Analysis of Phytochemical Constituents and Biological Activity of Taxus Wallichiana Zucc . Dolakha District of Nepal

Abstract Phytochemical and biological activities of methanolic extract of Taxus wallichiana Zucc. (Leaf, stem) were carried out. The brine shrimp bioassay showed T. wallichiana is pharmacologically active. The antibacterial potential was studied against one gram positive bacteria (Staphylococcus aureus) and one gram negative bacteria (Escherichia Coli) using Agar Well Diffusion Method. Stem of T. wallichiana showed significant zone of inhibition against gram positive bacteria while the leaf of T. wallichiana did not show significant zone of inhibition against both gram positive and gram negative bacteria. Antioxidant activity was evaluated by 2, 2-diphenyl-1-picryl hydrazyl (DPPH) free radical scavenging activity and FRAP assay. Both assay showed that T. wallichiana leaves has high antioxidant activities.


Introduction
Taxus wallichiana Zucc.known as Himalayan yew, belongs to the family Taxaceae (Shrestha et al.,1997).It is medium sized temperate, himalayan forest tree of medicinal importance (Baquar.1989).T. wallichiana Zucc.(Himalayan Yew) has a remarkable history of medicinal uses in contrast to the other yews.Depending on taxonomic treatment, T. wallichiana are found to have a wide growth range in Asia, stretching from Afghanistan through the Himalayas to Philippines.It is found growing in Afghanistan, Bhutan, China, India, Indonesia, Malaysia, Myanmar, Nepal, Pakistan, Philippines and Vietnam at the altitude range of 1500-3500 m asl.It has been used by the native population for treating common cold, cough, fever and painful inflammatory conditions.The leaves of this plant are used to make herbal tea for indigestion and epilepsy.Traditionally, it's bark and leaves are used in steam bath to treat rheumatism and the paste made from its bark is used to treat fractures and headaches (Gajurel et al.;2014).After the novel discovery of anticancer drug taxol from T. brevifolia, by Wani and Wall in 1971, tremendous work has been carried out on the chemical investigation of almost all parts (needles, bark, roots, seeds and heartwood) of several yew species, resulting in the isolation and characterization of over 300 taxoids (Appendino. (1995); Wani et al.1971;Baloglu and Kingston (1999) and Wall and Wani (1995).Although its antifungal, antiviral, analgesic, antipyretic, anti-allergic anti-inflammatory and tumor growth inhibitory activity has been reported, most of the research on Taxus have been based on its propagation and

Research Article
B. Subba.( 2018)  This paper can be downloaded online at http://ijasbt.org&http://nepjol.info/index.php/IJASBTanti-cancerous properties.Other equally important aspects of this evergreen gymnosperm, such as its antioxidant, antimicrobial potential, still needs advance and focused research efforts as only few has been reported (Erdemoglu and Sener (2001).
Studies report that Nepal Taxus wallichiana Zucc.has anticancer activity.However, no antioxidant and antimicrobial activity has yet been reported using T. wallichiana Zucc.T. wallichiana is renowned for its medicial potency, but we know that bioactive compounds found in plants may depend on the location where they grow.T. wallichiana used in this study was obtained from Jiri, Dolakha district of Nepal.This study of the phytochemical and biological properties of T. wallichiana will also provide information specific to plants grown in Jiri, Dolakha district of Nepal.The findings from this work may add to the overall value of the medicinal potential of T. wallichiana.

Plant Collection
Taxus wallichiana's leaves and stem were collected from Jiri, Dolakha district of Nepal.The plant was identified at the Central Department of Botany, TU Kathmandu, Nepal.

Preparation of Extracts
The collected plants were cleaned, air dried in shade.Exposure to the sunlight is avoided to prevent the loss and transformation of the active components.The completely dried samples were grinded into fine powder.The extraction of chemical constituents of plant material was carried out with methanol by the process of cold percolation.The powdered material was kept in clean and dry conical flask and dipped in methanol.It was left for 2-3 days at room temperature with shaking at intervals.Then it was filtered and filtrate was concentrated using rotator evaporator.This process was repeated for 6-7 times.The concentrated filtrate was air dried to obtain solid or semisolid residue.The same process was repeated for all plants.After completely drying they were kept into beaker.The dried extracts were used for different tests.

Phytochemical Screening
The phytochemical screening of all plant materials were done on the basis of procedure given by Prof. I. Culie (1982).

