Enhanced antioxidant activity in Curcuma caesia Roxb. microrhizomes treated with silver nanoparticles

Sonam Patel¹, Afreen Anjum¹, Veenu Joshi², Afaque Quraishi^1*

¹School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, C.G., India.

²Center for Basic Sciences, Pt. Ravishankar Shukla University, Raipur, C.G., India.

 Abstract

Curcuma caesia Roxb. is a highly valuable, endangered herb of therapeutic importance that resides in their rhizomes. In the present investigation, the effect of ½ strength liquid Murashige and Skoog (MS) medium supplemented with 1 mg/l Indole-3-butyric acid (IBA) and different sucrose concentrations (1.5%, 3%, 6%, 9%, or 12%) was studied on microrhizomes induction of C. caesia. The shoot length, root length and microrhizomes dry weight of C. caesia decreased significantly at 6% sucrose and above. When compared to the control (1.5% sucrose), the current water content significantly decreased at 6% sucrose. The optimum concentration for in vitro microrhizomes induction in C. caesia was 6% sucrose. Therefore for further experiments, the 6% sucrose was used. We also studied the effect of silver nanoparticles (AgNP) on microrhizome induction and antioxidant activity in C. caesia cultures. Field-grown C. caesia rhizomes extract was used in the green synthesis of AgNP. The synthesized AgNP was further characterized through scanning electron microscopy and X-ray diffraction. The AgNP, ranging from 0, 0.025, 0.05, 0.075 or 0.1 mg/l was supplemented in ½ strength liquid MS medium with 6% sucrose & 1 mg/l IBA. The MS medium with 0.05 mg/l AgNP found with significant morphological changes in C. caesia cultures (root number, root length and microrhizomes fresh weight). For the total phenolic and total terpenoids content estimation as well as for antioxidant activity analysis, the extracts of un-treated cultures (6% sucrose + 1 mg/l IBA, without AgNP), AgNP treated cultures (6% sucrose + 1 mg/l IBA with 0.025 & 0.05 mg/l AgNP) was used. The 0.025 and 0.05 mg/l AgNP enhanced the phenolic and terpenoid content in the cultures compared to the field-grown mother plant. The antioxidant activity of the cultures treated with AgNP also increased compared to un-treated cultures and field-grown mother plant. The Gas Chromatography-Mass Spectrometry (GC-MS) analysis revealed that the extract treated with 0.05 mg/l AgNP had increased production of monoterpene (camphor) and sesquiterpenes (β-elemenone & curcumenone). These increased terpenes could be responsible for the enhanced antioxidant activity of C. caesia cultures.

 Keywords: Antioxidant activity, Current water content, GC-MS, Indole-3-butyric acid, XRD.

Introduction

Curcuma caesia Roxb., also known as 'Kali Haldi' or 'Black Turmeric,' is an endangered herb belonging to the family Zingiberaceae. It is native to Northeast India and is also distributed in the Himalayan region, Northern Australia, and the tropical and subtropical regions of Asia, especially in Thailand, Indonesia, and Malaysia (Karmakar et al., 2011). The fresh and dried rhizomes of this herb are used for the treatment of various diseases, including asthma, anthelmintic, allergies, aphrodisiac, postpartum uterine abnormalities, bronchitis, cancer, splenomegaly, epilepsy, fertility, gonorrheal discharges, impotence, toothache, leukoderma, leprosy, piles, tumours, menstrual disorders, rubefacient, smooth muscle relaxant activity, vomiting, and wounds (Ravindran et al., 2007). C. caesia is characterized by tuberous bluish-black rhizomes with a camphoraceous aroma and medicinal properties (Donipati and Sreeramulu, 2015). C. caesia exhibits antimicrobial, anti-inflammatory, and antioxidant properties (Mukunthan et al., 2018; Borah et al., 2019; Benya et al., 2023). C. caesia contains various phytoconstituents, including curcuminoids, flavonoids, essential amino acids, and high alkaloid content (Baghel et al., 2013). These secondary metabolites are responsible for the pharmaceutical properties, fragrances, and flavouring associated with C. caesia.

