Preliminary Phytochemical Analysis and Antioxidant , Antibacterial Activities of Crocus alatavicus from Kazakhstan

Phytochemical analysis of C. alatavicus revealed the presence of phenols, flavonoids, anthocyanins, carotenoids, amino acids and carbohydrates. The flavonoid, amino acids and carotenoid contents were higher in aerial part (1.50%, 7.49% and 9.78 mg%, respectively) than in bulb (0.43%, 3.88% and 0.91 mg%, respectively). Total phenolic content (TPC), total antioxidant (TAA), 2.2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and antibacterial activities of water, methanol, ethanol and dichloromethane extracts from aerial part and bulb were tested. TPC ranged from 13.63 to 72.29 mg gallic acid equivalents (GAE)/g extract. The maximum TAA were observed in ethanol (61.34%) and methanol extracts (46.13%) from aerial part with a high TPC (72.29 and 62.37 mgGAE/g extract, respectively). Ethanol extracts from aerial part and bulb had good scavenger of DPPH radicals (65.5% and 54.08%, respectively) with an IC50 387 and 447 μg/ml. Ethanol extract from aerial part was most effective against gram-positive bacterial strains S. aureus, B. subtilis and B. cereus. Biological activities of the extracts were correlated with the TPC. It can be deduced that ethanol and methnol extracts of C. alatavicus contains useful potent bioactive compounds with antioxidant and antimicrobial activities.

C. alatavicus is an early spring ephemeral and geophyticgeocarpic species that grows in subalpine areas of the northern and western Tianshan Mountains (Baytenov, 2001;Zhang and Tan, 2009).It is an endemic of Kazakhstan flora belonging to the group of bulbous and tuberous plant.Wild habitat of this species is the Kora river middle flow, Koppal district, Taldykurgan region and South-East Kazakhstan.It widely distributed in Xinjiang that bordered by Mongolia, Kazakhstan, Uzbekistan and Kyrgyzstan (Saxena, 2015).The species mainly has potential in horticulture; it is the beautiful herb, highly rated for early blooming and easy cultivation (Ivaschenko, 2005).
ninhydrin reaction was analysed.The phenol-sulfuric acid method was used for determination of carbohydrates in the samples.The total carotenoid content was determined by the standard spectrophotometric method.

Determination of total phenolic content
Total phenolic content (TPC) of the different extracts of C. alatavicus was determined with the Folin-Ciocalteu reagent according to the procedure reported by Türkoğlu et al. (2010), using gallic acid as a standard.The standard gallic acid curve was produced within the concentration range from 0.01 to 0.2 mg.The results were calculated by the equation based on the standard curve: y = 1.1592x -0.0013, R 2 = 0.9986.TPC was expressed as mg of gallic acid equivalents (GAE) per g of dry extract.

Determination of total antioxidant activity
Total antioxidant activity (TAA) of the extracts was estimated by the β-carotene bleaching test following the procedure described by Wettasinghe and Shahidi (1999) with a slight modification.A stock solution of β-carotene-linoleic acid mixture was prepared as follows: 0.2 mg β-carotene was dissolved in 1 ml of chloroform (HPLC grade), 20 µl linoleic acid and 200 µl Tween 20 were added.Chloroform is completely evaporated at 40 o C for 10 min.Then, 50 ml distilled water was added with a vigorous shaking.4.8 ml of this reaction mixture was dispensed to test tubes and 200 µl portions of the extracts, prepared at 2 mg/ml concentrations were added.The mixture is then gently mixed and incubated at 50 o C for 2 h.Absorbance of the sample was measured every 30 min for 2 h at 470 nm using a spectrophotometer.The same procedure was repeated with synthetic antioxidant butylated hydroxytoluene (BHT) as a positive control.TAA was calculated using the following equation: AA = (1 -A0 -At / Ao o -At o ) x 100, AA is an antioxidant activity, A0 and A0 0 are the absorbance values measured at the initial incubation time of samples and control, respectively.While At and At 0 are the absorbance values measured in the samples and control at t = 120 min.

