Introduction
In the present scenario the life threatening microbial infections is on the rise on worldwide, irrespective of available and newer synthetic antimicrobial agents and moreover the usage of these agents are limited by the prompt development of multiple drug resistant after a very short exposure of these agents. The multiple drug resistant might be addressed by developing the drug originated from plant. Costus igneus famously also known as Insulin plant as twice daily intake of 2-3 leaves was shown to be effective on the management of diabetic mellitus patent, traditionally practiced in the state of Kannada, India.1, 2, 3, 4 Pharmacognatical analysis leaf of the various extracts found to possess important phytochemicals such as such as alkaloids, flavonoids, terpenoids, glycosides, steroids and saponins and the antidiabetic activity was correlated due to the presence of flavonoids and saponins.5 The present study was focussing the phytochemical and pharmacological screening on multiple extracts on rhizomes of Costus igneus on specific activities such as antioxidant, antibacterial, and antidiabetic activities to conclude the therapeutic potential of the Costus igneus rhizome.
Materials and Methods
Plant material
The Costus igneus fresh rhizomes were collected from Kodaikanal hills, Tamilnadu, India. The plant material was authenticated by School of Agriculture & Animal Sciences Gandhigram Rural Institute. The rhizomes were shade dried, powered by pulveriser and stored in an air tight plastic container, until further use.
Preparation of multiple Rhizome extracts of costus igneus
The 500g of the dried coarse powder of Costus igneus rhizome was first treated with petroleum ether, the defatted powder was extracted with series of extraction by ethyl acetate, methanol, ethanol and distilled water by cold maceration, each extract was filtered separately by whatman No. 1 filter paper, filtrates were evaporated to remove the solvent at 40 to 45°C by electrical water bath, obtained filter cakes of each extracts were freeze dried and stored separately at −20°C until use [Figure 1).
Gas Chromatography-Mass spectroscopy (GC-MS) Analysis
GC-MS is a hybrid analytical instrument that couples the separation capabilities of GC with the detection properties of the mass spectrometer (MS). The sample solution is injected into the GC inlet where it is vaporized and swept onto a chromatographic column by the carrier gas. The sample flows through the column and the compounds comprising the mixture of interest are separated by their relative interaction with the coating of the column and the carrier gas. The latter part of the column passes through a heated transfer line and ends at the entrance to the ion source where compounds eluting from the column are converted to ions. The next component is a mass analyzer, which separates the positively charged ions according to various mass-related properties depending upon the analyzer used. After the ions are separated they enter into the detector and the output from which is amplified to boost the signal.
GC-MS operating conditions
The initial column temperature was 35ºC with a hold time of 3 minutes. The temperature was programmed to rise by 8ºC/ minute with a final temperature of 280ºC. In the process, 1µl of the sample was injected into the port and immediately vaporized and moved down the column with helium as the carrier gas with a flow rate of 1 ml/minute. The MS Spectrum was taken at 70 eV. After the separation in the column, the components were identified and further analyzed by FID. The identification of the compounds was done by comparing in NIST MS 2.0 Structural library to find out the names, molecular weight, and structure.
Antioxidant activity in vitro (DPPH-Method)
It is determined by DPPH (1, 1-diphenyl2-picrylhydrazyl) by the principle based on the free radical scavenging activity by using ascorbic acid as a standard by measuring the absorbance at 517 nm by UV-Visible spectrophotometry against the blank methanol.6 Inhibition of free radical DPPH in percent was calculated as follows
IC50 values were calculated as the concentration of each sample required to give 50% DPPH radical scavenging activity from the graph (linear regression curve).
Antibacterial activity - in vitro (Disc-diffusion method)
Antibacterial study was carried out on 2 gram positive (B. subtiliesand S. aureus) and 2 gram negative (E.coliand P.aeruginosa) pathogenic organisms by disc diffusion method.7 The sample crude extracts of ethyl acetate, methanol, ethanol and aqueous extracts of 50 µg ig/ml and standard (ceftrioxone sodium 30 µg/ml) of each sample sterile disc was inoculated with 4 bacteria in sterile nutrient agar medium containing petri dish with the help of laminar flow, then all the petri dishes are transferred aseptically into incubator and incubated at 37°C for 24 h, after the incubation period of 24 h the zone of inhibition is measured, (in mm) tabulated and the results were compared with standard.
In vitro diabetic activity: Inhibition assay for α-amylase activity (DNS)
Acabose (standard) and various Costus igneus rhizome extracts (ethyl acetate, methanol, ethanol and aqueous (tests) of five concentrations (40, 80, 120, 160 and 200µg/ml) were prepared by dissolving in double distilled water. A total of 1000µl of each plant extract and 500 µl of 0.02M sodium phosphate buffer (pH 6.9 with 0.006M sodium chloride) containing α-amylase solution (0.5mg/ml) were incubated for 10 minutes at 25°C.After pre-incubation, 500µl of 1% starch solution in 0.02M sodium phosphate buffer (pH 6.9 with 0.006M sodium chloride) was added to each tube at 5s intervals. This reaction mixture was then incubated for 10 minutes at 25°C.1ml of DNS colour reagent was added to stop the reaction. These test tubes were then incubated in a boiling water bath for 5 minutes and cooled to room temperature. Finally, this reaction mixture was again diluted by adding 10ml distilled water following which absorbance was measured at 540nm.8
IC50 values were calculated as the concentration of each sample required to give 50% DNS α-amylase inhibitory activity from the graph (linear regression curve).
