Study on Phytochemical , Antibacterial , Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu

This study focuses on antibacterial, antioxidant and toxic potentials of Viscum album Linn, commonly known as European mistletoe associated with Acacia catechu (Khayer in Nepali). Methanol extract of the aerial parts of the Mistletoe was prepared by cold percolation method. The resulting extract was simultaneously subjected to phytochemical screening; anti-microbial activity; anti-oxidant potential and Brine shrimp toxicity test. The major biologically active phyto-constituents observed were alkaloids, glycosides, saponins, polyphenols, flavonoids, tannins, terpenoids and cardiac glycosides. Upon antibacterial activity screening, the plant extract was found to be highly effective against Pseudomonas aeruginosa with the zone of inhibition 16±1mm compared to 17±1mm of chloramphenicol (50 mcg). The antioxidant activity as EC50 value by DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging activity was found to be 1.58 mg/ml while the ferric reducing capacity was measured to be 282.83±19.55 mg FeSO4.7H2O eqvt/g dry wt. of the extract during Ferric Ion Reducing Antioxidant Power (FRAP) Assay. The LC50 value for Brine Shrimp Toxicity Assay was found to be 31.62 ppm. This study shows the medicinal value of the mistletoe associated with Acacia catechu. Further meticulous analysis of this plant might lead to identification of active biomolecules effective as drugs for various ailments.


Introduction
Viscum album Linn, commonly known as European mistletoe is a semi-parasitic shrub, growing on large variety of woody trees especially on their stems and branches [1].The plant is distributed along subtropical and temperate Himalayas at an altitude of 900-2100m [2].The most common host plants include Salix, Populus, Acer, Malus, Crataegus, Prunus, Sorbus, Abies, and Pinus [3].Viscum album in Nepal has been used as a medicinal herb since very long.Traditionally the paste of mistletoe bark is applied to relieve muscular swelling, boils and wounds and even in the management of joint dislocation; the berries act as laxative, tonic, diuretic and aphrodisiac while the plant has been used in complications like enlargement of spleen and tumors [2,4].The plant has also been used in case of epilepsy, vertigo, anxiety, exhaustion and hypertension [5].Bussing (2004) has compiled wide use of this plant in various countries as: In ancient Greek in spleen diseases and complications associated with menstruation while in Argentina; mistletoe has been used as sedative and as stabilizer in bone fractures; in India the mistletoe tea in diabetes; in Africa in treatment of various stomach troubles of children including diarrhea.The German Commission E Monographs, a guide to herbal medicine approved the use of the plant in rheumatism and tremor diseases.The evergreen mistletoe was regarded as a symbol of fertility and good luck and thus was believed to avoid any fertility problems [7].Despite the long traditional use of this herb in medicine, the chemical and biological study of this plant has only been initiated lately.The active chemical constituents reported from the mistletoe are Glycoproteins-mistletoe lectin I, II, and III; proteinviscotoxin; polysaccharides-galacturonan, arabinogalactan and alkaloids [5].Recently two novel amino-alkaloids namely 4,5,4'-trihydroxy-3,3'iminodibenzoic acid and 4,5,4',5'-tetrahydroxy-3,3'iminodibenzoic acid have been isolated and characterized [8].Other classes of secondary metabolites from Viscum album are flavonoids, phenylpropanoids, triterpenes, phytosterols, cyclic peptides and cyclitols [6].Being a parasitic plant, the phenolic content and the antioxidant capacity of mistletoe have been found to vary depending upon the host trees and the time of harvest [9,10].New compounds have been also reported in mistletoe growing on different hosts [11].The organic solvent fraction of mistletoe extract from its twigs and leaves showed good antimicrobial activity against both gram positive and gram negative bacteria [12,13] .The hexane extract of the plant has shown anti-fungal activity against C. albicans as well [14].

Materials and Methods
All the procedures were carried out in Natural Products Chemistry Laboratory, Faculty of Science, Nepal Academy of Science and Technology, Lalitpur, Nepal.

Plant Materials:
The aerial parts of Viscum album Linn.associated with Acacia catechu were collected from Sarlahi District, Nepal (1,000 -3,300ft).It was shade dried and crushed into powder using a crusher.The resulting powder was stored in plastic bag for further work at room temperature.

Extraction of phytochemicals:
For extraction of phytochemical compounds, cold percolation method followed by intermittent sonication was followed.First the solvent methanol was added in the ratio of 8:1 (plant powder weight in gram) and shaken well then kept on stand for 3 days at room temperature.From day four onwards, the resulting solution was subjected for intermittent sonication (Rocker Scientific Co. Taiwan) -a continuous cycle of sonication at 20 kHz at 60˚C for 15 minutes followed by 2 hour standing at room temperature, till day six.On day six, the supernatant was slowly poured into the round bottom flask through cotton plug filtration which was then evaporated at reduced pressure in rotatory vacuum evaporator (Hanshin Scientific Co., Korea).Resulting dry extracts were then sealed in small glass containers and stored at 4˚C until use.