Sugars are crucial as plant regulators, facilitating numerous physiological processes such as photosynthesis, seed germination, flowering, senescence, and more, especially during abiotic stresses (Sami et al., 2016). The external application of sugars in low concentrations regulates seed germination, flowering, and photosynthesis, while also delaying senescence under various stressful environmental conditions. Above a particular concentration, sucrose induces osmotic stress in the in vitro condition (Mehta et al., 2000). Previous research has demonstrated the beneficial impact of sucrose on the formation of storage organs like corms and tubers (Nayak and Naik, 2006).

In recent years, there has been significant interest in the green synthesis of silver nanoparticles (AgNPs) using plants. This method has gained attention due to its cost-effectiveness, non-toxic technology, biocompatibility, and environmentally friendly nature. Studies have shown that                      AgNPs synthesized using plant extracts are more stable than those synthesized by other organisms, such as fungi and bacteria (Mamidi and Polaki, 2019). Researchers have also been investigating the positive effects of AgNPs on plant growth and development. The present investigation focuses on the impact of sucrose and indole-3-butyric acid on microrhizome induction in C. caesia cultures. The effect of the nanoparticles on microrhizome induction in C. caesia cultures was also examined. The total phenolic and terpenoid content and the antioxidant activity in C. caesia microrhizome extracts after nanoparticles treatment was also investigated.

 Materials and Methods

The present work was conducted at the School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh (India). Curcuma caesia in-vitro cultures were used for the experimental work, which included the following procedure-

 Effect of sucrose on microrhizomes induction of C. caesia

To study the effect of sucrose concentration in the growth of microrhizomes of C. caesia, in vitro  experimental work was carried out. ½ strength liquid Murashige and Skoog (1962) (MS) medium  supplemented with 1 mg/l  IBA (Indole-3-butyric acid) was used  along with different concentrations of sucrose ranging from 1.5%, 3%, 6%, 9% or 12%. The selection of 1 mg/l IBA was based on the findings of Anjum et al. (2022). Culture tubes (25 x 150 mm) containing 15 ml liquid MS medium were plugged with cotton and sterilized at 121°C for 20 min in an autoclave. After autoclaving, the medium was stored in a clean, dust-free chamber  for a couple of days to check for contamination before use. Then, prior to inoculation, the medium and all other laboratory wares were disinfected by exposure to UV light inside the laminar air flow for 30-35 min. Inoculation was done after two days of medium preparation and six-month-old   cultures on the standard medium (Anjum et al., 2022)  was used for inoculation. The cultures were incubated for 30 days under white fluorescent light for 16 h dark and a photoperiod        of 8 h. Observation of the established cultures was taken after one month. From the observation, the best range of sucrose (6%) was used for further experiments.

 Green synthesis of silver nanoparticles ((AgNP)

Extract preparation

The field-grown rhizomes of C. caesia were dried in a hot air oven at 45^oC to remove the moisture                  completely. The rhizomes were powdered using a mixer grinder. For extract preparation, 5 g of C. caesia rhizome powder was added to 100 ml methanol in a 250 ml glass beaker and kept in a magnetic stirrer for 8 h at room temperature. Then the crude methanol extract was filtered through      Whatman filter paper (20–24 µm) paper, and the filtered extract was further used for AgNP preparation.

Synthesis of AgNP

To the 2 ml of C. caesia rhizome extract, 18 ml of 3 M silver nitrate (AgNO₃) solution was mixed in a 250 ml glass beaker, fully covered with aluminium foil to avoid any photoreactions. It was then thoroughly mixed using a magnetic stirrer for 2 h. The solution was checked every 30 min to monitor the colour change (from colourless to brown), which revealed the reduction of Ag⁺ into Ag⁰ nanoparticles. The synthesized nanoparticles were first characterized through a UV-visible spectrophotometer. After taking the spectrum, the solution was poured into a petri dish and placed in hot air over. After the solution was dried, the leftover precipitate was scratched to obtain a fine powder. The powder obtained was AgNP, which was used for further experiments.

The further characterization of synthesized nanoparticles was done through scanning electron microscope (SEM), X-ray diffraction (XRD).

Effect of AgNP on microrhizomes induction of C. caesia

We studied the effect of AgNP in the microrhizome induction of C. caesia. For this, ½ strength liquid MS medium was prepared supplemented with 6% sucrose and 1 mg/l IBA along with different ranges of concentration of AgNP: 0, 0.025, 0.05, 0.075 or 0.1 mg/l). The pH of the medium was adjusted to 5.8 with the help of a pH meter. The medium was poured into clean culture tubes and autoclaved with the rest of the materials required during the inoculation. The two-month-old microrhizome cultures were used for inoculation. After a month, the morphology of the cultures and fresh and dry biomass of the microrhizome was observed.