Figure 1
a: Ethyl acetate Extract of C. igneus of C. igneus b: Ethanolic Extract of C. igneus c: Methanolic Extract of C. igneus d: Aqueous Extract of C.igneus
Table 1
Qualitative phytochemical analysis
Table 2
GC-MS analysis chromatogram with pharmacological activity of EEIP
Table 3
In vitro Antioxidant activity by DPPH scavenging assay
Table 4
Antibacterial activity by disc diffusion method
Table 5
In Vitro Antidiabetic activity by α-amylase enzyme inhibitory activity
Results
Preliminary phytochemical screening
The Costus igneus rhizome extracts contained alkaloids, glycosides, saponins, flavonoids, terpenoids, and steroids as presented on the Table 1.
GC-MS phytochemical analysis
Among the Costus igneus rhizome extracts, it is evident that Costus igneus ethanolic rhizome extract possess all 3 in vitro pharmacological activity at moderate level, thereby we selected the same extract for GC-MS analysis in order to identify and correlate the specific phytochemicals possibly play a role in respective pharmacological actions. The results showed about 14 specific phytochemicals and its specific pharmacological actions as represented in the Figure 2 and Table 2.
Antioxidant activity - in vitro (DPPH-method)
Ascorbic acid showed the maximum antioxidant effect with the percentage inhibition of 93.6 % at 100 µg/ml concentration by the DPPH method, nearly close antioxidant effect was observed with Costus igneus rhizome extracts of ethyl acetate, ethanolic, methanolic and aqueous at the same concentration with the percentage inhibition of as 75%, 77.2%, 74.6 % and 64.2% respectively [Table 3 and Figure 3] and the IC50 value of ascorbic acid, ethyl acetate extract, ethanolic extract, methanolic extract and aqueous extract of Costus igneus rhizome was found to be 9.36 µg/ml, 12.49 µg/ml, 22.90 µg/ml, 44.45 µg/ml and 76.87 µg/ml respectively.
Antibacterial activity - in vitro (disc-diffusion method)
The antibacterial activity various rhizome extracts (50 µg/ml) of Costus igneus was compared with the standard drug (Ceftrioxone sodium 30 µg/ml) determined by disc diffusion showed highest zone of inhibition (22, 23, 20 and 26 mm) for the standard drug for B. subtilies, S. aureus, E.coli and P. aeruginosa respectively, the antibacterial activity of Costus igneus ethyl acetate, ethanol, methanol has moderate antibacterial activity as compare to the Ceftrioxone sodium (standard). Ethyl acetate has slightly higher antibacterial activity among the other extracts, however Costus igneus aqueous extract has no antibacterial against any of the bacteria [Table 4 and Figure 4].
α-Amylase inhibition activity by DNS method (in vitro)
The percentage inhibition for anti-diabetic activity was assessed at concentration ranges from 40 µg/ml to 200 µg/ml for standard and test and the maximal inhibition effect at 200µg/ml concentration was found to be 95.25%, 75.91%, 83.21, 68.24 and 62.77% for acarbose, ethyl acetate extract, ethanolic extract, methanolic extract and aqueous extract of Costus igneus rhizome extract respectively [Table 5 and Figure 5] and the IC50 value of acarbose, acetate extract, ethanolic extract, methanolic extract, and aqueous extract was found to be 10.04 µg/ml, 38.18 µg/ml, 20.11 µg/ml, 105.05 µg/ml and 153.99 µg/ml respectively. It implies Costus igneus rhizome ethyl acetate and ethanolic extracts has more potent, whereas methanol and aqueous extract of has mild in vitro anti-diabetic effect by inhibition of α-amylase activity.
Discussion
Insulin plant medicinal value was established traditionally for the treatment of diabetes and other ailments and substantiated by recent work on Costus igneus various leaf extract. The phytochemical analysis of Costus igneus rhizome extracts confirm the presence glycosides, saponins, flavonoids, terpenoids and steroids. Based upon rich pharmacological activities observed on ethanolic extract we have selected and performed GC-MS anlaysis for specific phytochemicals, results showed 14 important specific compounds as mentioned.
In vitro anti-oxidant effect was significantly high and almost same for all Costus igneus rhizome extracts by DPPH assay. The results also showed Costus igneus rhizome extract of ethyl acetate, ethanol and methanol possess the moderate antibacterial activity against gram positive (B. subtilies and S. aureus) and gram negative pathological organism (E.coli and P. aeruginosa) by disc diffusion method as compare to the standard drug (ceftriaxone sodium), but no antibacterial activity was found against aqueous extract. α-amylase inhibition activity by DNS method results infer, high anti-diabetic activity for Costus igneus ethyl acetate and ethanolic rhizome extracts whereas mild activity for methanolic and aqueous rhizome extracts.
Conclusion
Among the Costus igneus rhizome extracts, ethonolic extract has maximal antioxidant, antibacterial and antidiabetic activities also found to possess nearly similar phytochemicals as that of the leaf, especially the flavonoids were found to be more as compare to the remaining extracts. Thereby we would like to conclude that Costus igneus ethonolic extract might possess therapeutic potential on the antioxidant, antibacterial segment, and antidiabetic segments. Based on GC-MS Analysis in cognition with literature review we would like to conclude that antidiabetic activity might be mediated by gamma-sitosterol, antioxidant activity might be mediated by 7,9-Di-tert-butyl-1-oxaspiro [4.5] deca-6,9-diene-2,8-dione, 2,4-ditert-butylphenol and squalene. The antibacterial activity might be mediated by 7,9-Di-tert-butyl-1-oxaspiro [4.5] deca-6,9-diene-2,8-dione and 9,19 - Cyclocholestan-3-ol, 14-methyl- (3.beta.,5.alpha). However further in vivo and in vitro screening of Costus igneus ethanolic extract on specific phytochemicals are much needed to confirm the same.