DPPH radical scavenging assay
The hydrogen atoms or electron-donation ability of the extracts is measured from the bleaching of a purple-coloured methanol solution of DPPH according to the method Sanja et al. (2009).Stock solutions of the extracts were prepared at 1 mg/ml concentration by dissolving in methanol and various concentrations (100-500 µg/ml) of the extracts were tested.Ascorbic acid (10,15,20,25, 50 µg/ml) and Trolox (10, 25, 50, 75 µg/ml) were used as positive control.DPPH radical scavenging activity (% antiradical activity) was calculated using the following equation: % antiradical activity = (A0 -A1) / A0 x 100, A0 is the absorbance of the control and A1 is the absorbance of the extract/standard.A percent inhibition versus concentration curve was plotted and the amount of antioxidant necessary to decrease the initial DPPH concentration by 50% (IC50 value) was calculated.

Antimicrobial activity test
Antibacterial activity of some extracts was determined by the disk diffusion method (Singh et al., 2002).Among the extracts obtained by polar solvents only water and ethanol extracts were tested for antibacterial activity.The extracts obtained by C. alatavicus is also interested as a source of naturally active substances that have many useful biological properties.Accumulation of the bioactive compounds is associated with the geophytic life-form of the plant and its ephemeral development cycle.Formation of underground storage corms contributes plant to survive adverse environment and proceeds the growth and reproducing of vegetative and generative organs (Kamenetsky and Okubo, 2013).
The species was used as spasmolytic, anti-inflammatory, bactericidal and antiviral agents in traditional Chinese medicine (Duke and Ayensu, 1985).The flowers were used as diuretic, for treatment of abdominal illness and to improve hormonal regulation of women (Egamberdieva et al., 2012).Dried stigmas are applied for colouring food products and impart flavour for them (Golovkyn et al., 2001).
Little is known about the phytochemical constituents of this species.The leaves contain ascorbic acid and the stigmas of flowers contain yellow pigment available for food colorant (Ivaschenko, 2005).Anthocyanins such as delphinidin 3-O-βrutinoside and petunidin 3-O-β-rutinoside and flavonoids such as myricetin, quercetin, kaempferol have been investigated in flowers (Nørbaeket al., 2002).It was reported that content of biological active substances in bulb of C. alatavicus depens on the vegetation period and weather conditions and it contributes the resistance of the plant to hostility and microflora during the growth (Kukushkina and Sedelnikova, 2010;Sedelnikova and Kukushkina, 2014).Studies addressing its biological activities are currently nonexistent.Hence, the aim of this study was preliminary phytochemical screening of different plant part and estimation of in vitro antioxidant, antimicrobial activities and total phenolic content of various extracts from aerial part and bulb of C. alatavicus.

Plant material
Different parts (aerial part and bulbs) of C. alatavicus were collected from the natural environment of Almaty (Kazakhstan) in March, 2014.The Voucher specimen was deposited in the herbarium of Institute of Plant Biology and Biotechnology (Almaty, Kazakhstan).Aerial part and bulbs were cleaned, air dried in the shade at room temperature.

Preparations of the extracts
Powdered samples (10 g) were extracted with 100 ml of different solvents (distilled water, 96% ethanol, 99% methanol and 99% dichloromethane) at 60 °C (methanol at 35 °C) for 6 hours in a water bath shaker.After the filtration with Watman No:1 filter paper the solvents were concentrated under vacuum (48-49 °C) by evaporating to dryness.All the extracts were stored at -20 °C until use.

Phytochemical analysis
Preliminary qualitative tests for screening of major biological active compounds in powdered aerial part and bulb were carried out according to the Harborne (1984).Standard quantitative analysis of flavonoid, carotenoids, carbohydrates and amino acid was used.For the determination of the total flavonoid content of the samples the aluminum chloride colorimetric method was used with quercetin as a standard.Free amino acid content by relatively nonpolar dichloromethane also were tested.Four strains of bacteria including gram-positive Staphylococcus aureus, Baсillus subtilis, Baсillus cereus and gram-negative Escherichia сoli were used for tests.Sample solution were prepared by dissolving the dried extracts in their solvents (water, ethanol and dichloromethane) to give concentration of 1 mg/ml.25 and 50 µl of the extract was impregnated into standard disks.Antibacterial agent kanamycin (1 mg/ml; 20 µl/disk,) was used as a positive control and antimicrobial activity of each solvent used for extraction (ethanol and dichloromethane at 50 µl/disk) was tested.

Statistical analysis
All experiments were carried out in triplicate and expressed as average of three analyses ± standard deviation and Excel 2013 was used.

