Phytochemical Screening for Secondary Metabolites in Boswellia serrata Roxb . and Wrightia tinctoria ( Roxb . ) R . Br .

Boswellia serrata, an important source of oleo-gum resin commonly known as Indian olibanum and Wrightia tinctoria are well documented for their pharmaceutical properties. Indiscriminate removal, difficulty in vegetative propagation and poor germination accounts for the depletion of useful plant species. Plant tissue culture techniques are used as an alternative method for the production of specific metabolites and also for the propagation of plant species at a large scale. In the present study preliminary screening for the presence of secondary metabolites was reported in order to understand the levels of phytochemicals, so that in vitro production of secondary metabolites using cell cultures will be initiated in future studies. Both qualitative and quantitative analysis of the two plants show the presence of all the three major groups of secondary metabolites, like nitrogen containing alkaloids, phenolic compounds like flavonoids, tannins, terpenes like steroids, saponins etc. Significant levels of all three major secondary metabolites were present in both species. However, higher activities of phenols (277.0±4.36), tannins (240.67±5.21), alkaloids (963.3±11.7) and flavonoids (150.0±2.89) were observed in B. serrata. All the three major groups of secondary metabolites in both species demonstrate the rich content of many useful biochemicals like pharmaceuticals, flavors, fragrances, agricultural chemicals etc. However, higher quantities in B. serrata indicates its richness in medicinal properties over W. tinctoria.


Introduction
Since ancient times, people have been exploring the nature for plants in the search of new drugs.This has resulted in the use of a large number of medicinal plants with curative properties that can treat various diseases.Nearly 80% of the world's population relies on traditional medicine for primary healthcare, most of which involve the use of plant extracts.In India, almost 90% of the prescriptions were plant based in the traditional systems like unani, ayurveda, homeopathy and siddha (Lee, 1999).A large number of phytochemicals belonging to several classes have been shown to have inhibitory effects on all types of microorganisms in vitro (Cowan, 1999).Plant products have been part of phytomedicine since immemorial time.Knowledge on the chemical constituents of plants is desirable because such information will be valuable for the synthesis of complex chemical substances.Such phytochemical screenings of various plants are reported by many workers (Siddiqui et al., 2009;Kumar et al., 2010;Chitravadivu et al., 2009).
Boswellia serrata Roxb.(Burseraceae) is a perennial deciduous resin producing tree distributed in the tropical parts of Asia, Africa and Middle East (Anonymous, 1962;Gaofeng et al., 2006).The gum resin obtained from its bark is called 'Indian Olibanum' or 'Salai guggul'and is credited for its astringent, stimulant, expectorant, diuretic, antipyretic and antiseptic properties; it has also reported to be useful in ulcers, goiter, piles, diarrohea etc.In recent years the gum resin has been used extensively in pharmaceutical formulations for relieving aches and pain, particularly associated with arthritis (Singh and Atal, 1986;Chikamai, 2002).
Wrightia tinctoria (Roxb.)R.Br. is a small deciduous tree of the family Apocynaceae distributed in Central India, Burma and Timor (Chary, 1980).This plant is extensively used in the Indian system of medicine.Fresh leaves are pungent and are chewed for toothache relief (Kirtikar and Basu, 1975;Anonymous, 1976).Bark and seeds are anti-dysenteric, carminative, astringent, aphrodisiac, diuretic, used in flatulence, stomach pain and bilious affections.The plant is very useful as stomachic, in the treatment of abdominal pain, skin diseases, anti-diarrhoeal and anti-haemorrhagic (Shah and Gopal, 1988).Oil emulsion of W. tinctoria pods is used to treat psoriasis and also have fungicidal activity against Pityrosporum ovale recovered from dandruff (Krishnamoorthy and Ranganathan, 2000;Reddy et al., 2000).
In the present study qualitative and quantitative analysis for preliminary as well as secondary metabolites in Boswellia serrata and Wrightia tinctoria were reported.

Collection of plant material
Fresh leaf material of Boswellia serrata and root material of Wrightia tinctoria were collected during August mouth from Pydi Bhimavaram forest area, near Rajahmundry, Andhra Pradesh, India.Taxonomic identification of the plants was carried out with the herbarium present in the Department of Botany, Andhra University, Visakhapatnam.

