Analysis of Phytochemical Constituents, Pharmacological and Ethnobotanical Studies of Selected Folk Medicinal Plant, Curcuma amada Roxb. (Mango Zinger)

Curcuma amada , also known as Mango ginger, is a distinctive spice that shares morphological similarities with ginger but imparts a raw mango flavor. The primary use of Curcuma amada rhizome lies in pickle and culinary preparations. In traditional medicinal systems such as Ayurveda and Unani, Curcuma amada is highly valued for its diverse properties, serving as an appetizer, alexteric, antipyretic, aphrodisiac, diuretic, emollient, expectorant, and laxative. It is prescribed to address various conditions, including biliousness, itching, skin diseases, bronchitis, asthma, hiccough, and inflammation from injuries. Despite its extensive traditional use, there is a scarcity of studies exploring the antioxidant, antimicrobial, and cytotoxic activities of Curcuma amada rhizome extract. Antibacterial activity was assessed using the agar well diffusion method against Gram-positive Staphylococcus aureus and three Gram-negative strains-Klebsiella pneumonia, Escherichia coli, and Acetobacter aceti . The study aims to investigate the antimicrobial and cytotoxicity activities, as well as conduct in silico pharmacological analysis of Curcuma amada . In antibacterial tests, a notable zone of inhibition was observed against Acetobacter aceti and Escherichia coli , while none was observed against Klebsiella pneumoniae and Staphylococcus aureus . Cytotoxicity was assessed through a brine shrimp lethality bioassay, revealing results based on mortality numbers and the percentage of affected brine shrimp. In the in silico analysis, which evaluates pharmacological activity through pharmacokinetic analysis and molecular docking, four phytochemicals (E)-.beta.- Farnesene, eupatin, 3-Carene, and coumarin from the plant were selected. These phytochemicals were docked against the Estrogen receptor alpha (ERα), a protein associated with breast cancer. The docking results demonstrated promising binding scores, with E.beta.-Farnesene, eupatin, 3-Carene, and coumarin scoring -11.3, -6, -6.3, and -8.1, respectively.