Phytochemical screening
Qualitative analysis of the main biological active compounds revealed the occurrence of flavonoids, anthocyanins, phenols, carotenoids, amino acids and carbohydrates in aerial part of C. alatavicus (Table 1).The presence of flavonoids, phenols, carotenoids, carbohydrates and amine-containing compounds were found in bulb.
The results of quantitative analysis of some determined biological active compounds in different parts of C. alatavicus are given in Table 2.
The plant material of C. alatavicus has abundant presence of secondary metabolites such flavonoids, phenols, carotenoids.The carotenoids content was greater in aerial part (9.8 mg%) than in bulbs (0,9 mg%).Flavonoids are 1.50% in the powdered aerial part and 0.43% in the powdered bulbs.The results revealed that aerial part of plant contains more biological active substances that may have high biological activities.
Phytochemical constituents of this species insufficiently explored.Aerial part contains ascorbic acid and some glycosides.Presence and quantitative content of saponin, ascorbic acid, catechin, pectin and protopectin in bulb of C. alatavicus depends on vegetation period of the plant (Kukushkina and Sedelnikova, 2010).It was reported that high content of ascorbic acid in aerial part and in bulb is observed in summer and it twice decreases in autumn (Sedelnikova and Kukushkina, 2014).Some flavonoids such as derivatives of myricetin, quercetin and kaempferol have been identified by HPLC in flowers of C. alatavicus (Nørbaek et al., 2002).

Total phenolic content
The antioxidant activity, radical scavenging capacity and TPC of C. alatavicus has not previously been published.The reason to determine TPC in the plant extracts are directed that phenolics are the main group of compounds acting as antioxidants or free radical scavengers.
Methods for assessment of total phenolic content and determination of their antioxidant capacity are mostly based on oxidizing-reducing properties, possibility of phenolic compounds functioning as reduction agents and offering hydrogen radical or electron (Stratil et al., 2007).The results on TPC of C. alatavicus extracts obtained using different solvents are presented in Table 3.
The different solvents showed different content of phenolic.The highest phenolic contents were obtained in ethanol and methanol extracts from aerial part (72.29; 62.37 mg GAE/g extract, respectively).Ethanol and methanol extracts from bulb have shown higher amount of phenolic (43.65 and 36.06 mg GAE/g extract, respectively) than water and dichloromethane extracts from aerial part and bulb (23.55 and 24.26; 14.49 and 13.69 mg GAE/g extract, respectively).Rahaiee et al. (2015) reported that solvent type has significant effects on the TPC of the saffron extracts and 80% ethanol extract of saffron had the highest amount of TPC than aqueous and methanol (50%, 80%) extracts.
The different polarity of organic solvents influences to the efficiency of the extraction and the solubility of phenolic compounds.Previous study has shown that the phenolic compounds more extracted by ethanol and methanol than water and dichloromethane.Therefore, the lowest TPC was observed in water and dichloromethane extracts.

Total antioxidant activity
The β-carotene bleaching test is based on the loss of the yellow colour of β-carotene due to its reaction with radicals which are formed by linoleic acid oxidation in an emulsion (Kulisic et al., 2004).Table 4 shows the potential of C. alatavicus extracts and standard (BHT) to inhibit lipid peroxidation as evaluated by the bleaching of β-carotene.

