 11	8.10 ± 0.14	10.80 ± 0.08	11.23 ± 0.12	13.53 ± 0.12	6.10 ± 0.14	6.13 ± 0.17	9.87 ± 0.12	12.07 ± 0.12
	50	Accession 12	11.17 ± 0.05	12.83 ± 0.12	14.53 ± 0.12	17.23 ± 0.09	9.83 ± 0.12	7.37 ± 0.12	12.33 ± 0.09	15.27 ± 0.05
Curcumin	0.1 mg/cup		6.13 ± 0.04	7.13 ± 0.04	8.23 ± 0.16	8.86 ± 0.09	0	0	6.06 ± 0.09	6.66 ± 0.04
DMSO	50 µl/cup		0	0	0	0	0	0	0	0

Each value represents the mean ± standard error. All the experiments performed in triplicate

Elite chemotype selection

Elite chemotype selection was done mainly on the basis of chemical constituents and the phytoactive compounds found in the plants (Dey et al. 2020; Das et al. 2022). Previous research has indeed explored elite chemotype selection in various plant groups. However, the unique approach employed in this study, particularly concerning C. caesia, sets it apart. Prior to our investigation, no similar efforts had been undertaken for this plant species. This study introduces a novel perspective by conducting elite chemotype selection based on the content of crude extract, essential oil, and curcumin in wild accessions gathered from diverse agro-climatic zones within West Bengal. The outcomes of our study revealed that Accession 06 outperformed the others in terms of essential oil quantity, as well as the presence of other constituents and antimicrobial activity. Notably, there was substantial variation in curcumin content among all the accessions, with Accession 06 yielding the highest amount of curcumin compared to its counterparts (as depicted in Fig. 2). Consequently, Accession 06 was unequivocally designated as the elite chemotype among all the accessions, signifying its exceptional potential for further exploration and utilization.

Fig. 2.

Content of curcumin in Curcuma caesia rhizomes from twelve different accessions (01–12) collected from the different agro-climatic zone

GC–MS of essential oil of the elite plant

Gas chromatography is used to identify volatile components present in the sample, and mass spectroscopy is used for their molecular characterization. There are only a few reports are present on the volatile oil composition of C. caesia. According to Pandey and Chowdhury (2003), the essential oil of C. caesia is composed of camphor, ar-turmerone, (Z)-β-ocimene, ar-curcumene, 1,8-cineole, β-elemene, borneol, bornyl acetate and γ-curcumene as major constituents. Another report suggests that eucalyptol, epicurzerenone, and camphor are the major constituents of the same plant-mediated essential oil (Paw et al. 2020a, b). Whare as, Mukunthan et al (2014) reported that tropolone was found as a major compound in the essential oil of C. caesia and ledol, β-elemenone and α-bulnesene, spathulenol was found as minor compounds. In the present study, the essential oil from the rhizomes of accession 06 (elite type) of C. caesia has been analysed to characterize the components. 13 major bioactive compounds have been obtained from the spectrum, further characterized according to their bioactivity as reported in the literature (Fig. 3). Among 13 compounds, the first nine compounds were monoterpenes, and the other nine were sesquiterpenes (Table 5). In the present study, we have mainly searched for antimicrobial compounds to utilize them against the bacteria pathogenic to humans. Most of the compounds detected in the spectrum had antimicrobic potency. The compounds like α-Pinene, Camphen, L-β-Pinene, D-Limonene, Linalool, Camphor, Isoborneol, Germacrene D, Epicurzerenone, Germacrone were previously reported for their antimicrobial effectivity (da Silva Rivas et al. 2012; Hachlafi et al. 2021; Gogoi et al. 2020; Özek et al. 2010; Shokova et al. 2016; Farhat et al. 2017; Mevy et al. 2007; Lai et al. 2004; Wu et al. 2017). Some compounds like Camphene, β-Myrcene, D-Limonene, and endo-borneol have been reported previously for their antioxidant activity (Hachlafi et al. 2021; Gogoi et al. 2020; Anandakumar et al. 2021; Xu et al. 2021; Wu et al. 2017). Germacrene B, the oil's bioactive compound, has anti-leishmanial activity, as Simionatto et al (2009) reported. Other than these compounds, some others were also found to be active in some cases as represented in Table 5. As described previously, all the compounds found in the EO are biologically active which further makes the plant bioactive in nature. The multifunctional role of the plant is due to the presence of diverse phytocompounds. Hence, the EO can be used in medicine for its multifunctional significance.