Phytochemical Screening:
The procedures for phytochemical screening has been followed in reference to the book "Phytochemical Techniques" and the research paper [16,17]

Antibacterial Screening:
The antimicrobial activity of the crude extract was studied by Agar well diffusion Technique [18].

Determination of Anti-oxidant Activity: DPPH (1,1-diphenyl-2-picrylhydrazyl) Free Radical Scavenging Activity:
A range of concentration varying from 0-10mg/ml of the sample was taken for the assay.A 50µl of the crude extract was added to 450µl of Tris-HCl buffer (0.05M, p H 7.4) and 1ml of 0.1mM DPPH was added to the resulting mixture and incubated in dark at ambient temperature for 30min.Absorbance value of resulting solution at 517nm was recorded [19,20] ©NJB, Biotechnology Society of Nepal 62 Nepjol.info/index.php/njb The free radical scavenging activity (RSA) of the samples was calculated in percentage by using the formula: Radical Scavenging Activity (RSA) = The EC50 values of each extract were calculated by using the formula given below: Ferric Ion Reducing Antioxidant Power (FRAP) Assay: 10µl of the extract was added to 30µl of distilled water and 300µl of FRAP reagent; the resulting solution was incubated at room temperature for 5min.Then after the intensity of the blue colored complex formed was measured spectrophotometrically using Jenway UV-Vis Spectrophotometer at 593nm.The FRAP Reagent was prepared by mixing 25 ml of Acetate buffer (300mM Sodium acetate at p H 3.6) and 2.5ml of TPTZ (10mM 2,4,6-Tripyridyl-s-triazine in 40mM HCl) with 2.5ml of Ferric chloride solution (20mM FeCl3.6H2O in distilled water).All the chemicals were prepared fresh and the FRAP reagent was warmed at 37˚C before use.Then the reducing power of each extract was expressed as equivalent to that of 1mM of Fe (II) i.e.FRAP unit.All the tests were carried out in triplicates (n=3).[21][22][23] Brine Shrimp Lethality Assay: Crude plant extract was prepared in three different concentrations of 1000ppm, 100ppm and 10ppm.100ul of each of the extracts of each concentration were taken in wells of micro-titer plate.Freshly hatched brine shrimp (Artemia salina) nauplii were placed in each well at the rate of 5 in each and the plate was incubated at 22-28˚C for 24hrs.Potassium dichromate (K2Cr2O7 at 1mg/ml) was used as positive control [24].The percentage viability of the larvae was observed and the percentage mortality was calculated as:

Percentage of Mortality (PM) =
Then the LC50 values for each of the extracts were also calculated according to the formula given below:

Statistical Analysis
The final data were presented as mean ± standard deviation from the three independent assays.The DPPH radical scavenging activity and brine shrimp toxicity values were calculated using Microsoft Excel 2007 software while these data were exported to GraphPad Prism V.5.0 for generation of graphs and further analysis.

Results
The dried aerial parts upon extraction with methanol resulted into light brown dry powder.The yield percentage was 26.67% as shown in Table 1.The qualitative testing of bioactive phytochemicals in the methanolic extract of the aerial parts of Viscum album Linn.showed rich presence of alkaloids, saponins, phenolics and tannins.However, the flavonoid content in this extract was found to be very low.These phytochemical compounds are known to exert bioactive properties for medicinal plants.Other phytochemicals detected were steroids, terpenoids and cardiac glycosides as shown in Table 2.The bacterium Pseudomonas aerouginosa was the most susceptible bacterium to the plant extract for which the zone of inhibition was marked to be 16mm while the rest of the microorganisms were not inhibited to significant extent, shown in Table 3. Chloramphenicol (50mcg) was employed as the positive control which showed significant inhibition to all the test organisms.The crude methanolic extract was then subjected to in vitro test to evaluate antioxidant activity using two different tests: DPPH Free radical Scavenging Assay and FRAP assay.During DPPH Assay the EC50 value of the methanolic extract of Viscum album was found to be 1.58 mg/ml which was only 50 times lower than that of the standard -Gallic acid as shown in Table 4.The value of FRAP assay was expressed as mg FeSO4.7H2O per gm dry wt. of the sample.In table 5, the FRAP activity value of the plant found to be 282.83±19.55mg FeSO4.7H2O/gmdry wt.has been shown.In the brine shrimp toxicity assay, the degree of lethality was found to be directly proportional to the concentration of the extract.All the brine shrimp larvae were found to be alive after 24 hours in the extract concentration of 10ug/ml while higher concentration was toxic enough to kill all the larvae before 24hrs.(Table6) The plant extract showed significant toxicity which was measured to be an LC50 of 31.62 ppm (Table 7).