Radical scavenging activity
The DPPH test is based on the ability of the stable DPPH free radical to react with hydrogen donors.In this test, a solution of radical is decolourized after reduction with an antioxidant or a radical (Parejo et al., 2000).
The results of the experiments also indicated that ethanol extracts of C. alatavicus have the high free radical scavenging activity.The comparable free radical scavenging rates of the extracts at the concentration of 500 µg/ml are shown in Fig. 1.
The highest scavenging activity was observed in ethanol extracts from aerial part and bulb (65.5% and 54.08%).Methanol extracts showed lower inhibitory activity (48.73% and 35.04%), but higher than activity of dichloromethane extracts (17.9% and 13.9%, respectively).
The IC50 value is one of the important parameter in determination of antioxidant capacity.IC50 value was determined from the plotted graph of scavenging activity against various concentrations of the extracts.IC50 values of C. alatavicus extracts were calculated to compare the antioxidant activity and listed in Table 5.
The lowest IC50 indicated the strongest ability of the extracts to act as DPPH radicals scavengers.Out of the all extracts minimum IC50 (387 µg/ml) was determined in ethanol extract from aerial part.Dichloromethane and water extracts showed the high IC50 values (from 801 to 3,221 µg/ml).Antiradical activities of commercial antioxidants of vitamin C and Trolox were higher (IC50 26.35 and 37.16 µg/ml).
The results obtained in this study show that DPPH scavenging activity of C. alatavicus is less than C. sativus, but higher than other species of Crocus as compared to the published experimental data.So, IC50 value of C. sativus ranged from 231.75 to 210.79 µg/ml for methanol extract, 255.44 µg/ml for water extract and 299.4 µg/ml for ethanol extracts (Karimi et al., 2010;Sariri et al., 2011).Whereas methanol extracts of C. baytopiorum, C. flavus, C. biflorus showed 78.21%, 90.51% and 76.52% radical scavenging activity at 1.6 mg/ml concentration (Acar et. al., 2010).Trice less concentration of ethanol extracts from different part of C. alatavicus initiate activity about 65.5% and 54.98% in this study.

Antibacterial activity
The various extracts from C. alatavicus showed selective and varying degree of activity against different bacteria.The results of the zone inhibition determined by the disk diffusion method are presented in Table 6.
It is clear from the results that all tested bacterial strains were susceptible at 20 µl concentration of antibiotic kanamycin.Among the solvents, ethanol at 50 µl showed antibacterial activity against strains of E. сoli, B. subtilis and B. cereus, but did not show activity against stains of St. aureus.Dichloromethane was low effective only against E. сoli with 13.08 mm diameter zone of inhibition.Plant extracts exhibited selective activity against different bacteria; did not show activity against gramnegative bacteria E.сoli, but were effective against gram-positive S. aureus, B. subtilis and B. cereus.Ethanol extracts with a high phenolic content showed a high inhibition activity.In addition, ethanol extract from aerial part at 50 µl concentration showed antibacterial activity against B. subtilis, B. cereus and S. aureus.Diameter of zone inhibition ranged from 17.32-21.72mm, compared to control (70% ethanol) 10 mm.Minimal concentration of the extract (25 µl) did not show activity against all test-cultures.Ethanol extract from bulb was effective against B. subtilis and B. cereus at all tested concentrations.Diameter of zone inhibition ranged from 15.66 to 20.67 mm.Water and dichloromethane extracts with a low phenolic content were not effective against all the tested bacteria.Dichloromethane extract from bulb showed antibacterial activity against only B. cereus at 25 µl concentration (zone of inhibition -20.03 mm).It has been reported that aqueous extracts of saffron onion at different concentration also have not antibacterial activity against B. anthracis, S. enteritidis, S. aureus, Proteus, E. coli by the disk diffusion test (Soureshjan and Heidari, 2014).Aqueous extract of C. sativus also showed least activity, but hexane and methanol extracts showed a promising antibacterial activity (Hussain et al., 2014).
The results of this study reveal selective antibacterial activity of ethanol extracts of C. alatavicus with a high phenolic content against gram-positive bacteria S. aureus, B. subtilis and B. cereus.

Conclusions
The results obtained by the phytochemical screening of C. alatavicus revealed the predominant content of secondary metabolites in aerial part of the plant.The highest TPC and DPPH-radical scavenging activity and TAA were detected in ethanol and methanol extracts.These results indicated that antioxidant activities C. alatavicus are correlated with the amount of phenolic in the plant extracts.Ethanol extract from aerial part compared to the other extracts is the most effective against tested gram-positive strains.Hence, C. alatavicus growing in Kazakhstan area may be considered for accessible source of natural active compounds with antioxidant properties as well as antimicrobial activity.Further studies will be done to develop the effective methods of in vitro propagation of the valuable endemic species of crocus.

Table 2 .
Quantitative contents of some biological active compounds of C. alatavicus

Table 3 .
Total phenolic content of the extracts from different parts of C. alatavicus

Table 4 .
Total antioxidant activity (% ± SD) of C. alatavicus extracts and standard by the β-carotene bleaching method

Table 5 .
IC50 values of C. alatavicus extracts and standards

Table 6 .
Antimicrobial activity of the various extracts from C. alatavicus Note: -= no activity; data are mean of three replications