Fig. 3.

GC–MS chromatogram of essential oil from elite chemotype of C. caesia

Table 5.

Compounds detection and their activities from the essential of Curcuma caesia

Sl. no.	Compound name	Molecular formula	RT	Biological activity	References
1	α-Pinene	C10H16	9.07	Antibacterial and antifungal activities	da Silva Rivas et al. (2012)
2	Camphene	C10H16	9.80	Antibacterial, antifungal, anticancer, antioxidant, antiparasitic, antidiabetic, anti-inflammatory, and hypolipidemic activities	Hachlafi et al. (2021)
3	L-β-Pinene	C10H16	11.32	cytogenetic, gastroprotective, anxiolytic, cytoprotective, anticonvulsant, and neuroprotective effects	Salehi et al. (2019a, b)
4	β-Myrcene	C10H16	12.42	Antioxidant and antimicrobial activities	Gogoi et al. (2020)
5	D-Limonene	C10H16	14.2	antioxidant, antidiabetic, anticancer, anti-inflammatory, cardioprotective, gastroprotective, hepatoprotective, immune-modulatory, anti-fibrotic, anti-genotoxic activities	Anandakumar et al. (2021)
6	Linalool	C10H18O	18.24	antimicrobial, antifungal and antimalarial activities	Özek et al. (2010)
7	Camphor	C10H16O	20.23	Antimicrobial, antiviral, antitubercular, antifungal, antitumor, anticonvulsant and myorelaxant Activity	Shokova et al. (2016)
8	Isoborneol	C10H18O	20.93	Increases acetylcholinesterase activity, antimicrobial activities	Farhat et al. (2017)
9	endo-Borneol	C10H18O	21.43	antioxidant, antidiabetic and cholinesterase inhibitory activities	Xu et al. (2021)
10	Germacrene D	C15H24	35.42	Anti-bacterial and anti-fungal activities	Mevy et al. (2007)
11	Germacrene B	C15H24	38.65	anti-leishmanial activity	Simionatto et al. (2009)
12	Epicurzerenone	C15H18O2	40.63	Antimicrobial activities	Lai et al. (2004)
13	Germacrone	C15H22O	43.95	Anti-inflammatory, depressant, vasodilator, antibacterial, choleretic, antitussive, antitumor, antifeedant, antifungal and hepatoprotective activities	Wu et al. (2017)

