Evaluation of Phytochemicals, Antioxidant, Antibacterial and Antidiabetic Potential of Alpinia galanga and Eryngium foetidum Plants of Manipur (India)

Background: Alpinia galanga and Eryngium foetidum are two commonly used traditional aromatic plants of Manipur which is traditionally used in Aroma therapy. Rationale of pharmacological potentials of these plants are still unclear, even if few preliminary studies are available in literature for individual plants. Objective: This study was conducted for comparative assessment of antioxidant, antibacterial, and antidiabetic potential of A. galanga and E. foetidum. Materials and Methods: The rhizome of A. galanga and leaf of E. foetidum were extracted in methanol, ethanol and water. Phytochemicals of each extracts of Alpinia galanga and Eryngium foetidum were analyzed. The antioxidant potential of all the extracts was assessed by measuring total phenolic content, total flavonoid content and free radical scavenging potential was assessed by 1,1-diphenyl-2-picrilhydrazyl (DPPH) assay, antibacterial activity was assessed against various pathogenic and nonpathogenic bacteria in vitro by Kirby-Bauer agar well diffusion method and antidiabetic activity was assessed by α-amylase inhibition. Results: Both the plant showed presence of all the tested phytochemicals. It was observed that methanolic extracts of both the plants have higher phenolic content than ethanolic and aqueous extracts, however ethanolic extracts E. foetidum shows higher flavonoid contents. Both the plant shows similar DPPH scavenging and metal chelating activity. It was also observed that the antidiabetic potential of A. galanga is greater than E. foetidum. The methanolic and ethanolic extracts of the plants shows quite similar and good antibacterial potential than the aqueous extracts. Conclusion: The present study suggests that both Alpinia galanga and Eryngium foetidum plants of Manipur could be used as herbal remedies for the treatment of diabetes mellitus as well as managing oxidative stress and oxidative stress related disorders.


INTRODUCTION
Manipur state is situated at north-eastern region of India geographically distributed from 23 o 56' N to 25 o 44'N and from 93 o 02'E to 94 o 47'E. 1 Manipur is famous for its rich biodiversity of flora and fauna, beautiful environment surrounded by numerous hills and mountains like Imphal valley and for its precious source of medicinal plants. This provides a great opportunity to explore and formulate therapeutic medicines. The phytochemicals present in these therapeutic plants have received a great attention in preventing diseases caused by oxidative stress which releases reactive oxygen species such as singlet oxygen and other free radicals as a result of side-effect of aerobic metabolism. DNA damage, tissue injury, cardiovascular malfunctions, tumour promotion and early ageing like numbers of uncountable disorders includes the involvements of damaging activity of these free radicals. 2,3 Various indigenous formulations have been produced from many medicinal plants found in this region also successfully used as anti-diabetic and anti-microbial agents. The plants Alpinia galanga and Eryngium foetidum are among them, these plants are not only a source of food, flavour but are also a good source of natural and herbal medicine. The plant Alpinia galanga is a perennial herb belonging to the Zingiberaceae family. A. galanga commonly known as great galangal also called as Kulanjan in Hindi and Kanghoo in Manipuri. It is distributed throughout the Western Ghats, Gujarat, Mysore, Goa, Manipur, and other Southeast Asian countries. One of the most active compounds found is terpinen-4-ol and also contains alkaloids, saponins, glycosides, terpenoids, phenolics, flavonoids, phytosterols, carbohydrates are important phytoconstituents present in this plant. 4 Galango flavonoid a newly reported flavonoid was isolated from ethanolic extract of Alpinia galangal. 5 The rhizome extract of A. galanga has been reported to possess several medicinal active ingredient that have a potential of antitumour, antioxidant, antifungal, antibacterial, gastro protective, hypoglycaemic, hypolipidemic, anti-inflammatory substance. 6 It has been also reported by many researcher that methanolic and aqueous extracts of Alpinia galanga showed significant free radical scavenging activity against DPPH radical and also possess strong superoxide anion scavenging activity as well as metal chelating activity. Moreover the ethanolic extract of galangal was also found to be effective in inhibiting α-glucosidase and α amylase (in vitro) as well as a good antimicrobial activity. 7,8 Eryngium foetidum is another perennial herb of Manipur extensively used as a medicinal and aromatic plant. It belongs to the Apiaceae family and distributed throughout many state of India namely Manipur, Tamil Nadu, Kerala, Karnataka, Assam, Andaman and Nicobar Islands. 9 The plant is commonly used as home remedies to treat burns, ear ache, fevers, hypertension, constipation, seizures, asthma, stomach ache, worms, infertility complications, snake bites, arthritis, diarrhea and malaria by tribal people of Manipur. 10 The various bioactive component of E. fotidum was discovered till date includes phenols, flavonoids, tannins and polyphenols. The polyphenolic compounds of E. foetidum acts as anti-inflammatory, hepato protective, antioxidant, antithrombotic, anticarcinogenic, free radical scavenger, antimutagenic, antimicrobial and antihyperglycemic activity in vitro against α-amylase enzyme. 11,12,13 E. foetidum leaf extract possesses suppressive effects against proinflammatory mediators. Thus, E. foetidum has a high potential to be used as a food supplement to reduce risk of cancer associated with inflammation. 14