Discussion
Preliminary Phytochemical screening of the aerial parts of V. album Linn.associated with Acacia catechu, especially the stem parts showed presence of rich bioactive secondary metabolites like Alkaloids, Cardiac Glycosides, Saponins, Phenolics, Tannins and Steroids.This result was in positive correlation with findings from Bussing (2004) and Kunwar (2010).
These rich bioactive molecules have potential to be used in various ailments as tannins are capable of binding protein molecules which might have been exploited in treatment of diarrhea and skin bleedings by traditional healers.Anabolic steroids are known to retain nitrogen in osteoporosis and various wasting illnesses [25].Alkaloids are known for their inherent toxicity to cells [26].Flavonoids and phenolics are universally known as potent anti-oxidants and antiinflammatory agents and therefore have been found useful against tumors and cancers [27].Regulations of Na+/K+-ATPase-pumps are assisted by cardiac glycosides while saponins are well known for their immune-modulatory and anti-neoplastic effects [28,29].The rich presence of the phytochemicals of medical value thus proves the effective use of this plant in various formulations in ethno medicine.
The exciting results from the qualitative test of bioactive phytochemicals continued to show their strong presence in antibacterial activity assay as well.The crude extract was able to form a clear and distinct halo zone of inhibition measuring a diameter of 16mm (Table 3).This was quite comparable with that of the positive control used.Ps. aeruginosa is considered one of the most rapidly growing bacteria in its resistance to existing antibiotics [30].This bacterium has been one of the major causes of hospital acquired infections all around the globe [31].So, the excellent inhibition of this bacterium by V.   1 explains that the radical scavenging activity is in positive correlation with the concentration of the extract used.So, the RSA value can be enhanced further by increasing the purity of the extract.Similarly the FRAP activity has been calculated as 282.83±19.55 which is also very high value which indicates significant reduction potential of the extract.The polyphenols and flavonoids might be responsible for this active anti oxidant activities of this plant.This therefore provides evidence for the use of this plant in treatment of free radicals associated diseases like diabetes.
Toxicity is yet another important parameter to qualify any drug or drug formulation/preparation regarding safety.Preliminary monitoring of the toxicity of natural products has been carried out via Brine Shrimp Toxicity assay.The freshly hatched nauplii between 24-48 hours of hatching are considered highly sensitive to toxins and therefore this stage of brine shrimp larvae are used for toxicity assay.V. album extract was found to have LC50 31.62 ± 0.00 ppm which is below 1000ppm and the standard chart explains that only those plant extracts having LC50 lesser than 1000ppm are practically cytotoxic.This observed toxicity might be attributed to high alkaloid content of the plant.Duke, 2010 has mentioned toxicity of the lectin fraction, viscotoxin and the juice of the plant with the LC50 value of 80µg/kg, o.7mg/kg, 32mg/kg of mouse while the capability of mistletoe lectins in inactivating ribosomes thereby halting protein synthesis has been discussed by Bussing, 2004.Therefore care must be taken while formulating this herb for drug preparation.Instead the cytotoxicity of this plant might be exploited for their use as anticancer drugs.However, Viscum album Linn should be subjected to further rigorous bioassays for confirmation of specific toxicity.

Conclusion
The Viscum album aerial parts associated with Acacia catechu showed rich presence of bioactive phytochemical constituents like alkaloids, glycosides, saponins, flavonoids and phenolics.The significant inhibition of Ps. aeruginosa presents this plant a promise for screening active constituent against its resistant forms.The significant toxicity of the extract demands careful analysis of the plant before use in medical formulations in one hand while in other it also directs its potential use for targeting cancer cells.
. Mayer's Test and Dragendorff's Reagent Test were employed for detection of alkaloids while carbohydrates and cardiac glycosides were detected with Molisch's Test and Fehling's Test.Proteins and amino acids were tested by applying Biuret Test and Ninhydrin Test.
Liebermann-Burchard's Test gave results for presence of Phytosterols.Ferric Chloride Test, Alkaline reagent Test and Magnesium-Hydrochoric acid Reduction Test detected phenols and tannins.Bramer's Test was used for specific detection of tannins.Liebermann's Burchard test and Salkowski's Test detected terpenoids.Shinoda Test for flavonoids and Kellar-Killiani Test for cardiac glycosides were used.Saponins, Gums and Mucilages were also subsequently detected.

Table 1 :
Percentage Yield and Physical characteristics of the crude methanolic extract

Table 2 :
Preliminary Phytochemical Screening of methanolic extract The anti-bacterial activities of methanolic extract of Viscum album were evaluated against 6 bacteria (5 gram negative and one gram positive).The results are ©NJB, Biotechnology Society of Nepal 63 Nepjol.info/index.php/njbpresented as zone of inhibition by the extract (mm in diameter) against the growth of the test microorganisms.

Table 3 :
Anti-bacterial sactivity of the crude methanolic extract

Table 4 :
EC50 values of Crude methanolic extracts in DPPH Free

Table 5 :
Estimation of Antioxidant Activity of Crude Methanolic

Table 7 :
LC50 values in Brine shrimp cytotoxicity assay