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Author(s): Afreen Anjum1, Afaque Quraishi*2



    1School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, INDIA
    2School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, INDIA
    *Corresponding Author Email-

Published In:   Volume - 4,      Issue - 1,     Year - 2022

Cite this article:
Afreen Anjum, Afaque Quraishi (2022) Investigation of biochemical changes in the leaves of Curcuma caesia Roxb. under sucrose-induced osmotic stress environment. NewBioWorld A Journal of Alumni Association of Biotechnology,4(1):1-6.

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NewBioWorld (2022) 4(1):1-6


Investigation of biochemical changes in the leaves of Curcuma caesia Roxb. under sucrose-induced osmotic stress environment


Afreen Anjum and Afaque Quraishi*


School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, INDIA,

*Corresponding Author Email-



Article history:


30 March 2022

Received in revised form

28 June 2022


30 June 2022


Curcuma caesia Roxb., also known as black turmeric, is a rare rhizomatous herb in the Zingiberaceae family. In the present study, we used the two different sucrose concentrations (3% and 9% sucrose) in Murashige and Skoog medium supplemented with 8 mg/l benzyladenine, 8 mg/l kinetin, 100 mg/l citric acid, 200 mg/l adenine sulfate and 2 mg/l indole-3-acetic acid to investigate the biochemical differences in the leaves of C. caesia. Biochemical parameters such as total sugar, proline, protein, chlorophyll, and hydrogen peroxide (H2O2) content, as well as superoxide dismutase (SOD) and catalase (CAT) activity, were analyzed to study the differences. All the parameters were performed on the 0th and 30th day after inoculation (DAI). It has been found that as sucrose concentration raised from 3% to 9% sucrose, the SOD activity, as well as the total sugar, proline, protein, chlorophyll, and H2O2 content increases significantly at 30th DAI. These increased enzymatic and non-enzymatic defense systems, thus helping the plant to maintain its growth under a stressed environment.


Curcuma caesia,

Osmotic stress,

Reactive oxygen species,




 DOI: 10.52228/NBW-JAAB.2022-4-1-2


Curcuma caesia (C. caesia) is an endangered rhizomatous herb with high medicinal and economic value (Sharma et al. 2017). Volatile essential oil of rhizomes of this species consists of about 30 components representing 97.48% of oil with camphor (28.3%) and others as their major constituent (Verma et al. 2010). The rhizome of C .caesia is widely used in making Ayurvedic drugs in India (Mishra 2013). It possesses anti-neurodegenerative, antidiabetic, antiulcer, smooth muscle relaxant, anticonvulsant, and anxiolytic effects and cures metabolic disorders like leukoderma, asthma, piles, tumor, bronchitis, etc. (Devi et al. 2015). Tribes of Chhattisgarh use a paste of fresh rhizomes in curing skin diseases (Mahato and Sharma 2017).

Presently, C. caesia is considered to be an endangered species by the Central Forest department of India (Sharma et al. 2017). Plant density is declining due to poor regeneration and wild harvesting in Madhya Pradesh and Chhattisgarh states and if this condition continues this species will soon get completely vanished from the natural forest of Central India (Mishra 2013). In vitro propagation and conservation methods can salvage the C. caesia from extinction (Krishnan et al. 2011). Pharmaceutically important secondary metabolites of medicinal plants can be enhanced by in vitro elicitation for their higher exploration (Chauhan et al. 2018). The most popular carbon source utilized in plant tissue culture media, sucrose, is also known to cause abiotic osmotic stress when applied in excess  (Javed 2002c; Ahmad et al. 2007). The plant underwent certain biochemical or molecular modifications to maintain osmotic balance in the stressful environment, which resulted in a shift in the solute content in the apoplast of the plant (Munnik and Meije 2001). Under stressful conditions, it is also known that plants activate many signaling molecules that aid in maintaining their metabolism and allowing them to live (Zhang et al. 2006). Literature is available on in vitro propagation studies (Haida et al. 2022), antioxidant properties of rhizomes of C. caesia (Karmakar et al. 2011), and in vitro microrhizome induction (Sarma et al. 2021). Not many reports are available on biochemical studies of sucrose-induced osmotic stress on C. caesia. Therefore in the present study, biochemical changes were used to investigate the sucrose-induced osmotic stress.