In vitro conservation

In vitro shoot multiplication

Shoot multiplication of C. caesia was experimented with in the presence of different cytokinins like 6-Benzylaminopurine (BAP), Kinetin (KIN), and 6-(γ,γ-Dimethylallylamino)purine (2IP) individually and in the presence of their combination. MS medium without PGRs was used as control, which resulted in no multiplication after the final regeneration of the plant. Different concentrations (1.0, 2.0, 3.0, 4.0 mg/l) of each PGRs are used in the present study to conclude the best composition of PGRs for this respective plant's growth. In the presence of different concentrations of BAP, maximum shoot multiplication was observed in the presence of 3.0 mg/l concentration of BAP. In the presence of BAP (3.00 mg/l) maximum shoot 7.28 ± 0.18 per explant has resulted. In the presence of a low concentration of BAP, small number of shoot induction has resulted. Furthermore, when the concentration was increased to 4.00 mg/l, shoot multiplication also resulted in a decrease. Hence, it can be concluded that an increased concentration of BAP can lead to lower multiplication after a certain concentration. Another cytokinin kinetin is used to study the multiplication and resulted in a maximum multiplication of 6.06 ± 0.42 per explant at a concentration of 2 mg/l. Further increases in concentration resulted in a decrease in the multiplication of the plant. But in a combination of BAP and KIN, the most significant multiplication has resulted in 9.48 ± 0.18 shoots per explant maximum in the presence of 3.00 mg/l BAP with a combination of 1.00 mg/l KIN. Other two different cytokinins also tested for the growth and multiplication of the plant that is TDZ and 2IP. In the presence of TDZ, a maximum number of 5.23 ± 0.42 shoots per explant has resulted, which is significantly lower than KIN and BAP. A combination of BAP and TDZ (3.00 + 1.00, 3.00 + 2.00, and 3.00 + 3.00 mg/l) also experimented and as a result, 6.86 ± 0.13 shoots per explant resulted, which is significantly lower than the combination of BAP and KIN. In the presence of 2IP, 4.14 ± 0.12 shoots per explant were recorded at a concentration of 2 mg/l, which is quite lower than the other three PGRs. Therefore, the combination of BAP and KIN (3.00 + 1.00 mg/l) was selected for shoot multiplication media for further experiments. This particular combination resulted in 9.48 ± 0.08 shoots per explant after 60 days of introduction into the medium. Furthermore, auxins were combined with the cytokinins to evaluate maximum shoot and root regeneration per explant after final regeneration. NAA and IAA have been used to study maximum shoot multiplication. As a result, NAA was found to be more efficient than IAA. The presence of NAA with previously concluded cytokinin medium (3.00 + 1.00 + 0.5 mg/l BAP + KIN + NAA) resulted in 19.28 ± 0.37 shoots per explant which is significantly better than the composition of BAP + KIN + IAA (3.0 + 1.0 + 0.5 mg/l) (Table 6). According to Bharalee et al. (2005), MS medium supplemented with 4.0 mg/l BAP in combination with 1.5 mg/l NAA resulted from 3.50 shoots per explant which is quite lower in amount. Furthermore, some researchers accommodate experiments on the micropropagation of this respective plant and found 5.70 shoots per explant when introduced in the MS medium containing 0.9 mg/l NAA; 1.2 mg/l with 1.9 mg/l BAP, respectively (Singh et al. 2015). However, according to Ghosh et al. (2013), when the plant is implanted on MS medium supplemented with 1.5 mg/l BAP and 1.0 mg/l of NAA, maximum shoots of 15.40 were found per explant, which is similar to our result. Previous research findings highlight a significant relationship between the concentrations of BAP and shoot regeneration. Notably, a heavy increase in BAP concentration led to a drastic decrease in shoot regeneration. Conversely, when PGRs were supplied at a lower, optimized concentration, shoot regeneration rates improved significantly. Therefore, it can be concluded that achieving higher micropropagation rates is feasible by utilizing a growth medium containing 3.00 mg/l BAP, 1.0 mg/l of NAA, and 0.5 mg/l NAA under optimized conditions. Subsequently, the explants were successfully acclimatized in a controlled environment and later transferred to the field for maturation, with a remarkable 100% survival rate (as depicted in Fig. 4).

Table 6.

Effect of different cytokinins, auxin and their combination on shoot multiplication of C. caesia