CHEMICALS AND REAGENT
Chloroform, DMSO, Sulphuric acid, Dilute ammonia, Ferric chloride, Hydrogen chloride, Deionised distilled water, Folin-Ciocalteau reagent, Draggendorff 's reagent, Sodium carbonate and Gallic acid are used for phytochemical screening and antioxidant assay. Mueller-Hinton broth and agar media are used for antibacterial assay. All the reagent and media were purchased from Hi Media. Starch and α-amylase enzyme are used for antidiabetic assay.

PLANT MATERIAL
The whole plant of Eryngium foetidum and Alpinia galanga were collected from a local area of Imphal, Manipur in December, 2013. The plant samples were identified by the taxonomist at D.M. College of Science, Imphal, Manipur. The rhizome part of the plant A. galanga and leaf of E. foetidum were separated and cleaned in running tap water followed by distilled water and dried in shade for 15 days. The parts under study i.e leaf of Eryngium and rhizome of Alpinia were then ground in a coarse powder using grinder and preserved in air-tight containers.

EXTRACTION OF PLANT MATERIALS
The extraction of plant parts was carried out according to the method suggested by Fatope et al. 1993. 15 In this, 20 g fine powder of rhizome part of the plant A. galanga and leaf of E. foetidum were percolated at room temperature with 400 ml in methanol for methanolic extraction, in ethanol for ethanolic extraction and in water for aqueous extraction respectively (so as to achieve a solvent extraction in ratio 1: 20) in 600 ml screw caped conicals. The conical were screw caped and placed in shaker at room temperature for 24 hrs at 100 rpm. After 24 hrs the extracts were filtered using muslin cloth and then re-filtered using Whatman filter paper No. 1. The filtrates were concentrated using water bath at 35-40 o C. The extracts were labeled accordingly and preserved in refrigerator at 4 o C, till further work. Extract was dissolved in Dimethyl sulfoxide (DMSO) to made concentration of the stock solution of 0.5 g/ml.

PHYTOCHEMICAL SCREENING
Phytochemical are non-nutritive chemical present in plants that contribute to the medicinal properties of that plant, which have disease preventive properties. Phytochemical screening of different extracts of Alpinia galanga and Eryngium foetidum were carried out according to the methods of Ayoola et al. 2008. 16 The different plant part extracts prepared were screened for terpenoids, flavonoids, saponins, tannins and phenols respectively:

Test for Terpenoids (Salkowski Test)
To 0.5 g of each of the plant part extracts were added with 2 ml of chloroform. Then into it 3 ml of concentrated sulphuric acid was carefully added, to form a layer. A reddish-brown colouration of the interface indicated the presence of terpenoids.

Test for Flavonoids
In 0.5 ml of filtrate of each of the plant parts extracts, 5 ml of dilute ammonia was added, followed by addition of 1ml of concentrated sulphuric acid to it. The presence of flavonoids was detected by yellow colouration of the solution that disappears on standing.

Test for Saponins
To 0.5 g of each plant extracts 5 ml of distilled water was added in test tubes. The solution was shaken vigorously and observed for a stable persistent froth. The frothing was mixed with 3-4 drops of olive oil and again shaken vigorously after that it was observed for the formation of emulsions.

Test for Tannins
About 0.5 g of each plant extracts was boiled in 10ml of water in each test tube and then filtered. Then few drops of 0.1% ferric chloride was added and observed for brownish-green or a blue-black colour formation which indicate the presence of tannins.