Materials and Method

Three-month-old cultures which were grown on Murashige and Skoog (MS) medium (Murashige and Skoog 1962), supplemented with 8 mg/l benzyladenine, 8 mg/l kinetin, 100 mg/l citric acid, 200 mg/l adenine sulfate, and 2 mg/l indole-3-acetic acid (standard medium) (Anjum et al. 2022) (Fig. 1), they were inoculated on the standard medium along with 3% and 9% sucrose. Biochemical examination of both of the aforesaid mediums was performed at 0th and 30th DAI (the day after inoculation). Three replicates were used in each test, which was repeated twice. The following experiments were carried out:

Total sugar content

Dubois et al. (1956) described the method of detecting the total sugar concentration in a sample. 0.025 gm of leaf sample were crushed in 5 ml of 80% ethanol and centrifuged at 4000g for 10 minutes. The supernatant was made up to 10 ml. The reaction is then finished by mixing 0.1 ml extract with 4 ml anthrone reagent diluted in sulfuric acid and placing it in a bath at 100oC for 10 minutes. The absorbance is measured at 625 nm after the solution has cooled. The total sugar content was measured in mg g-1 of fresh weight. Pure glucose was used as the standard.

Proline content

Bates et al. (1973) described a method to calculate proline content. According to this, 5 ml of 3% aqueous sulfosalicylic acid was used to crush 0.1 gm of leaf sample. It is then centrifuged for 10 minutes at 5000g. To the 1 ml of filtrate, 2 ml glacial acetic acid and 2 ml acid-ninhydrin solution are added. The mixture was heated to 100°C for 1 hour and after that cooled in an ice bath. At last, 4 ml toluene was added and mixed thoroughly. The lower aqueous phase was removed and absorbance was taken at 520 nm. The proline content was expressed in mg g-1 fresh weight.

Superoxide dismutase (SOD) activity

Enzyme extracts were prepared using 0.2 gm of  leaf sample that was completely pulverized using a cold mortar and pestle in an ice bath in 0.1 M potassium phosphate buffer with 0.5 mM EDTA (ethylene-diamine tetra-acetic acid) (pH 7.5). The sample is then centrifuged at 15000g for 10 minutes at 4oC. The filtrate is used for enzymatic assay. For determining SOD activity, Dhindsa et al. (1981) were used. The reaction mixture consist of 50 mM potassium phosphate buffer (pH 7.8), 13 mM methionine, 75 µM nitroblue tetrazolium chloride (NBT), 0.1 mM EDTA and 50 mM sodium carbonate. The reaction was begun by adding 2 µM riboflavin to the mixture, which was then incubated for 10 minutes under fluorescent lights before being left in the dark to cease the reaction. The mixture's absorbance was measured at 560 nm. Due to the highest rate of NBT reduction, the reaction mixture without enzyme acquired the most color. The amount of enzyme that inhibits 50% NBT photo-reduction was determined to be one unit of SOD. Enzyme units are measured in micrograms of protein as well as per gram of fresh weight. The Enzyme unit was calculated using the formula:

Hydrogen peroxide (H2O2) content

Velikova et al. (2000) described a method for determining H2O2 content. At 0oC, 0.25 gm of leaf tissue was crushed in 3 ml of 0.1 % TCA (tricarboxylic acid) with 0.1 g of activated charcoal. After that, it is centrifuged for 15 minutes at 1200g. To the 0.5 ml of supernatant, 0.5 ml of 10 mM potassium phosphate buffer (pH 7.0) and 1 ml of 1M potassium iodide were added. Absorbance was taken at 390 nm. H2O2 content was expressed in µM H2O2 g-1 FW.

Catalase (CAT) activity

The activity of CAT was assessed using the procedures described by (Mahely and Chance 1959). The reaction mixture consist of 2.5 ml potassium phosphate buffer (pH 7.4), 0.1 ml 1% H2O2, and 50 µl enzyme extract. The activity was determined by measuring the decrease of H2O2 absorbance at 240 nm, which was calculated using the extinction coefficient of H2O2, which is 36 mM-1 cm-1. The reaction is started by adding the H2O2. For 0, 1, and 3 minutes, absorbance (A) was recorded. The enzyme activity was measured in min-1 mg-1 of protein. CAT activity was calculated using the formula:

where, €= Extinction coefficient; d= Size of cuvette side, t= 1 or 3 min, c= Protein content in test sample in mg.


Protein content

Bradford method (Bradford 1976) is used to determine protein content. To 0.1 ml of extract, 3 ml Bradford reagent was added and absorbance was measured at 595 nm. Protein concentrations were measured in mg gm-1 of fresh weight. Using bovine serum albumin as a standard curve, the protein content was measured.