Introduction
Since ancient times, medicinal plants have served as a rich reservoir of antimicrobial agents (Mahesh and Satish, 2008).In rural areas, traditional medicine derived from plant materials is widely utilized due to its easy accessibility and cost-effectiveness compared to modern medicine (Rashedul et al., 2010).Plants synthesize diverse secondary metabolites, including phenols, alkaloids, terpenoids, and glycosides, which play pivotal roles as sources of microbicides, pesticides, fungicides, and pharmaceutical drugs (Srinivasan et al., 2001).Traditional medicine predominantly relies on plant products as the primary source of pharmaceutical agents (Srinivasan et al., 2001).
The Zingiberaceae family, renowned for its wealth of phytochemical substances, is particularly abundant in curcuminoids with diverse chemical structures and varied physicochemical characteristics (Revathy et al., 2011).These plants, widely used in traditional medicine, exhibit a broad range of biological activities (Anand 2008).Numerous Curcuma species, including Curcuma longa, Curcuma zedoaria, Curcuma aromatic, and Curcuma amada, have been reported in literature for their antifungal, antibacterial, and anti-inflammatory properties (Apisariyakul et al., 1995;Yoshioka et al., 1998).The rhizomes of these plants, used for centuries in traditional medicine, are valued for their medicinal effects (Wibowo et al., 2012).
Curcuma amada, commonly known as mango ginger, belongs to the Zingiberaceae family and is closely related to turmeric (Curcuma longa).Its rhizomes share similarities with common ginger but lack the pungent taste, offering a distinct raw mango flavor.Coined by Linnaeus in 1753, the genus name Curcuma likely originates from the Arabic word 'kurkum,' meaning yellow color (Salvi et al., 2000;Shirgurkar et al., 2001).As an aromatic, perennial rhizomatous plant, Curcuma amada is part of a larger family comprising 70-80 species of rhizomatous annual or perennial plants (Purseglove 1974;Aminul 2004).The genus is extensively dispersed throughout the tropical regions of Asia, Africa, and Australia, having its origins in the Indo-Malayan area (Sasikumar 2005).In north India, they are used to make chutneys, and in south India, pickles.Plant-derived extracts have long been seen as a valuable medicine for preventing and treating chronic illnesses, as well as for preserving general immunological function (Alzoreky, N. et al., 2003).In food processing, extracts from spices, fragrant herbs, and medicinal plants are used to provide taste and other useful qualities.Phytochemicals that are found naturally in fruits, vegetables, and spices have the ability to either prevent or lower the risk of several illnesses (Bartz, J. A. et al., 2002).The scientific confirmation of the widespread usage of polyphenols, terpenoids, alkaloids, flavonoids, and other secondary metabolites in plants will be achieved through their existence (Nasar-Abbas, S. et al., 2004).The Curcuma genus encompasses more than 80 rhizomatous herb species, with origins in the Indo-Malayan region and widespread distribution across the tropical regions of Asia, Africa, and Australia (Policegoudra, R. et al., 2008).Curcuma amada, with a distinctive appearance similar to ginger, is commonly utilized for pickling, adding a raw mango flavor to cuisine, and for medicinal purposes (Sasikumar 2005).In the ancient Indian system of medicine, Ayurveda, the rhizome is valued for its properties as an appetizer, alexiteric, antipyretic, aphrodisiac, and laxative (Shankaracharya 1982).It finds application in the treatment of various conditions such as biliousness, itching, skin diseases, bronchitis, asthma, hiccups, and injuriesinduced inflammation.The Unani system attributes diuretic, maturant, emollient, expectorant, antipyretic, and appetizer qualities to Curcuma amada, recommending it for ailments like inflammation in the mouth and ear, gleet, ulcers on male sex organs, scabies, lumbago, and stomatitis (Warrier 1994).
Worldwide, infectious diseases pose significant health risks (Madhuri, S. et al., 2009).Staphylococcus aureus is a prevalent microorganism causing skin diseases, while Escherichia coli is a well-known member of the human intestinal microbiota and a versatile gastrointestinal pathogen.Zingiberaceae family plants, including Curcuma amada, are renowned for their preservative and medicinal properties, suggesting the potential presence of antimicrobial compounds.A study on mango ginger's antibacterial properties, using both aqueous and organic solvent extracts, demonstrated efficacy against Escherichia coli, Bacillus subtilis, and Staphylococcus aureus (Chandarana et al., 2005).Notably, the heated aqueous extract exhibited higher antibacterial activity compared to the unheated counterpart.Solvent extracts, particularly 1, 4-dioxan and DMF, showed lower efficacy against bacteria.Besides antibacterial effects, phenolic fractions of mango ginger effectively inhibited H+, K+-ATPase activity and the growth of Helicobacter pylori.Specifically, cinnamic and ferulic acids in Curcuma amada were identified as significant contributors to these inhibitory effects.Various extracts, including hexane, chloroform, ethyl acetate, acetone, and methanol, displayed high antibacterial activity against several bacteria (Policegoudra et al., 2007a, b).Recently, difurocumenonol and amadannulen, two bioactive molecules, were isolated from mango ginger, exhibiting diverse bioactivities such as antibacterial activity, platelet aggregation inhibitory activity, cytotoxicity, and antioxidant activities like DPPH radical scavenging, lipid peroxidation inhibition, metal chelation, and superoxide radical scavenging (Policegoudra et al., 2007a, b).

Sample collection
The plants rhizome was collected from different area at Kushtia District in Bangladesh.In our study, we work on mango ginger which smells like a raw mango.We have collected this mango ginger from Sheikhpara, Tribeni, Modhupur, Bittipara located area.

Sample preparation
The rhizome of Curcuma amada were washed thoroughly with double distilled water properly to remove dust, sands and other unnecessary particles and slice it properly by knife to dry almost 7-10 days at room temperature.After drying the samples, materials are blended by mechanical grinder to convert powder form.Then 5 g powder was measured using weight balance and takes it a conical flask.Then 25 ml Ethyl acetate are taken in sample to form solution and shake this solution by shaker for proper mixing this solution.Then, filter this sample to require the extract of this sample.Solvent extraction was carried out for 48 h in total.After each solvent extraction step, the extracts were filtered and concentrated by using rotary evaporator.The concentrated extracts were freezedried to remove the solvent and stored in refrigerator.