DPPH Radical Scavenging Activity (RSA) Assay
The free radical scavenging activity of samples and standard ascorbic acid solution in methanol was determined based on their ability to react with stable 1, 1-diphenyl-2picrylhyrazyl (DPPH) free radical (Blois, 1958).The plant samples at various concentrations (15-250 μg/ml) were added to a 100 μM solution of DPPH in ethanol.After incubation at 37 °C for 30 min, the absorbance of each solution was determined at 517 nm.The measurement was performed in triplicates.The antioxidant activity of the samples was expressed as IC50 (inhibitory concentration), which was defined as the concentration (in μg/ ml) of sample required to inhibit the formation of DPPH radicals by 50%.Ascorbic acid was used as positive control.Free radical scavenging activity was calculated by using following equation: Where, AO = Absorbance of DPPH solution and AT = Absorbance of test or reference sample.The % scavenging was then plotted against concentrations used and from the graph IC50 was calculated.

FRAP Assay
The antioxidant activity by FRAP assay was conducted according to the procedure given by Benzie and Strain with slight modification (Benzie and Strain, 1996).The FRAP reagent was prepared by mixing acetate buffer of pH 3.6 (300 mM), TPTZ (tripyridyltriazine) solution of 10 mM and ferric chloride solution of 20 mM in the ratio of 10:1:1.Antioxidant activity was calculated with the standard calibration of ferrous sulfate.The leaf extracts (5 mg/ml) was prepared by adding methanol and was used as sample.Finally, absorbance was taken at 593 nm keeping the temperature 37 °C.

Microorganism
The microorganisms used in this study were identified strains obtained from Central Department of Microbiology, TU, Nepal.In this study one was gram positive (Staphylococcus aureus) and one gram negative (Escherichia coli) were used.

Antimicrobial Assay
The antimicrobial activity of the plant extracts were carried by disc diffusion method (Bauer et al., 1996).A suspension of tested micro organisms was spread on Muller-Hilton Agar (MHA) medium.The sterile filter paper discs (6 mm in diameter) were individually impregnated with different concentration of plant extract prepared in dimethyl sulphoxide (DMSO) and then placed into the agar plates which had previously been inoculated with the tested micro organisms.The plates were subsequently incubated overnight at 37 o C.After incubation the growth inhibition rings were quantified by measuring the diameter of the zone of inhibition in mm.For control dimethyl sulphoxide (DMSO) discs were used.All tests were performed in triplicate.

Brine Shrimp Bioassay
The brine-shrimp toxicity assay for each extract was carried out according to Mayer et al (Mayer et al.,1982).Briefly, sample solutions were prepared by dissolving 200 mg of each plant extract in DMSO up to the mark in 10 ml volumetric flasks.To calculated volume of the sample solution for 10, 100 and 1,000 μg/ml dose levels in three replicates was introduced freshly hatched ten brine-shrimp nauplii in artificial sea water (total volume 10 ml).A control tube for each dose level was also prepared.After 24 h of illumination under a table lamp (60 Watt), the number of survivors was counted.No death was observed in the control tubes.The LC50 (Lethal concentration for 50% mortality) values was determined using the probit method (Finney1971), as the measure of toxicity of the extracts.

Phytochemical Screening
The different phytochemicals in the crude methanol extracts were identified by the color reaction with different reagents.
The results of phytochemical screening are shown in Table 1.
From the above result it was observed that polyphenols, terpenoids and flavonoids were present in both extracts, while glycosides were present only in leaves extracts.The alkaloids were absent in both extracts.Similarly, saponins were present only in stem extract.
Various studies have revealed phenolic compounds have been reported to possess biological properties such as anticancer, antioxidant, anti-inflammatory, etc.Similarly, flavonoids to kill or inhibit many bacterial strains.The glycosides are found antibacterial, antitumor, antioxidant.Likewise, the alkaloids are also found anticancer, antibacterial, analgesic and antimalarial.Further saponins of plant are known to enhance antibody production.The present study insight the presence of various phytochemicals, thus these phytochemical can be attributed to the potential antioxidant and antibacterial properties in the tested sample.

Brine-Shrimp Bioassay
The newly hatched brine-shrimp nauplii were exposed to the plant extracts and their biological activities were evaluated on the basis of their toxicity towards the nauplii.The LC50 values (µg/mL) for different fractions were determined and those having values less than 1000 are supposed to be pharmacologically active.Results obtained during brine shrimp bioassay are given below in Table 2.
The results of brine shrimp bioassay showed that methanolic extract of T. wallichiana leaf was pharmacologically active while the stem extract is inactive.

