In vitro Antioxidant and Antibacterial Activity and Phytochemical Screening of Mango Ginger (Curcuma amada Roxb.) in Mizoram

Author: Evarin Debbarma*, Saikhom Herojit Singh and Ngangom N.M.

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Abstract

Mango ginger (Curcuma amada Roxb.) is a perennial, rhizomatous, fragrant herb, having morphological resemblance with ginger but imparts a raw mango flavour. This plant has been utilised for the treatment of many ailments in traditional medical systems (Ayurveda and Unani) from ancient times. Curcuma amada possesses several pharmaceutical properties such as antimicrobial, anti-inflammatory, analgesic, anticancer, anti-hyperglyceridemic, antioxidant activity etc. Owing to these properties, an experiment was conducted at Mizoram University, Aizawl (2021), to evaluate the phytochemical constituents of Curcuma amada Roxb. (Zingiberaceae). The study revealed the potential antioxidant and radical scavenging activity (IC50 223.2 µg/ml) of C. amada rhizome extracts by DPPH assay, indicating its protective role against oxidative damage and as an important natural antioxidant. The above said pharmaceutical activity may be shown due to the presences of various bioactive compounds (screened using respective scientific methods and protocols) including phenols (11.47±0.004 mg/100 mg), flavonoids (5.05±0.068 mg/100mg), ascorbic acid (5.05± 0.068 mg/100mg), tannins, saponin, glycosides etc. The antibacterial activity, determined by the disc diffusion assay, of C. amada rhizome extract exhibits an inhibition zone of 12±0.2 mm, 11±0.5 mm, 10±0.2 and 8.6±0.7 mm diameter against E. coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis culture respectively. The diverse array of phytochemicals present in the plant thus suggests its therapeutic potentials which may be explored in drug manufacturing industry as well as in traditional medicine.

Keywords

Curcuma amada, mango ginger, phytochemical screening, quantitative analysis, antioxidant activity, antibacterial activity

Conclusion

The study revealed the potential antibacterial, antioxidant and radical scavenging activity of extracts of Curcuma amada rhizomes, indicating its protective role against microbial infections, oxidative damage and as an important natural antioxidant. C. amada possesses several pharmaceutical properties such as antimicrobial, anti-inflammatory, analgesic, anticancer, anti-hyperglyceridemic, antioxidant activity etc. The above said pharmaceutical activity may be shown due to the presences of various bioactive compounds including tannins, saponin, flavonoids, phenolics, glycosides etc. It may be stated that the phytochemical examination provided valuable information about the various phytoconstituents found in the plant, which will aid future researchers in selecting the appropriate extract for further exploration of isolating the active principle.