Media Preparation
In this study, we used 100 ml of Tryptone Soy Agar (TSA) media for inoculation in antimicrobial activity tests.The preparation involved suspending 4.5 grams of TSA in 100 ml distilled water, boiling to dissolve the medium, autoclaving for sterilization, cooling to 45-50°C, and then pouring into sterile Petri plates.

TSB broth preparation for Bacterial growth
We prepared 100 ml TSB broth in each conical flask, and I used four conical flask for four types of microorganism's growth.Weight 3 gm of Tryptic Soy Broth (TSB) powder and dissolve it 100 ml distilled water Then mix it properly by glass rod and autoclave it 121º C for 45 min.

Disc diffusion method
Microbial strains underwent assessment for their sensitivity to antimicrobials utilizing the disc diffusion technique, as detailed by Bauer et al. in 1966.This method served to appraise the in vitro antibacterial and antifungal efficacy of the test sample against specific human pathogenic microorganisms.Tryptone Soy Agar (TSA) and other agar media were employed for the antibacterial and antifungal evaluations, respectively.The test solutions, derived from 5 g of Curcuma amada powder and 25 ml of ethyl acetate, were meticulously dispensed in varying volumes (50, 100, 150, and 200 μl) onto individual 6 mm diameter discs.
To initiate the procedure, 1000 µl of broth culture was evenly distributed to cover the entire surface area of the media.Subsequently, the plates were incubated at a temperature of 37±1°C for a duration of 24 to 48 hours.Following the incubation period, the resulting zones of inhibition were measured in terms of diameter (mm).

Collection of Plant Materials
The fresh Curcuma amada plant rhizome was collected from different area at Kushtia District in Bangladesh (Fig 1 ).

Preparation of Solvent Extracts
The rhizomes of Curcuma amada underwent a meticulous cleaning process, involving repeated washing with running water and a final rinse with sterile water.Subsequently, they were finely chopped, subjected to shade drying, and mechanically ground into a coarse powder using a grinder (refer to Fig 2).The resulting powder was sieved, and 50 grams of the dried material were subjected to extraction using ethyl acetate in a Soxhlet apparatus.The obtained extract underwent filtration through Whatman No. 1 filter paper, followed by concentration using a rotary flash evaporator.The concentrated extract was then stored in airtight bottles at 5º C for future use.These extracts were later evaluated for both antimicrobial and cytotoxic activities.

Cytotoxicity Test
The brine shrimp lethality assay was carried out based on the methodology outlined by Meyer et al. (1982), with minor adjustments.Brine shrimp eggs underwent hatching in 1% NaCl saline water, maintaining a pH of 8.5 and ensuring continuous aeration for a duration of 36 hours.Following hatching, 10 active nauplii were introduced into individual test tubes containing 20 gm/L saline water.Various concentrations of the rhizome extract (0, 25, 50, 100, and 200 ppm) were introduced into the test tubes, which were subsequently incubated for 24 hours under optimal conditions.The calculation of the LC50 (50% lethal concentration) involved a comparison between the mortality rates of nauplii in the experimental groups and the control group.

Molecular Docking
We used the CB-Docking a blind docking web server for finding out the interaction between our compound and receptors (https://www.nature.com/articles/s41401-019-0228-6.pdf)., for C. amada we selected the ERα receptor for breast cancer.The CB-Dock web server employs an innovative curvature-based cavity detection technique to predict binding sites for a given protein, calculating their centers and sizes.Subsequently, it conducts docking using Autodock Vina, a widely-used docking program.Through meticulous optimization, this approach demonstrates an impressive ~70% success rate for top-ranking poses, exhibiting a root mean square deviation (RMSD) within 2 Å from the X-ray pose.Notably, it outperforms existing blind docking tools in our benchmark tests.CB-Dock also provides an interactive 3D visualization of results.The determination of the best-docked complex with an appropriate pose is based on the energy score.