 Water bath-assisted extraction

After the observation, the dried microrhizomes were powdered and extracted using a water bath sonicator for 1 h at a temperature between 30-35^oC. The solid-to-liquid ratio was 1:25 (1 g dried powder with 25 ml methanol). The obtained extract was centrifuged at 10,000 rpm for 15 min, and the supernatant was collected and used for further experiments.

 Total phenolic content

The total phenolic content of the extract (field grow mother plant, 6% sucrose + 1 mg/l IBA + 0 AgNP, 6% sucrose + 1 mg/l IBA + 0.025 mg/l AgNP, 6% sucrose + 1 mg/l IBA + 0.05 mg/l AgNP) was determined quantitatively by an assay that utilizes the Folin Ciocalteu reagent (Ainsworth and Gillespie, 2007). 100 µl of the extract was mixed with 200 µl 10% Folin Ciocalteu reagent and vortexed thoroughly. Then, 800 ml of 700 mM sodium carbonate was added and incubated at room temperature for 2 h. Then, absorbance was taken at 765 nm using Micro Scan (Electronics Corporation of India Limited and expressed in mg gallic acid equivalents (GAE)/g dry weight (DW).

 Total terpenoids content

Total terpenoid content of the extract (field grow mother plant, 6% sucrose + 1 mg/l IBA + 0 AgNP, 6% sucrose + 1 mg/l IBA + 0.025 mg/l AgNP, 6% sucrose + 1 mg/l IBA + 0.05 mg/l AgNP) was estimated using method of Ghorai et al. (2012). 200 µl extract mixed with 1.5 ml chloroform and vortex and rested for 3 min. After that, 100 µl H2SO4 was added and again incubated for 2 h in the dark at room temperature. After incubation, reddish-brown precipitation formed. Then all supernatant was decanted carefully, and 1.5 ml of 95% methanol was added to the precipitate and vortex thoroughly till the precipitate dissolved completely. Absorbance was recorded at 538 nm using Micro Scan (Electronics Corporation of India Limited) and expressed in mg abscisic acid equivalents (AAE)/g DW.

 In vitro antioxidant activity

DPPH radical scavenging activity

For screening the antioxidant activity of the extract (field grow mother plant, 6% sucrose + 1 mg/l IBA + 0 AgNP, 6% sucrose + 1 mg/l IBA + 0.025 mg/l AgNP, 6% sucrose + 1 mg/l IBA + 0.05 mg/l AgNP), the protocol of DPPH radical scavenging activity given by Blois (1958) was followed. To 1 ml of extract (100 µg/ml), 1 ml of DPPH (0.1 mM) was added, and the tubes were incubated in the dark for 30 min. Absorbance was recorded at 517 nm using Micro Scan (Electronics Corporation of India Limited). Ascorbic acid was used as standard.

Ferric-reducing antioxidant power assay (FRAP)

The reducing power was determined by following the procedure given by Oyaizu (1986). 2.5 ml  of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of 1% potassium ferricyanide were added to of 2.5 ml extract (100 µg/ml). The mixture was placed in the water bath for 20 min at 50^oC and was cooled immediately. After cooling, 2.5 ml of 10% trichloroacetic acid was added, then samples were centrifuged at 3,000 rpm for 10 min. 5 ml supernatant was then mixed with 5ml distilled water, followed by the addition of 1 ml of 0.1% ferric chloride. The absorbance was taken at 700 nm after 10 min using Micro Scan (Electronics Corporation of India Limited). Butylated hydroxytoluene was used as standard.

 Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

GC-MS analysis was performed using extracts of un-treated cultures and treated cultures with 0.05 mg/l AgNP. GC-MS analysis was done using hired facilities of Sophisticated Analytical Instruments Facility, Indian Institute of Technology-Madras, Chennai, Tamil Nadu.

Statistical analysis

For the microrhizomes induction, each sucrose range had ten replicates repeated three times. Each range of AgNP had ten replicates repeated three times. Antioxidant tests, total phenolic, and total terpenoid content analysis were conducted using three replicates repeated twice. The obtained data were analyzed using analysis of variance (ANOVA) with SPSS software version 20, and mean differences were calculated using Duncan's multiple range test (DMRT) at a significance level of p≤0.05.