Chlorophyll content

Hiscox and Israelstam (1979) method for determining chlorophyll (Chl) content was used. 1 gm of a fresh leaf was crushed in 5 ml of 80% acetone that had been pre-chilled. The material was then centrifuged at 4°C for 20 minutes at 5000g. The supernatant was then collected and absorbance were made at three different wavelengths: 630, 645, and 663 nm. Furthermore, the Chl content was determined using the following equation:



Chl a = 11.64 × A663− 2.16 × A645+ 0.01 × A630


Chl b = 20.97 × A645− 3.94 × A663+ 3.66 × A630


Chl a + b = 20.2 × A645 + 8.02 × A663



Statistical analysis of data

The data obtained were analyzed using analysis of variance (ANOVA) using SPSS software version 10 (SPSS Inc 1999) and the mean differences were calculated by Duncan's multiple range test (DMRT) at a significance level of p = 0.05

Results and Discussion

Sucrose is the most often utilized carbon source in plant tissue culture media because it promotes optimal development when supplied at a specific dosage (Swedlund and Locy 1993). It acts as an osmotic agent when administered at a specific concentration, but when present over a certain level, it can cause osmotic stress in the in vitro condition (Mehta et al. 2000; Kim and Kim 2002). In this experiment, we employed a higher sucrose concentration (9%) in the medium. The exogenous addition of a higher concentration of sucrose induces a significant increase in the total sugar content (4.05 fold) at 30th DAI in the leaves of C. caesia, whereas the total sugar content did not increase at 30th DAI in the leaves grown on medium with 3% sucrose (Fig. 2A) when compared to control (cultures on both the medium at 0th day). Similarly, a significant increase (3.17 fold) observed in the proline content at 30th DAI in the leaves of C. caesia grown on 9% sucrose, whereas leaves grown on medium containing 3% sucrose did not show any differences in the proline content at the same 30th DAI (Fig. 2B). This finding supports Javed and Ikram's (2008) findings that free proline and total soluble carbohydrate increased in two wheat genotypes, S-24 and MH-97, along with increased sucrose concentration in Linsmaier and Skoog medium (Linsmaier and Skoog 1965). The addition of exogenous sugar boosted proline concentration because a-ketoglutarate, a five-carbon precursor necessary for proline accumulation, is produced by sugar oxidation (Stewart et al. 1966). Sucrose when added beyond the normal limit, changed the osmotic potential, causing carbohydrates and proline to accumulate in greater amounts, allowing the plant to take up more water and develop faster (Javed 2002c; Ahmad et al. 2007). SOD is a key intracellular antioxidant enzyme that protects cells from oxidative stress caused by reactive oxygen species. SOD is well-known for acting as the first line of defense against the stress caused by reactive oxygen species (Gill and Tuteja 2010). The SOD activity was observed to increase significantly by 21.6 fold with increased sucrose concentration (Fig. 2C), which agrees with Wang and Li (2008), who discovered higher SOD activity in white clover (Trifolium repens) leaves under water stress conditions, also Moharramnejad et al. (2016) discovered the similar pattern in SOD activity of maize seedling shoots under stress condition. In the present study, H2O2 content also increased significantly by 2.38 fold in the leaves grown on 9% sucrose at 30th DAI whereas H2O2 content did not increase in the leaves grown on 3% sucrose at 30th DAI (Fig. 2D). In the case of CAT activity, the non-significant difference observed between the leaves grown on medium with 3% and 9% sucrose (Fig. 2E). Kolarovic et al. (2009) found an increase in CAT activity in maize under stressful conditions. However, some studies have found that under water stress, such as in sunflowers, CAT activity remains similar or even decreases (Luna et al. 2004). Total chlorophyll content also increased significantly by 4.8 fold in the leaves of 9% sucrose and also showed a significant increase (2.25 fold) in the leaves grown on 3% sucrose at 30th DAI as compared to control (Fig. 2G) and previous studies (Winicov and Button 1991; Chang et al. 1997) also reported the same, i.e., increased chlorophyll production in dicot chlorophyllic cells under saline stress, while Valenzuelaa et al. (2005) found the same trend in graminaceous chlorophyllic cells under osmotic stress. Like the other defensive biomolecules, the protein was also found to be significantly elevated (6.46 fold) at 30th DAI in the leaves grown on a medium with 9% sucrose, whereas almost the same protein content as that of control was maintained in the cultures grown on 3% sucrose at 30th DAI (Fig. 2F). Noman et al. (2018) showed a significant increase in shoot proteins in water-stressed wheat (Triticum aestivum). Under the osmotic stress environment, these enzymatic and non-enzymatic scavenging systems accumulate thereby showing their increased amount at high sucrose concentration, which helps the plant in lowering the osmotic potential and thus maintain its growth (Ikeda et al. 2002).


The present study shows that high sucrose concentration induces osmotic stress in C. caesia which causes a rise in the enzymatic and non-enzymatic scavenging components that helps in avoiding or lowering stress. This study thus helps in understanding the physiological and biochemical changes in C. caesia during osmotic stress and how they adapt themselves to that environment.


Figure: 1 Three-month-old cultures on standard medium

     Figure: 2 Effect of sucrose on: A total sugar content, B proline content, C superoxide dismutase activity, D hydrogen peroxide content, E catalase activity, F protein content, and G total chlorophyll content. Values are represented as mean ±standard error.


Conflict of interest

Authors had no conflict of interest.


Whole hearted thanks to School of Studies in Biotechnology, Pandit Ravishankar Shukla University, Raipur (C.G.)



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