Extraction of plant material
The fresh leaf material of Boswellia serrata and root material of Wrightia tinctoria collected were washed thoroughly with running tap water and air dried under shade.After complete shade drying the plant material was grinded and the powder was kept in small plastic bags with paper labelling.The grinded leaf and root material of 5 gm crushed in 25 ml of sterile water, boiled at 50-60 °C for 30 minutes in water bath and it was filtered through Whatman No.1 filter paper.Then filtrate was centrifuged at 2500 rpm for 15 minutes and the filtrate was stored in sterile bottles at 5 °C for further use.The filtrate was used for the phytochemical screening.
The extraction was done by using soxhlation extraction method with analytical grade methanol as refluxing solvent.At the completion of extraction process, the plant extract was recovered from the mixture by distillation and stored at 4 °C until further use.The percentage of plant extract content was calculated by using standard formula.Methanolic extract was used for the quantitative estimation of phenols, flavonoids, tannins and alkaloids.

Phytochemical screening
Preliminary qualitative phytochemical screening was carried out with the following methods.
Steroids: 1 ml of the extract was dissolved in 10 ml of chloroform and equal volume of concentrated sulphuric acid was added by sides of the test tube.The upper layer turns red and sulphuric acid layer showed yellow with green fluorescence.This indicates the presence of steroids (Gibbs, 1974).
Terpenoids: 2 ml of extract were added to 2 ml of acetic anhydride and concentrated H2SO4.Formation of blue/ green rings indicates the presence of terpenoids (Ayoola, 2008).
Fatty Acids: 0.5 ml of extract was mixed with 5 ml of ether.This extract was allowed for evaporation on filter paper and dried.The appearance of transparency on filter paper indicates the presence of fatty acids (Ayoola, 2008).
Tannins: 2 ml of extract were added to few drops of 1% lead acetate.A yellowish precipitate indicated the presence of tannins (Treare and Evans, 1985).
Saponins: 5 ml of extract were mixed with 20 ml of distilled water and then agitated in a graduated cylinder for 15 minutes.Formation of foam indicates the presence of saponins (Kumar et al., 2009).
Anthocyanins: 2 ml of aqueous extract were added to 2 ml of 2N HCl and ammonia.The appearance of blue-violet color indicates the presence of anthocyanins (Paris and Moyse, 1969).
Leucoanthocyanins: 5 ml of aqueous extract were added to 5 ml of isoamyl alcohol.If upper layer appears red in colour it indicates for presence of Leucoanthocyanins (Paris and Moyse, 1969).
Coumarins: 3 ml of 10% NaOH were added to 2 ml of aqueous extract; formation of yellow colour indicates the presence of coumarins (Rizk, 1982).
Emodins: 2 ml of NH4OH and 3 ml of Benzene were added to the extract.Appearance of red colour indicates the presence of emodins (Rizk, 1982).
Alkaloids: Aqueous extract was mixed with 2 ml of 1% HCl and heated gently.Mayer's and Wagner's reagents were then added to the mixture.Turbidity of the resulting precipitate was taken as evidence for the presence of alkaloids.
Phenols: Aqueous extract was mixed with 2 ml of 2% solution of FeCl3.A blue-green or black coloration indicates the presence of phenols.
Flavonoids: Aqueous extract was mixed with few fragments of magnesium ribbon and concentrated HCl was added drop wise.Pink scarlet colour appeared after few minutes, which indicated the presence of flavonoids.

Quantitative analysis Total phenolic content
The total phenolic content was determined spectrophotometrically by the method described by Sadasivam and Manickam (1996).To 2 ml of phenol extract, 1.0 ml of Folin Ceocalteau reagent was added.After 3 minutes, 13 ml of distilled water were added.Next, 2 ml of sodium carbonate (7.5%) solution were added and the volume was adjusted to 20 ml.The above mixture was kept for 1 hour for colour development and absorbance was recorded at 630 nm.The concentration of total phenolic content in plant extracts was calculated from the calibration curve of gallic acid and it was expressed as gallic acid equivalents/gram fresh weight.Each experiment was repeated three times.