 Results and Discussion

Effect of 1 Indole-3-butyric acid (IBA) with different sucrose concentrations on microrhizomes induction of C. caesia

To induce microrhizomes in C. caesia, six-month-old cultures of C. caesia cultures grown on standard medium were used as explants. Sucrose of varied concentrations, 1.5%, 3%, 6%, 9%, or 12% was added to the ½ strength liquid MS medium supplemented with 1 mg/l IBA (Table 1). The cultures treated with 1.5%, 3%, or 6% sucrose had no effect on shoot number, similar to the control (1.5% sucrose). When the concentration of sucrose rose beyond 6%, the shoot number decreased significantly. The least shoot number was observed on medium with 12% sucrose (Table 1). The shoot length was significantly increased with the increasing sucrose concentration from 1.5% to 3%, while there was a decrease in the shoot length when the concentration further increased to 6%, and 9%. The shoot length was statistically the same at 9% and 12% sucrose (Table 1). In the case of root number, there was a significant decrease with the increasing sucrose concentration (1.5% to 12%). There was an increase in the root length when the concentration rose from 1.5% to 3% sucrose; on increasing the concentration beyond 3%, the root length continuously  decreased with the increased sucrose concentration (3% to 12%) (Table 1). There was no significant difference in the fresh weight of microrhizomes, while the dry weight of the microrhizomes increased significantly at 6% sucrose and above (Table 1). Similarly, the maximum in vitro tubers of Chlorophytum borivilianum was found in MS media that contained 60 g/l of sucrose (Chauhan et al., 2018). A higher intake of sucrose might stimulate enzymatic activity resulting in starch synthesis and accumulation in the storage tissues. Reduced shoot growth observed in culture media with high sucrose concentration, along with a swollen basal region, suggested that sucrose must have been transported to the stem for rhizome formation (Chirangini                    et al., 2005).

 Current water content (CWC)

The CWC of the C. caesia cultures at 1.5% and 3% sucrose was the same as that of the control (1.5% sucrose) (Fig. 1). The CWC decreased significantly at 6% sucrose compared to the control. This decrease was more intense at 12% sucrose. Sucrose induces abiotic osmotic stress when added beyond the normal limit (Kim and Kim, 2002). Relative water content in the leaves of maize cultivars decreased after drought treatment (Valentovic et al., 2006). Thus, in the present investigation, based on the morphological parameters (shoot length, root number, and root length) and CWC, 6% of sucrose was best for C. caesia micro rhizome formation in vitro. Therefore, a further experiment was performed with 6% sucrose.

 Green synthesis of silver nanoparticles (AgNP)

The AgNP was successfully synthesized using field-grown C. caesia rhizomes extract.

Characterization of AgNP

 UV-visible spectroscopy

UV-visible spectrum refers to the absorbance spectra in the UV-visible region. When the light beam passes through the solution, part of the light is absorbed, and the rest is transmitted through the solution. The transmittance is defined as the ratio of light entering the sample to the light that exits the sample at a fixed wavelength. The negative logarithm of transmittance is called absorbance. In the present study, the maximum absorption spectra of the synthesized AgNP were obtained at 400 nm (Fig. 2), which showed the formation of AgNP. It concludes that the AgNP was effectively synthesized using C. caesia rhizome powder methanol extract. The formation of AgNP through the reduction of silver ions by plant extract was observed via a UV-visible spectrophotometer (Cittrarasu et al., 2019). When AgNO₃ was added to C. longa extract and the suspension stirred for 24 h at room temperature, the emulsion's colour changed from yellow to dark brown. The surface plasmon resonance (SPR) phenomenon is responsible for the colour variations in aqueous solutions (Shameli et al., 2012). UV–visible spectroscopy showed that the SPR sharp peak at 350-430 nm wavelength indicates the formation of AgNP (Naik et al., 2002). Previous research has demonstrated that the spherical Ag-NP contribute to the absorption bands in the UV-visible spectrum at about 400-420 nm (Stepanov, 1997; Shameli et al., 2012). These absorption bands are thought to have extra-fine quality and small size of the Ag-NP. AgNP absorption spectra can be between 330 and 700 nm, with a sharp peak at 432 nm. This peak denotes the formation of AgNPs as it falls within the region of the SPR for AgNP (Logeswari et al., 2013).