Test for Phenols (Ferric chloride test)
Small quantity of extract was dissolved in 2 ml of distilled water, and in to it few drops of 10% aqueous ferric chloride solution were added. A blue or green colour was produced which indicating the presences of phenols.
Test for Alkaloids 0.2 g of plant extracts were diluted to 10 ml with acid alcohol. It was boiled and filtered. To 5 ml of the solution 2 ml of dilute ammonia was added then 5 ml of chloroform was added to it and shaken gently. In this solution mixture 10ml of acetic acid was added. This was divided into two portions. The upper layer was discarded; Draggendorff 's reagent was added to the other layer. The formation of reddish-brown precipitate with Draggendorff 's reagent was regarded as positive for the presence of alkaloids.
Test for Anthraquinones 0.2 g of each of the plant extracts was boiled with 10 ml of H 2 SO 4 and was shaken with 5 ml of chloroform. The chloroform layer was pipetted into another test tube and 1ml of dilute ammonia was added. The resulting solution was observed for colour change.
Test for Cardiac Glycosides (Keller-Killiani Test) 0.2 g of each of the plant extracts was mixed with 5 ml of distilled water. 2 ml of glacial acetic acid containing one drop of ferric chloride solution was added to each extract. This was underlayed with 1ml of conc.H 2 SO 4 . A brown ring at the interface indicated the presence of a deoxy-sugar characteristic of cardenolides. A violet ring appeared below the brown ring, while in the acetic acid layer a greenish ring may formed just above the brown ring that gradually spread throughout this layer.

DETERMINATION OF TOTAL PHENOLIC CONTENT
The total phenolic content of different extracts of rhizome of Alpinia galanga and leaves of Eryngium foetidum was evaluated using a method The metal chelating capacities of the extracts were evaluated using the following equation:

ANTI-DIABETIC ACTIVITY ASSAY (IN VITRO)
Anti-diabetic potential of verious extracts of rhizome of Alpinia galanga and leaves of Eryngium foetidum were assay in vitro by a method proposed by Prabhakar et al. 2013. 22 The in vitro anti-diabetic activity was determined by assaying the inhibitory activity of enzyme α-amylase which involves in the breakdown of starch to produce glucose. In this method, 1 ml of extracts was added to 1 ml of the enzyme α-amylase in a test tube and incubated for 10 minuts at 37 o C. Then 1 ml of 1% starch solution was added into it and again incubated for 15 minutes at 37 o C. Then 2 ml DNSA reagent was added in to it, in order to terminate the reaction. The reaction mixture was then incubated in boiling water bath for 5 minutes, and then allows it to cool at room temperature. The absorbance of the reaction mixture was then measure at 546 nm in spectrophotometer. The standard (control) of the reaction without the extract represents the 100% enzyme activity. The % age inhibition of enzyme activity of α-amylase was determined by:

Well diffusion method
The antimicrobial activity of the extracts was determined by Kirby-Bauer Method in agar well diffusion. 23 In this method initially the stock cultures of bacteria were revived by inoculating in broth media and grown at 37 o C for 18 hours in incubator. Then agar plates of the Muller Hilton's Agar media were prepared. Each plate was inoculated by 18 hours fresh bacterial suspension (10 5 -10 6 colony forming unit "CFU"/ml) or fresh bacterial culture by swabbing evenly on the surface of solid agar media. After 20 minutes, wells of 6 mm diameter were made in solid agar medium with the help of gel puncher and filled with 50 µl of various test samples i.e. DMSO dissolved solvent extracts of rhizome part of the plant A. galanga and leaf of E. foetidum. The positive control wells were filled with Amikacin 30 (Standard drug). All the plates were incubated at 37 o C for 24 hours and then during observation after 24 hours, the diameter of zone of inhibition was measured in millimetre. All the equipments used like media, Petri plates, cotton swab etc. must be sterilised before performing the experiments. As well as proper labelling of the petri plates must be done before performing. All the determination of antibacterial activities of plant extracts were also done in triplicates.

STATISTICAL ANALYSIS
The results are represented as mean of three replicates followed by standard deviation i.e. Mean ± SD. The data were subjected to student t-test analysis and the significance of the difference was determined by Graph pad. P-value<0.05 were considered to be significant. described by Kim et al. 2003. 17 According to this, 0.5 ml of extract was added to 4.5 ml of distilled water and then 0.5 ml of Folin Ciocalteu reagent was added to it. After mixing, the mixture was maintained at room temperature for 5 minutes followed by the addition of 5 ml of 7% sodium carbonate and 2 ml of distilled water in to the mixture. After mixing the solution was incubated for 90 minutes at 23°C. The absorbance was measured by spectrophotometer at 750 nm. The total phenolic content was expressed in mg/g of Gallic acid equivalent, as standard. Determination of total phenolic content was performed in triplicate.