Total flavonoid content
Total flavonoid content was measured by aluminum chloride colorimetric assay described by Marinova et al. (2005).A calibration curve was constructed by using standard Catechin (250 µg/ml) solution.1 ml of plant extract was added to 10 ml volumetric flask containing 4 ml of distilled water.To the above mixture, 0.3 ml of 5% NaNO2 was added.After 5 minutes, 0.3 ml of 10% AlCl3 were added.At 6 th min, 2 ml of 1 M NaOH were added and the volume was made up to 10 ml with distilled water.The solution was mixed well and the absorbance was measured against prepared reagent blank at 510 nm.Each experiment was repeated three times.

Tannin content
The tannin content was determined by the method given by Sadasivam and Manickam (1996).To 1 ml of plant extract 3.5 ml of distilled water and 0.5 ml of Folin-Denis reagent were added.Contents were allowed to mix then 1 ml of saturated sodium carbonate solution was added.The final volume was made up to 10 ml with distilled water.The solution was mixed well at room temperature and the absorbance was measured against prepared reagent blank at 760 nm.Tannin content in plant extracts was calculated from the calibration curve of tannic acid (10-100 µg) and it was expressed as tannic acid equivalents/gram weight.Each experiment was repeated three times.

Alkaloid content
Total alkaloid content in the extract was estimated by spectrophotometric method based on using Dragendorff's reagent.The amount of bismuth present was estimated after precipitating the alkaloids with Dragendorff's reagent (Sreevidya and Mehrotra, 2003).Each experiment was repeated three times.

Statistical analysis
Each experiment was repeated three times; the data were subjected to one way ANOVA using Minitab version 15.A significance level of 0.05 was used for all statistical tests.

Results and discussion
The percentage of extract after soxhlation was found to be 13.3% for Boswellia serrata and 1.33% for Wrightia tinctoria respectively.The phytochemical screening and analysis of the two medicinally important species showed that leaf and roots were rich in steroids, terpenoids, fatty acids, tannins, saponins, coumarins and emodins.Flavonoids, phenols and alkaloids were also present (Tab.1).
Quantitative analysis of B. serratareveals the phenolic content of (277.0±4.36),tannins (240.67±5.21),alkaloids (963.3±11.7)and flavonoids (150.0±2.89).Similarly, the Steroids are important sources of sex hormones (Santhi et al., 2011) and also possess anti-bacterial activities (Raquel, 2007).Presence of steroidal compounds in several plants was reported by Savithramma et al. (2011).Terpenoids and tannins possess analgesic and anti-inflammatory activities (Nand et al., 2012).Tannins have stringent property, hasten the healing of wounds, inflamed mucous membrane (Okwu and Josiah, 2006) and bind to proline rich proteins and block the protein synthesis (Yadav and Agarwala, 2011).They also inhibit the growth of many fungi, yeasts, bacteria and virus (Chung et al., 1998).Coumarins are potential antioxidant and their antioxidant activity is due to their stability to scavenge free radicals and to chelate metal ions (Tseng, 1991).The presence of above compounds in B. serrata and W. tinctoria showed their effective medicinal properties.
For the past two decades most of the research efforts were focused on the production of secondary metabolites using cell suspension cultures, hairy root cultures (Chandra et al., 2006(Chandra et al., , 2008;;Karuppusamy, 2009).Phytochemical screening of the two species B. serrata and W. tinctoria provided information about the richness of several secondary metabolites, which can be helpful for in vitro production of secondary metabolites on a large scale, which is not explored so far for these two species.

Conclusions
The traditional system of medicine like ayurvedic, homeopathy, naturopathy etc. use medicinal plants as raw materials.B. serrata and W. tinctoria are such medicinal plants, having wide applications.B. serrata as a source of oleogum resin is rich in phenolics, tannins, alkaloids and flavonoids etc.These phytochemicals are credited for their astringent, stimulant, expectorant, diuretic and antipyretic properties.Similarly W. tinctoria is also known for its medicinal properties like in the treatment of psoriasis, stomach pains, toothache, dysentery etc. due to the presence of all the three major secondary metabolites.Since about 30% of worldwide sale of drugs is based on natural products and knowledge about traditional indigenous medicine is important, exploitation of pharmacological properties of the above two species is needed and will be continued in further investigations.
Tab. 2. Quantitative screening of phytochemicals (phenols, tannins, alkaloids, flavonoids) in methanolic leaf extract of B. serrata and root extract of W. tinctoria