Scanning Electron Microscopy (SEM)

Scanning Electron Microscopy (SEM) is a technique that provides an image of the surface morphology of the nanoparticle by providing information about the samples' size, shape, and other physical and chemical properties. An electron beam is generated and passed through the sample, and the scattered electrons from the particle’s surface are detected, which creates a high-resolution image. Black scattered electrons can reveal differences in chemical composition because heavier elements reflect more electrons, making them look brighter in the image. Figure 3 displays the    SEM image of synthesized silver nanoparticles of size ranging from 60-80 nm. SEM analysis was used to examine silver nanoparticles extracted from Syzygium aromaticum, which revealed the formation of spherical nanoparticles with a diameter range of 40–50 nm (Geoprincy et al., 2013).

 X-Ray Diffraction

XRD is a technique used to identify the crystalline phases present in a material and can determine the element proportions. The interaction between the X-ray beam and the atomic planes results in partial transmission of the beam, and the rest is absorbed, refracted,     scattered, and diffracted by the sample. X-rays are diffracted by each element in a different way, depending on the atomic arrangement and the type of atoms. In our study, the XRD pattern of synthesized AgNP showed sharp peaks at 2θ angles of 35.58°, 35.60°, 35.68°, 43.87°, and 47.87° (Fig 4). The sharp peak can be attributed to the crystalline structure of AgNP. The XRD peaks at 2θ of 38.18°, 44.25°, 64.72°, and 77.40° were each assigned to one of the face-centered cubic silver crystals (Ahmad et al., 2009). In a study performed by Shameli et al. (2012), XRD peaks of AgNP which were synthesized using C. longa, obtained at 2θ of 38.18°, 44.25°, 64.72°, and 77.40°, which attributed to face-centered cubic crystals structure of synthesized AgNP.

 Effect of AgNP on microrhizomes induction of C. caesia

After the selection of the best range of sucrose (6%) for the induction of microrhizomes in C. caesia, the cultures were treated with a varied range of AgNP, ranging from 0, 0.025, 0.05, 0.075  or 0.1 mg/l (Table 2). The root number increased significantly when AgNP concentration increased from 0.025 to 0.05 mg/l. When the concentration was increased beyond 0.05 mg/l, the root number decreased. The root length had no significant difference in microrhizomes treated with 0.025 mg/l AgNP compared to the un-treated cultures (without AgNP) (Table 2). But when the concentration was raised from 0.025 to 0.050 mg/l^, root length increased significantly, again decreasing at 0.075 mg/l AgNP. There was no significant difference in the microrhizomes fresh weight of the cultures treated with 0, 0.025, and 0.05 mg/l AgNP concentration. However, in Curcuma longa, compared to the control treatment, zinc oxide NP significantly enhanced the productivity, yield, and curcuminoid content (Khattab et al., 2023).

 Total phenolic content

TPC of the extract treated with 0.025 and 0.05 mg/l AgNP had 0.62 folds and 0.59 folds higher phenol content than the field-grown mother plant (Fig. 5). However, this content was comparatively less than the un-treated cultures. On the other hand, Salih et al. (2022) investigated the impact of different concentrations (0.0, 2.5, 5, 10, 25 mg/l) of biogenic AgNP on the antioxidant activity of Solanum tuberosum in vitro. They reported that 5 mg/l AgNP showed the  highest value of TPC, which was 281.7 mg GAE/g DW. Hasan et al. (2022) hydroponically exposed Lactuca sativa seedlings to different concentrations of Ag⁺ ions and AgNP for 25 days. Ag⁺ ions raised the TPC by 18% and flavonoid content by 12% of seedlings, respectively, while AgNP boosted TPC by 12%.

 Total terpenoid content

The extract treated with 0.025 and 0.05 mg/l AgNP had the highest terpenoid content (Fig. 6). This content was 3.5 folds and 1.67 folds higher than the field-grown mother plant and un-treated cultures, respectively. Solanki et al. (2023) conducted research to study the synergistic effect of AgNP and fungal symbionts in enhancing the secondary metabolites in leaves of black rice (Oryza sativa). Maximum production of secondary metabolites found at 80 ppm of AgNP. AgNP treatment could significantly increase terpenoids such as β-cymene, ϒ-terpinene, terpinene-4-ol, α-elemene, linalool, caryophyllene, β-ocimene, trans linalool, and myrcene.