Antibacterial Assay
In biological screening, the ability to kill or inhibit the growth of pathogenic bacteria was evaluated by antibacterial assay of the plant extract.Agar well diffusion method was used in the study of evaluation of antibacterial activity and measured in the form of zone of inhibition (ZOI) given by Dingle et al (1953).Antibacterial activity was shown by the methanolic extract of stem of T. wallichiana against gram positive bacteria on the dose dependent manner while no antibacterial activity was observed against E. coli.The negative control 5% DMSO did not produce any zone of inhibition whereas the positive control neomycin produced zone of inhibition.Results of antibacterial assay are shown in Table 3. Literature survey has revealed that no significant work has been done on the anti-bacterial activities of the T. wallichiana.However, Nisar et al, reported the antimicrobial activity of methanol extracts of the leaf, bark and heartwood of T. wallichiana against six bacterial and six fungal strains using the hole diffusion and macro-dilution methods (Nisar et al., 2008).Taxol and related bioactive taxoids from T. wallichiana may be responsible for the observed antimicrobial activities (Prasain et al., 2001).These differences may be due to the altitude variation, time of collection of that plants and laboratory conditions.

Antioxidant Activity
The antioxidant activity of the methanolic solution of samples were explored by using 2,2-diphenyl-1-picryl hydrazyl (DPPH) radical scavenging assay and FRAP (Ferric reducing antioxidant power) assay.

DPPH Assay
The methanol extractives of stem and leaves of T. wallichiana were assessed for free radical scavenging activity.The graph of concentration against the corresponding % radical scavenging activity of different samples were plotted (Fig. 1) and concentration providing 50% inhibition was determined, which was shown in the Table 4.  IC50 value of the standard i.e., Ascorbic acid was found to be 21.20 µg/ml.T. wallichiana leaves extract has lower IC50 value than stem extract.The antioxidant activity of the plant may be due to the phytochemicals like polyphenols, flavonoids and terpenoids etc.

FRAP Assay
The FRAP assay measures the total antioxidant activity on the basis of the ability to reduce a ferric salt Fe(III)(TPTZ)2Cl3 to Fe(II) ions.The FRAP assay was carried out under acidic conditions (pH 3.6) in order to maintain the iron solubility.With reference to the calibration curve obtained at 593 nm for ferrous sulphate solution (R2 = 0.992) (Fig. 2).
The FRAP values of extracts of leaves and stem of T. wallichiana were found 2.25 ± 0.57 and 2.23 ± 0.29 mM Fe+2/liter respectively, Table 5.The results obtained from the assay are given below Table 5.The highest antioxidant activity was found for leaves extract in both methods.It becomes evident that the antioxidant activities of the extracts are due to the presence of flavonoids and polyphenols in the plant.Although there is no report on the antioxidant activity of T. wallichiana so far, the present study reveals the mild antioxidant and antimicrobial activity of T. wallichiana.These results can be correlated with fact that the natural products profile and consequently the bioactivity is known to vary with the climate and geographic location of the plants.

Conclusion
Phytoconstituents and biological activity of T. wallichiana collected from Jiri, Dolakha District of Nepal has been successfully carried out.Furthermore, to the best of my knowledge, this is the first study of the antioxidant and antibacterial potential of the stem and leaves extracts of T. wallichiana.A wide range of phytochemicals is present in stem and leaves extract of plant.From Brine shrimp bioassay, leaves of T. wallichiana, was found to be pharmacologically toxic against brine shrimp nauplii.The stem of extract also showed significant zone of inhibition against gram positive bacteria.The leaf extract was shown to be inactive based on the antibacterial assay.However, further pharmacological and toxicity studies are necessary to confirm this suggestion.Bioassay guided compound isolation should be carried out to characterize the compounds in T. wallichiana that act as antioxidant and antibacterial agents.

Table 1 :
Results of Phytochemical Screening +sign indicates the presence of phytochemicals while -sign indicates the absence of phytochemicals, (L)=leaves, (s)= stem

Table 2 :
LC50 value of different plant extracts

Table 3 :
Mean Zone of inhibition (ZOI) shown by T. wallichiana stem against tested bacteria

Table 4 :
Antioxidant activities of T. wallichiana extracts

Table 5 :
Antioxidant power of methanol extracts of T.