MS basal media with 0.3% sucrose						Response (%)	Regeneration of explant
Cytokinin (mg/L)				Auxin(mg/L)			Shoot per plant	Shoot length (cm)
BAP	KIN	TDZ	2IP	NAA	IAA
0	0	0	0	0	0	88.39 ± 0.35f	2.00 ± 0.46p	3.37 ± 0.21no
1						70.63 ± 0.29k	6.01 ± 0.39hij	4.84 ± 0.35ijk
2						90.04 ± 0.37e	7.16 ± 0.18fgh	6.36 ± 0.49gh
3						73.56 ± 0.31j	7.28 ± 0.04fgh	6.72 ± 0.12 g
4						62.40 ± 0.39m	4.56 ± 0.15klmn	4.21 ± 0.21klmn
	1					57.99 ± 0.33n	3.70 ± 0.34no	4.23 ± 0.08klmn
	2					66.10 ± 0.64l	6.06 ± 0.42hij	4.14 ± 0.12klmn
	3					78.57 ± 0.61i	5.14 ± 0.46jkl	3.85 ± 0.16jkl
	4					63.25 ± 0.37 m	4.36 ± 0.19klmno	4.38 ± 0.36klm
		1				52.22 ± 0.57o	4.63 ± 0.27klmn	4.06 ± 0.09klmn
		2				48.25 ± 0.51p	5.23 ± 0.42jkl	4.49 ± 0.37jkl
		3				40.70 ± 0.44r	4.04 ± 0.48mno	3.83 ± 0.32lmno
		4				29.57 ± 0.83t	3.76 ± 0.14no	3.48 ± 0.13mno
			1			38.39 ± 0.85 s	3.69 ± 0.29no	3.24 ± 0.13mno
			2			42.25 ± 0.27q	4.14 ± 0.12lmno	4.01 ± 0.62klmno
			3			30.41 ± 0.71t	3.39 ± 0.26o	4.38 ± 0.31klm
			4			25.45 ± 0.33u	3.55 ± 0.23no	5.37 ± 0.27ij
3	1					94.42 ± 0.62b	9.48 ± 0.18d	14.10 ± 0.46a
3	2					91.17 ± 0.34cde	8.19 ± 0.32ef	12.08 ± 1.11b
3	3					88.39 ± 0.35f	7.33 ± 0.28 fg	11.20 ± 0.13b
3		1				83.18 ± 0.22 g	6.86 ± 0.13gh	10.01 ± 0.48c
3		2				81.37 ± 0.14 h	6.45 ± 0.27ghi	9.51 ± 0.14c
3		3				79.16 ± 0.26i	5.40 ± 0.21ijk	8.32 ± 0.17de
3	1			0.5		98.15 ± 0.25a	19.28 ± 0.37a	9.21 ± 0.11 cd
3	1			1		94.14 ± 0.13b	16.03 ± 0.57b	7.64 ± 0.59ef
3	1			1.5		91.35 ± 0.42 cd	9.28 ± 0.26de	7.15 ± 0.12 fg
3	1				0.5	90.45 ± 0.58de	11.93 ± 0.94c	8.32 ± 0.08de
3	1				1	91.84 ± 0.33c	12.27 ± 0.43c	7.49 ± 0.28ef
3	1				1.5	91.24 ± 0.26cde	9.30 ± 0.13de	7.17 ± 0.12 fg

Each value represents the mean ± standard error, n = 30 (3 sets, 10 samples in each set). The mean followed by the same letters in each column is not significantly different at P < 0.05 according to Tukey’s tests

BAP- 6 Benzylaminopurine, KIN Kinetin, TDZ Thidiazuron, 2IP isopentenyl adenine, NAA α-naphthalene acetic acid, IAA Indole-3-acetic acid

Fig. 4.

A Initial inoculation of sprouts from the rhizome of Curcuma caesia in MS medium with PGRs. B Shoot regeneration after 12 days of inoculation. C Micropropagated plant after 25 days of inoculation. D Well-developed plants after 35 days of inoculation. E The flowering of C. caesia in the experimental garden. F Uprooted flowering plant with the entire rhizome. G A mature plant of C. caesia in the experimental garden. H Rhizome (colour development) from a mature plant

In vitro rooting

Root induction and growth of C. caesia have been experimented on MS medium supplemented with auxins (IBA, IAA and NAA). Different concentrations of all three PGRs (0.5, 1.0, and 1.5 mg/l) were tested for maximum root induction in the present study. A 100% response has resulted in the presence of 0.5 mg/l NAA (Table 7). Whereas 83.54% and 89.56% of responses were found in the presence of IBA and IAA, respectively. In the context of root induction, our study reveals that NAA (Naphthalene acetic acid) outperforms IAA (Indole-3-acetic acid) and IBA (Indole-3-butyric acid) when supplemented in the MS (Murashige and Skoog) medium. While the response percentages are similar for IBA and IAA, the number of roots per shoot regeneration is significantly higher when IAA is used. In the presence of NAA, an impressive 24.01 ± 0.24 roots were regenerated, which is notably higher than the 12.39 ± 0.28 roots observed in the presence of IAA and the 8.47 ± 0.31 roots in the presence of IBA. Furthermore, the length of the roots also varied significantly depending on the PGR used. The highest root length of 7.76 ± 0.59 cm was achieved with half-strength MS medium supplemented with 0.5 mg/l NAA, a length significantly greater than that observed with IAA or IBA. The concentration of PGRs in the medium increased, and there was a decrease in both root number and length, as evidenced in our study. However, it's crucial to highlight that a higher number of roots corresponded to an increased length. This study provides valuable insights for selecting the optimal medium composition for effective root induction, which is essential in plant tissue culture and propagation.