 Antioxidant tests

DPPH radical scavenging activity

The extract treated with 0.05 mg/l AgNP had the DPPH radical scavenging activity similar to the standard ascorbic acid (Fig. 7). The activity of the extract treated with 0.05 mg/l AgNP was  significantly higher than the field-grown mother plant and un-treated cultures. However, the activity of extract treated with both the range of AgNP (0.025 and 0.05 mg/l) possessed similar % inhibition of DPPH radical. Selvan et al. (2018) synthesized AgNP by using aqueous extracts of garlic, green tea, and turmeric and assessed the antioxidant potential of the synthesized AgNP. Compared to other nanoparticles, the AgNP synthesized using turmeric extract exhibited excellent antioxidant activity in terms of DPPH assay. Elegbede et al. (2018) synthesized nanoparticles using xylanases     from Trichoderma longibrachiatum and Aspergillus niger. The 100 µg/ml of AgNP had maximum DPPH free radical scavenging activities compared to other tested ranges of AgNP and un-treated cultures.

 Ferric-reducing antioxidant power assay (FRAP)

FRAP of the extract treated with 0.025 and 0.05 mg/l AgNP was found to be similar (Fig. 8). The FRAP of both the treated extracts had a similar reducing power as that of standard butylated hydroxytoluene. The reducing power of the un-treated cultures and extract treated with 0.025 mg/l AgNP was the same but was significantly higher than the field-grown mother plant. Zhang and Jiang (2020) used chitosan/tea polyphenols-silver nanoparticles composite film (CS/TP-AgNP) in their studies. CS/TP-AgNPs III (8 ml AgNP) showed the highest FRAP activity, followed by CS/TP-AgNPs II (4 mL AgNPs) > CS/TP-AgNP I (2mL AgNP) nanocomposite film. The least FRAP activity was in un-treated cultures.

 GC-MS

GC-MS analysis identified some important monoterpenes i.e. camphor (1.98%) and ethyl N-(o anisyl)formimidate (3.72%), important sesquiterpenes i.e. 1,5-cyclodecadiene, 1,5- dimethyl-8-(1- methylethenyl)-, [S-( (1.51%), β-elemenone (3.4%), pentadecanoic acid (8.32%), curcumenone (9.2%) and (4aR,5S)-1-hydroxy-4a,5-dimethyl-3-(propan2-ylidene)-4,4a, (14.1%) in the control samples (without AgNP) (Fig. 9b). GC-MS analysis of the extract treated with 0.05 mg/l AgNP identified monoterpenes such as camphor (4.15%) and sesquiterpenes such as 1,5-cyclodecadiene, 1,5- dimethyl-8-(1- methylethenyl)-, [S-( (1.69%), β-elemenone (5.18%), (4aR,5S)-1-hydroxy-4a,5-dimethyl-3-(propan-2-ylidene) (23.84%) and curcumenone (26.03%) (Fig. 9c). The treatment with 0.05 mg/l AgNP resulted in the enhanced production of monoterpene (camphor) and sesquiterpenes (β-elemenone & curcumenone). The area % of camphor, β-elemenone and curcumenone increased by 2.09, 1.52 and 2.82 times respectively in the extract treated with 0.05 mg/l AgNP compared to un-treated extract. Whereas as compare to field-grown mother plant curcumenone area % increased by 17.46 and 6.17 times in the extract treated with 0.05 mg/l AgNP and un-treated extract respectively (Fig. 9a). The sesquiterpenes are known to possess antitumor properties as had an important role in eliminating reactive oxygen species (Anjum and Quraishi, 2023). Chung et al. (2018) AgNPs at 5 mg/l elicited cell suspension cultures of bitter gourd had enhanced amount of phytoconstituents than the control. These changes were responsible for high pharmacological activities (antioxidant, antidiabetic, antibacterial, antifungal and anticancer) in the AgNPs (5 mg/l)-elicited cell suspension cultures of bitter gourd. Keshari et al. (2020) reported that the AgNP (synthesized by Cestrum nocturnum extract) have more antioxidant activity as compared to vitamin C. The AgNP has 29.55% DPPH radical scavenging activity while vitamin C has 24.28% antioxidant activity.