Antimicrobial activity of Curcuma amada against some selected bacterial strains
This experiment was conducted for observing the zone of inhibition against Acetobacter aceti and Escherichia coli which are Gram negative bacteria, but zone of inhibition is not observed against

Results of Cytotoxicity Activity
The toxicity assessment level of Curcuma amada was measured by counting dead larvae of brine shrimp after 24 h of incubation.To assessment the toxicity level of this leaf extract, different concentration of leaf extracts was used such as-25 ppm , 50 ppm, 100 ppm , 200 ppm.Through the calculation of LC 50 , the concentration, 100ppm and 200ppm showed most toxicity value.2).While these computations may not yield the precise lethal concentration for a particular compound or extract causing 50% population mortality, they undoubtedly provide significant preliminary data for subsequent toxicity testing assays.Notably, most studies conducted the experiment in triplicate to ensure statistically reproducible results, as highlighted by Hamidi et al. in 2014.The assessment of toxicity, quantified by LC50 values, is a widely acknowledged parameter.In accordance with Clarkson's toxicity criteria, extracts are categorized based on their concentrations: extracts with LC50 values exceeding 1000 μg/ml are deemed non-toxic, those with LC50 values ranging from 500 to 1000 µg/ml are considered low toxic, concentrations with LC50 values falling between 100 and 500 μg/ml are classified as medium toxic, and those with LC50 values ranging from 0 to 100 µg/ml are characterized as highly toxic (Clarkson et al., 2004).The LC50 value for the E. foetidum leaf extract is measured at 532.81 µg/ml, placing it within the category of LC50 values ranging from 100 to 500 μg/ml.Consequently, it can be inferred that Curcuma amada exhibits low toxicity.

Results of Chemical Profiling and ADMET
In our study, we curated information of 135 compounds from different parts of the C. amada that are extracted, elucidated, and reported in many previous studies.The details of these compounds are depicted in Table 4 with their chemical name, SMILE, and drug likeness.Maximum one violation was considered for selection of these compounds.

Discussion
Numerous researchers worldwide have extensively investigated the robust antimicrobial properties inherent in plant extracts (Reddy et al., 2001;Ateb and Erdo, 2003).Effectiveness against diverse pathogens has been demonstrated in the crude extracts derived from medicinal plants (Kubo et al., 1981).An exploration of the antimicrobial potential of the ethanolic rhizome extract of Curcuma amada was conducted, employing the disc diffusion method with various pathogenic microorganisms.
The examination revealed that the ethanolic extract from Curcuma amada exhibits both antibacterial and antifungal activities (Chhetri, 2008;Okigbo, 2009).The results of antimicrobial activities of against selected bacterial species such as Klebsiella pneumonia, Escherichia coli, Acetobacter aceti, and Staphylococcus aureus are observed through disc diffusion method.In antibacterial studies, extract was most effective against Escherichia coli and Acetobacter aceti which are gram negative but show no effects on Klebsiella pneumoniae and Staphylococcus aureus bacterial strains (Table 1).
To assessment the toxicity level of this Curcuma amada Rhizome extract, different concentration of rhizome extracts was used such as-25 ppm, 50 ppm, 100 ppm, 200 ppm.Through the calculation of LC 50 , the concentration, 100ppm and 200ppm showed most toxicity value.At the mortality rate was observed 10% and 20% at 100ppm and 200ppm, respectively.Depending on the conditions of the experiment been obtained by a plot of percentage of the shrimps killed against the logarithm of the sample concentration (Meyer, Brine shrimp: a convenient general bioassay for active plant constituents, 1982) (Ahmed, 2010;Moshi, 2010).LC 50 values were estimated using a probit-regression analysis (Table 2).The LC 50 (Lethal concentration of 50%) value of the Curcuma amada on the animal model Artemia salina (Brine shrimp).The lower the lethal concentration the higher the toxicity level (Figure 3.3).
Molecular docking is a computational method that assesses the interaction between a ligand and receptor protein at the atomic level, offering insights into the conformation and behavior of small molecules within macromolecules.Chemical profiling furnishes detailed information regarding the indigenous chemical compounds and their properties in a given source.Utilizing virtual screening algorithms, significant chemical compounds can be efficiently identified from a myriad of candidates.In contemporary drug discovery, virtual screening has garnered substantial attention due to its robustness, cost-effectiveness, and time-saving advantages over empirical screening methods.
In the context of our study, four phytochemicalsnamely, E)-.beta.-Farnesene,eupatin, 3-Carene, and coumarin-derived from the plant were chosen and subjected to molecular docking against the Estrogen receptor alpha (ERα), a protein implicated in breast cancer.The docking analysis unveiled promising binding scores, with maximum scores of -11.3 (E-.beta.-Farnesene),-6 (eupatin), -6.3 (3-Carene), and -8.1 (coumarin).This suggests the potential bioactivity of these compounds.Consequently, the extract from C. amada holds promise as a prospective drug, offering a safer alternative in its purified form to synthetic chemical-based chemotherapeutics.