References

INTRODUCTION Plants are regarded as living biochemical factories that produce a wide range of chemical molecules known as phytochemicals or secondary metabolites with different biological activities as a result of their metabolic processes (Kaur et al., 2018; Mahadevi & Kavitha, 2020) and these phytochemical constituents occur in different plant parts such as leaf, stem, root, flower, bark etc. (Gordon, 2001). Primary constituents include carbohydrates, amino acids, proteins and chlorophyll, whereas secondary metabolites include alkaloids, terpenoids, steroids, flavonoids, saponins etc. (Dhawale, 2003). About 80% of the world’s population rely on traditional medicine that involves use of plant and herb extracts for their primary healthcare system (Sandhya et al., 2006). Due to their wide availability and fewer side effects, herbal medicines are acceptable for treating a broad range of infection and diseases (Chattopadhyay et al., 2004). Curcuma genus of the Zingiberaceae family consists of about 80 species, of which 40 are indigenous to India. Extensive research has been conducted on Curcuma longa (turmeric) and Zingiber officinale (ginger), but the medicinal properties of Curcuma amada (mango ginger) has yet to be fully explored (Saipriya, et al., 2017). Curcuma amada originated in the Indo-Malayan region and widely distributed in the tropics from Asia to Africa and Australia (Sasikumar, 2005) with a geographical distribution ranging from India, Indo-China, Thailand, Indonesia, Malaysia and northern Australia (Policegoudra et al., 2011). C. amada is found in the wet (semi-evergreen) mixed forests of West Bengal (Mallick, 2019), and is cultivated in the North Eastern states, Gujarat, Uttar Pradesh, Kerala, Karnataka and Tamil Nadu (Policegoudra et al., 2011). C.amada is an aromatic herb known as Amba haldi or Mango ginger. It closely resembles the morphology of ginger; its rhizome gives a characteristic odour of raw mango flavour due to the presence of terpene hydrocarbons cis-ocimene and car-3-ene which makes mango ginger a unique spice (Gholap et al., 1984). It is used as a major ingredient in pickles, candies, salads, sauces and chutneys (Yogamaya et al., 2012). Therapeutically, it is used to treat a range of mood and medical disorders in traditional and Ayurvedic medicine (Policegoudra et al., 2011). Curcuma plants have a camphoraceous aroma and contain many functional compounds such as phenolics, flavonoids and different antioxidant enzymes (Krishnaraj et al., 2010) and various species belonging to this genus are well known for their multiple use as medicine, cosmetic, dye, flavouring agent and nutraceuticals. Likewise, the antioxidant and anti-inflammatory activities of C. amada is due to the presence of phenolic compounds (Mara et al., 2006). The essential oil of rhizome exhibits antimicrobial, antifungal and anthelmintic activity against tape worms and such pharmaceutical properties may be shown due to the presences of various bioactive compounds including curcumin, demethoxycurcumin, bisdemethoxycurcumin, phenol and terpenoids (Policegoudra et al., 2011). Curcuma amada rhizomes are exported from India as medicinal plant parts (Hasan et al., 2009). Few considerable studies have been conducted on C. amada regarding its phytochemicals as well as further screening of the found constituents. Although there has been some documentation on the traditional uses and its benefits yet the indigenous knowledge on the various aspects of usage has been passed down from generation to generation orally. There is a need for the proper documentation and elaborate study on this plant species. Huge potential is observed in future studies and research which can be carried out with respect to the fields of medicine, pharmaceutical, pharmacology, oil industries, food industries, perfumery etc. which in the long run will not only elevate better treatment options for ailments but also give an immaculate area for economic growth of the local people with proper cultivation practices and better marketing approaches. Therefore, this study aims at understanding and analysing the primary photochemical constituents of C. amada. MATERIAL AND METHODS Collection of plant material: C. amada rhizomes were collected from Amtali village, Takarjala, West Tripura during March, 2021. The rhizome samples were thoroughly cleaned under tap water. Fresh rhizomes of C. amada were used for further experiments. Phytochemical screening: Standard phytochemical screening protocols were used to detect the presence of bioactive agents. These tests were identified by visual inspection of colour changes or precipitate formation after the addition of particular reagents to the solution. Test for Tannin (Braymer’s Test): 2 ml of extract was mixed with few drops of 5% FeCl2 solution and blue colour was observed to indicate the presence of tannins. Test for Saponin (Foam Test): 2 ml of extract was mixed with 5 ml of distilled water in a test tube and shaken vigorously. Formation of stable foam was observed to indicate the presence of saponin. Test for Flavonoid (NaOH Test): 2 ml of extract was added into 2 ml of 10% NaOH solution. Yellow to orange colour was observed to indicate the presence of flavonoids. Test for Protein (Xanthoproteic Test): 2 ml of extract was added into 2 ml of HNO3 and boiled in water bath. Orange colour was observed to indicate the presence of protein. Test for Carbohydrate (Benedict’s Test): 2 ml of extract was mixed with 2 ml of Benedict’s reagent and boiled in water bath. Yellow, green or red precipitate was observed to indicate the presence of carbohydrate. Test for Glycosides (Keller-Kiliani Test): 2 ml of extract was mixed with 2 ml of glacial acetic acid containing 2 drops of 2 % FeCl2 solution. The mixture was poured into another test tube containing 2 ml of concentrated H2SO4. Brown ring at the interface was observed to indicate the presence of cardiac glycosides. Quantitative analysis: Determination of Flavonoid content: To determine the total flavonoid content, AlCl2 method was used. 1g of fresh sample was weighed and crushed by mortar and pestle. 