DETERMINATION OF TOTAL FLAVONOID CONTENT
The flavonoids content of different extracts of rhizome of Alpinia galanga and leaves of Eryngium foetidum was measured based on methods described by Ebrahimzadeh et al. 2008. 18 According to this method, 0.5 ml of each extracts was mixed with 1.5 ml of methanol and then 0.1 ml of 10% aluminium chloride was added, followed by the addition of 0.1 ml of 1M potassium acetate and then 2.8 ml of distilled water was added to the total mixture. Then mixture was incubated for 30 minutes at room temperature. The absorbance of the reaction mixture was measured at 415 nm in spectrophotometer. The total flavonoids content was expressed in mg/g of Quercetin equivalent, as standard. All the determination was perform in triplicate.

DETERMINATION OF DPPH (1,1-DIPHENYL-2-PICRILHYDRAZYL) RADICAL SCAVENGIN ACTIVITY
The DPPH radical scavenging activity of different extracts of rhizome of Alpinia galanga and leaves of Eryngium foetidum were determined using the method proposed by Von Gadow et al. 1997. 19 In this, 50 μl of samples of each extracts was placed in separate cuvettes, then in to it 2 ml of 6 X 10 -5 M methanolic solution of DPPH radical was added immediately and absorbance was measured immediately at 517 nm. Then after 16 minutes of incubation decrease in absorbance was again measured for the entire samples. Methanolic solution of Ascorbic acid of 1 μl/mg concentration was used as standard. All the determination was performed in triplicates. The DPPH Radical Scavenging Activity was measured in percentage of reduction of the colour. The percentage inhibition of the DPPH radical by the sample extracts was calculated by the formula suggested by Yen and Duh 1994. 20 Control was taken at time zero that mean, before the antioxidant present in plant extracts starts its activities.
A A(t) = Absorbance of antioxidant at time, t = 16minute.

METAL CHELATING ACTIVITY
The metal (ferrous ion) chelating activity by different extracts of rhizome of Alpinia galanga and leaves of Eryngium foetidum were estimated by a method suggested by Dinis et al. 21 with minor modification. In this, about 100 µl the each plant part extracts samples were added to 50 µl solution of 2 mM FeCl 2. Then the addition of 200 µl of 5 mM ferrozine started the reaction and then the mixture was shaken vigorously and left at room temperature for 10 minutes. Absorbance for reduction of colouration of the solution by each samples were measured at 562 nm in spectrophotometer. EDTA solution in place of sample extracts was taken as positive control and reagent mixture without sample was used as negative control. The metal chelating activity is expressed in percentage reduction of colour. All the determination was performed in triplicate.

RESULTS AND DISCUSSION
The different solvents extracts of Alpinia galanga and Eryngium foetidum exhibited presence of all the phytochemicals tested and varying degree of antioxidants activity in vitro. Flavanoids, phenols, saponins, tannins, anthocyanines, sterols, triterpenoids and anthraquinones were found to be the most common phytochemicals in both the plants. These findings suggest that these plants could be a promising source of bioactive compounds having therapeutic potentials. Both the plants showed comparable phytochemicals, antioxidant, antibacterial and antidiabetic potential (Tables 1-3).
These results indicate that these plant extracts could be a potential source of natural antioxidants as well as they have promising antidiabetic and antibacterial potentials.

CONCLUSION
Results of present study showed that the plant extracts have strong antioxidant, antidiabetic and antibacterial activities. The highest scavenging activity was seen in E. foetidum ethanolic extract, highest alpha amylase enzyme inhibition in A. galanga methanolic extract. E. foetidum contained higher flavonoids as well as phenolics contents than galanga. The highest was seen in ethanolic E. foetidum and lowest in A. galanga aqueous. But in terms of metal chelating activity, A. galanga extracts showed more than E. foetidum. The ethanolic extract of galanga was also advantageous as an antioxidant in food due to its mild odour compared with the essential oil. This knowledge of the phytochemical constituents of these plants would be valuable in future in order to discover the herbal folkloric remedies in low cost and high efficacy. Further studies are required to isolate active compounds of these extracts to elucidate medicinal potential of individual active ingredients which may give a promising drug molecule to be served for managing oxidative stress and related disorders. This will help to develop new, effective, safe and affordable herbal medicine for the treatment of diabetics and other oxidative stress related disorders.