 Conclusions

In the present investigation, the 6% sucrose with 1 mg/l IBA was optimum for in vitro microrhizomes induction in Curcuma caesia. The effect of AgNP on microrhizome induction and on antioxidant activity in C. caesia cultures was evaluated. The 0.025 and 0.05 mg/l AgNP enhanced the phenols and terpenoid content in the cultures compared to the field-grown mother plant. The antioxidant activity of the cultures treated with AgNP also increased compared to un-treated cultures and field-grown mother plant. The application of 0.05 mg/l AgNP to C. caesia cultures elicited the terpenoids content (especially curcumenone) which might be responsible for enhanced antioxidant activity.

 Acknowledgements The authors are thankful to the Head, School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur (India), for providing laboratory facilities. We are also grateful to the Pt. Ravishankar Shukla University, Raipur (India), for the university research fellowship (797/Fin/Sch/2021 date 20.10.2021) to Ms. Afreen Anjum. The authors are thankful to the Sophisticated Analytical Instruments Facility, Indian Institute of Technology-Madras, Chennai (TN), for GC-MS analysis.

 Conflict of interest The authors declare no conflicts of interest.

 Author contribution SP: Experimentation and Original draft, AA: Methodology, Data curation, Formal analysis, and Editing, VJ: Review and Editing, AQ: Conceptualization, Supervision, Review, and Editing.

 Funding The authors are thankful to the Pt. Ravishankar Shukla University, Raipur, Chhattisgarh (India), for the financial support in the form of a research fellowship and contingency grant (797/Fin/Sch/2021 date 20.10.2021).

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Fig. 1 Effect of ½ strength liquid Murashige and Skoog (1962) medium with Indole-3-butyric acid and different sucrose concentrations on -the current water content of Curcuma caesia cultures.

Fig. 2 UV-Visible absorption spectra of the synthesized silver nanoparticles (using field-grown Curcuma caesia rhizomes extract) with λ max= 400 nm.

Fig. 3 SEM image of the synthesized silver nanoparticles using field-grown Curcuma caesia rhizomes extract.

Fig. 4 XRD pattern of the synthesized silver nanoparticles using field-grown Curcuma caesia rhizome extract.

Fig. 5 Effect of AgNP-treated extract from field-grown mother plant of Curcuma caesia on  total phenolic content in cultures after one month.

 Fig. 6 Effect of AgNP-treated extract from field-grown mother plant of Curcuma caesia on total  terpenoids content in cultures after one month.

Fig. 7 Effect of AgNP-treated extract from field-grown mother plant of Curcuma caesia on  DPPH radical scavenging activity in cultures after one month.

Fig. 8 Effect of AgNP-treated extract from field-grown mother plant of Curcuma caesia on     ferric reducing antioxidant power assay in cultures after one month.

Fig. 9 Chromatogram of Curcuma caesia a Field-grown mother plant b Un-treated cultures c Cultures treated with 0.05 mg/l AgNP.

Fig. 10 (a) Curcuma caesia cultures with different sucrose concentrations; C. caesia cultures with (b) 1.5% sucrose (Control) (c) 1.5% sucrose + 1 IBA (d) 3% sucrose + 1 IBA (e) 6% sucrose + 1 IBA (f) 9% sucrose + 1 IBA (g) 12% sucrose + 1 IBA (h) C. caesia cultures with different AgNP concentrations; C. caesia cultures with (i) 6% sucrose + 1 IBA + 0 AgNP (j) 6% sucrose + 1 IBA + 0.025 AgNP (k) 6% sucrose + 1 IBA + 0.05 AgNP (l) 6% sucrose + 1 IBA + 0.075 AgNP,  and (m) 6% sucrose + 1 IBA + 0.1 AgNP

Table 1 Effect of ½ strength liquid Murashige and Skoog (1962) medium with Indole-3-butyric acid and  different sucrose concentrations on the morphology of Curcuma caesia cultures.

Values are represented as mean ±standard error. Means denoted with different letters are significantly different at p<0.05 (DMRT) compared using ANOVA.

Table 2 Effect of silver nanoparticles (AgNP) with 6% sucrose and 1 mg/l IBA on morphology of Curcuma caesia cultures.

Values are represented as mean ±standard error. Means denoted with different letters are significantly different at p<0.05 (DMRT) compared using ANOVA.