0.3 ml of NaNO2 and 4 ml of H2O was added and kept for 5 minutes. After 5 minutes, 0.3 ml of 10% AlCl3 was added and left for 6 minutes. At 6 minutes, 2 ml of 1M NaOH was added and volume was made up to 10 ml with distilled water. The absorbance was recorded at 510 nm using a digital spectrophotometer. Determination of Carbohydrate content: Total carbohydrate was determined by using Anthrone reagent and HCL following the Anthrone method and absorbance was measured using a digital spectrophotometer at 630 nm. Determination of antioxidant content: Total antioxidant was determined using DPPH (2,2-diphenyl-1-picrylhydrazyl) and methanol following DPPH assay and absorbance was measured using a digital spectrophotometer at 517 nm. The free radical scavenging activity (percentage antiradical activity) was calculated by the equation Determination of Phenol Content: The total phenolic content was determined according to Folin-Ciocalteu method (FCM) described by Siddhuraju and Becker, 2003 using catechol as a standard. Different aliquots (0.5 & 1 ml) are taken in test tubes diluted in 3 ml of water and 0.5 ml of Folin-Ciocalteu reagent is added. After 3 minutes, 2 ml of 20% Na2CO3 is added in each tube and mixed the content thoroughly. Colour was developed and absorbance was measured at 650 nm using digital spectrophotometer against blank reagent. Standard graph was prepared using different concentrations of catechol. Determination of Protein content: The total protein content was determined by Folin Lowry Method using bovine serum albumin as standard. The absorbance was measured at 660 nm using a digital spectrophotometer. Determination of Ascorbic acid: The total ascorbic acid content was determined by Volumetric method using reagent 2,6-dichlorophenolindophenol and the result is expressed in mg/100 ml sample. Determination of Anthocyanin content: Anthocyanin was estimated by taking a gram of sample and adding 50-60 ml of Methanolic HCl (85:15v/v). The produced extract was stored for a day under airtight condition and then diluted to 100 ml with Methanolic HCl. The colour density was measured using a digital spectrophotometer at 445 nm. Antibacterial studies: Evaluation of antibacterial activity of C. amada rhizome extract was carried out by disc diffusion assay described by Lennette, 1985. The rhizome extracts were diluted with Dimethyl sulphoxide (DMSO) and aliquots were loaded on a disc and the antibacterial activity was evaluated against five standard bacterial strains which included the Gram positive Staphylococcus aureus (MTCC-96) and Bacillus subtilis (MTCC-441) and Gram negative Pseudomonas aeruginosa (ATCC-15442), Bacillus Pumilus (ATCC-14884) & E. coli. The bacterial strains were inoculated on freshly prepared agar plates with a loop, evenly spread by a spreader and incubated overnight at 37°C. The diameter of zone of inhibition produced by the inoculums were measured in mm. RESULT AND DISCUSSION Phytochemical analysis revealed the presence of tannin, saponin, glycoside, protein, carbohydrate and flavonoid. Saponin is a natural antioxidant which also promotes tumour cell death (Podolak et al., 2010; Tapondjou et al., 2011; Bi et al., 2012). Saponins have anti-hypercholesterolemic activities as well as antibacterial characteristics. Tannin has been employed as an active ingredient in medicine and beverages due to its antioxidant properties (Amarowicz and Troszynska, 2003; Amarowicz et al., 2005). Tannins have been shown to prevent the growth of harmful fungus. Strong lipid peroxidation inhibitors have been found in glycosides such as quercetin monoglycosides, diglycosides and flavonol glycosides (Plumb et al., 1999). Physiochemical parameters of the rhizome of Curcuma amada Roxb. are tabulated in Table 1. Antioxidant activity: Plants having antioxidant and radical scavenging properties are valuable in medical applications and pharmaceutical industries. The antioxidant capacity of C. amada was assessed in this work using the DPPH radical scavenging method and compared to the activity of ascorbic acid, a well known antioxidant (Fig. 1). The different concentrations of 50, 100, 150, 200, 250 and 300 µg/ml showed different levels of radical scavenging activity of 17.7, 26.0, 29.1, 38.5, 53.1 and 73.9 % of inhibition respectively with an IC50 value of 223.2 µg/ml while ascorbic acid content has an IC50 value of 107.5µg/ml. Total flavonoid content: The extracts' total flavonoid content was calculated as a percentage of quercetin equivalents per 100 mg of the sample (Fig. 2). The total flavonoid estimation of rhizomes of C. amada showed the content value of 5.05± 0.068 mg/100mg. Total phenol content: The total phenol estimation of C. amada rhizomes showed the content value of 11.47±0.004 mg/100 mg (Fig. 3). Total protein content: The total protein content of the rhizomes of C. amada showed the content value of 12.47 ± 0.03 mg/g (Fig. 4). Total carbohydrate content: The total carbohydrate content of C. amada rhizomes showed the value of 14.55 ± 0.06 mg/100mg (Fig. 5). Total anthocyanin content: The total anthocyanin content of C. amada rhizome showed a value of 5.29 ±0.001 mg/100g. Total ascorbic acid content: The total ascorbic acid content of rhizome of C. amada showed a content value of 5.05± 0.068 mg/100mg. Antibacterial activity: The antibacterial activity of the extracts varied depending on the extract concentration and was measured in terms of diameter, is shown in Table 2. The maximum inhibition zone observed was 12 mm against bacteria Escherichia coli followed by 11 mm and 10 mm against Pseudomonas aeruginosa and Staphylococcus aureus respectively and the minimum inhibition observed was 8.6 mm against Bacillus subtilis. Spices are economically significant and high in phenolic chemicals and flavonoids, which are easily absorbed by our body and does not cause harm (Chandarana et al., 2005). In the present study we have observed that Curcuma amada extract contains carbohydrate, flavonoid, phenol, tannin, anthocyanin and glycoside compounds, all of which are known to have therapeutic effects against disease causing microorganisms. The findings suggest that the rhizome of Curcuma amada holds promise as a potent source of pharmaceutically important compounds. The most major classes of secondary metabolites and bioactive substances found in plants are flavonoids and phenolic compounds (Surapaneni and Vishnu 2009). Flavonoids found in the non-aerial portions of plants, such as rhizome, plays an important function in metabolism and development in living systems. Phenolic compounds are a type of antioxidant agents that operate as free radical terminators. Their bioactivities may be connected to their ability to chelate metals, inhibit lipoxygenase and scavenge free radicals (Roya & Fatemeh, 2013). The study has revealed the total phenolic and flavonoid content of 11.47 and 5.05 mg/100 mg respectively which gives them their antioxidant properties. Earlier reports have shown that some flavonoids, such as quercetin are anticarcinogenic and can stop cancer cells from growing (Elattar & Virji, 2000; Ranellett et al., 1999).

How to cite this article

Evarin Debbarma, Saikhom Herojit Singh and Ngangom N.M. (2022). In vitro Antioxidant and Antibacterial Activity and Phytochemical Screening of Mango Ginger (Curcuma amada Roxb.) in Mizoram. Biological Forum – An International Journal, 14(1): 1704-1709.