Table 7.

In vitro rooting of C. caesia in the presence of different auxins

Basal medium	PGR (mg/L)			Response	Root number	Root length
	IBA	IAA	NAA
MS	0	0	0	69.02 ± 0.52i	4.16 ± 0.14j	2.73 ± 0.09i
	1			100.00 ± 00a	24.01 ± 0.24a	7.76 ± 0.59a
	2			99.58 ± 0.30a	19.46 ± 0.11b	6.39 ± 0.37b
	3			98.14 ± 0.21b	17.87 ± 1.13c	5.47 ± 0.13d
		1		83.54 ± 0.56e	8.47 ± 0.31f	4.79 ± 0.11e
		2		79.36 ± 0.62f	7.28 ± 0.22 g	3.96 ± 0.08 g
		3		72.68 ± 0.49 h	6.19 ± 0.12hi	3.16 ± 0.39 h
			1	89.56 ± 0.37c	12.39 ± 0.28d	5.91 ± 0.23c
			2	85.23 ± 0.16d	9.85 ± 0.59e	4.92 ± 0.29e
			3	74.05 ± 0.65 g	6.99 ± 0.11gh	4.48 ± 0.27f

Each value represents the mean ± standard error, n = 30 (3 sets, 10 samples in each set). The mean followed by the same letters in each column is not significantly different at P < 0.05 according to Tukey’s tests

IBA indole-3-butyric acid, NAA α-naphthalene acetic acid, IAA Indole-3-acetic acid

Genetic stability study through SCoT

Genetically uniform or true-to-type plant production is the primary goal of plant tissue culture. However, several causes, like chromosomal aberration, gene mutation, gene alteration, and gene amplification, can cause different levels of plant genotypic and phenotypic variations (Subrahmanyeswari et al. 2022; Ghosh et al. 2021). To study the monomorphic nature of plants, we have performed SCoT primers-mediated amplification. SCoT primers were used in duplicate in this study. Some of the primers failed to reproduce, which may be due to several factors like primer length and annealing temperature. Primers reproduced successfully (SCoT – 01 and SCoT – 11) were used to study the monomorphic nature between wild in vivo plants and tissue culture-generated ex vitro plants. Fifteen SCoT primers amplified 72 loci ranging between 200–2500 bp, and a total of 648 bands were scored from one mother plant and eight randomly selected regenerated plants (Fig. 5). All 648 bands indicated the monomorphic nature of the plants, and no variation has been seen. Hence, the regenerated plants are true-to-type and clonally uniform by nature.

Fig. 5.

Genetic fidelity of regenerated C. caesia (1–8) and their mother plant (9) based on SCoT primers A SCoT 1, B SCoT 11 with a ladder of 100–3000 bp (M)

Conclusion

The study has successfully developed a straightforward, validated, and optimized protocol for the extraction of essential oil and crude extracts from C. caesia. This process utilized hydrodistillation and solvent-mediated extraction techniques and was further validated through the quantification of curcumin using HPTLC fingerprinting. Significantly, both the essential oil and crude extract exhibited promising antimicrobial activity against multidrug-resistant urinary tract infection (UTI) and respiratory tract infection (RTI) pathogens. This finding suggests their potential as future medicinal alternatives to chemical drugs. Moreover, the concept of elite chemotype selection holds immense promise for agriculture, as it can lead to the cultivation of high-quality plants capable of consistently producing abundant phyto-constituents year-round. The identified elite chemotype could also facilitate the commercialization of this compound, which boasts antimicrobial, anti-inflammatory, anti-diabetic, and neuroprotective properties, particularly curcumin. Given that these cell lines are propagated in vitro under controlled environmental conditions, essential oil and curcumin content variations are highly unlikely. Furthermore, by establishing high-yielding chemotypes through reproducible tissue culture techniques, the uncontrolled collection of C. caesia from the wild could be reduced, contributing to the conservation of this valuable plant species.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.