Conclusion
Antimicrobial resistance is a global problem.Hence, this study was aimed to focus the antimicrobial properties of Curcuma amada (mango ginger) rhizome extract on some selected bacteria which are gram positive and gram negative.The Curcuma amada (mango ginger) rhizome extract shows low level and no observable antimicrobial activity against the selected bacterial strain.So, in case of infection of one of these bacteria, the use of mango ginger powder is not so much effective.The compound exhibits promising bioactive properties with considerable potential for pharmaceutical applications.However, prior to its utilization in humans, it is imperative to conduct essential procedures such as isolating the pure compound, performing toxicological studies, and assessing its pharmacological activity.Further research is warranted to comprehensively evaluate the antimicrobial and cytotoxic effects of mango ginger extracts.Additionally, our investigation revealed the low toxicity of C. armada against A. salina.Furthermore, potential inhibitors of Estrogen Receptor Alpha (ERα) were identified in C. amada.Through virtual screening, four compounds were pinpointed out of 135, and their binding properties were validated via ADMET analysis and Molecular Dynamics (MD) simulations.The MD simulations elucidated crucial binding interactions, showcasing binding scores of -11.3, -6, -6.3, and -8.1 for (E)-.beta.-Farnesene,eupatin, 3-Carene, and coumarin, respectively.These interactions are vital for inhibiting Estrogen Receptor Alpha (ERα) activity.
In conclusion, this study lays the groundwork for developing novel drugs that inhibit Estrogen Receptor Alpha (ERα) to combat breast cancer, leveraging the natural source of C. amada.

Figure 2 .
Figure 2. Curcuma amada dried rhizome and powder Collection of organisms Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Acetobacter aceti strains were collected from microbiology laboratory of Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia, Bangladesh.
Staphylococcus aureus and Klebsiella pneumoniae which one is Gram positive and another is Gram negative bacteria (Fig 3.1).

Figure 3 . 2 .
Figure 3.2.Toxicity assessment of Curcuma amada rhizome extract by Artemia Salina Data Analysis Depending on the experimental conditions, survivors were quantified either through the use of a magnifying glass or a microscope within a specified time frame of 24 hours.The determination of the median lethal concentration (LC50) for the test samples involved plotting the percentage of shrimp killed against the logarithm of the sample concentration, following the methodology outlined by Meyer et al. in 1982.Estimation of LC50 values employed probit regression analysis, as described by Finney et al. in 1971.Finney's statistical approach has been integrated into various software packages, including Stata, MATLAB, R, and IBM SPSS, facilitating automated LC50 calculations along with confidence intervals (refer to Table2).While these computations may not yield the precise lethal concentration for a particular compound or extract causing 50% population mortality, they undoubtedly provide significant preliminary data for subsequent toxicity testing assays.Notably, most studies conducted the experiment in triplicate to ensure statistically reproducible results, as highlighted byHamidi et al. in 2014.

Table 1 .
Antimicrobial activity of Curcuma amada at varied concentrations.The protein Estrogen receptor alpha (ERα) is responsible for breast cancer, so we selected this ERα for our target protein(Chou et al., 2020).

Table 2 .
Toxicity value of C. amada extract by Artemia Salina

Table 6 .
Selected Compounds and Target Protein Receptor