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Malik et al / Physical Chemistry 19(1) (2019) 28-33

STUDY OF PHYTOCHEMICALS IN STEM AND ROOTS OF EQUISTUMDEBILE ROXB.

Tabassum Malik*1,3, Shaukat Ali3, Muhammad Suleman Malik2, Abdul Naeem1,

1NCE in Physical Chemistry, University of Peshawar 2Department of Biochemistry, Quaid-i-Azam University, Islamabad, 45320

3 Department of Chemistry Islamia College, University, Peshawar, KP, 25120 Pakistan *Corresponding author E-mail:

A R T I C L E I N F O A B S T R A C T Article type

Equisetumdebile Roxb. commonly known as field horsetail, having wide prospective in medical field. Equisetum genus has many applications in the field of the medicine that’s why in present work we determine the phytochemicals present in E. debile Roxb.because no one reported the phytochemical composition of this plant collected from the region of Swat, Pakistan. Plant sample was shade dried and extract (aqueous and n-hexane) of stem and roots of E. debile Roxb. was obtained. Qualitative analysis for phytochemical were performed. The phytochemical screening indicates the presence of carbohydrates, tannins, phenolic compounds, reducing sugar, fats and oils, anthraquinone glycosides, alkaloids, monosaccharides and saponin glycosides. As the plant have bioactive phytochemicals that’s why this therapeutic plant can be used in medical industry.

Research article

History

Received 17-10-2018

Accepted 15-05-2019

October2019 Issue 1

Keywords

phytonutrients,

Equisetum debile Roxb.

satgandi booti

medicinal herb

horse tail

bioactive compounds

© 2019 NCEPC, University of Peshawar: All rights reserved

Cite This Article As Tabassum Malik, Shaukat Ali, Muhmmad Suleman Malik, Abdul Naeem. Study of Phytochemicals In Stem And Roots of EquistumDebile Roxb. Physical Chemistry 19(1) (2019) 28-33

Introduction Therapeutic plants are gift of nature to living world and plays vital role in health regardless of modernization in the field of medicine (Krishnaiah et al., 2009; Zengin et al., 2011). Research has shown that consumption of the diet rich in fruits, vegetables, seeds, nuts, whole grains and herbs at regular basis have beneficial effect on the health of human beings due to different activities of

phytochemicals present in them (Maraldi et al., 2014). Phytochemicals of plants are non-nutritive components but havegood action againstcertain diseasesandpreventivein nature (Chede, 2013). It is established fact that the phytochemicals alone or in group have positive effect and play major part metabolic abnormalities (Upadhyay and Dixit, 2015). In plant kingdom widely distributed and variant

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group is phytonutrients (Okwu et al., 2007). Certain functions related to plant’s metabolism are performed by phytochemicals. There are two major groups in which the phytonutrients of plants are divided. Primary phytonutrients, firstgroup, includes amino acids, proteins, carbohydrates, and chlorophylls. Secondary phytochemicals, secondgroup,is consists of flavonoids, steroids, terpenoids, and alkaloids, so on (Dhawale, 2013). The interest in therapeutic plant is increasing due to their role in ethno-medicine for the treatment of common diseases. Medicinal plants with high phenolic and flavonoids content have antioxidant potential and play important role in preventing diseases related with age (Azwanida, 2015). Equisetumdebile Roxb. (Chinese herb/horse tail), member of family Equisetaceae, commonly known as sumbak, widely distributed in the shaded hills and stream banks of Southeast Asia and South of China (Umoren et al., 2016; Xu et al., 2006). The genus Equisetum includes about twenty-five species, the single survivors of a very ancient group of plant (Husby, 2013). This herb has been used for treatment of diarrhea, urethritis, conjunctivitis, and hepatitis by local healers (Xu et al., 2006). The EquisetumdebileRoxb., cooling medicine, is used for treatment of fractures in bones and gonorrhea. Extract of plant is used for various cancers of breast, stomach, kidneys, tongue, intestine, and liver and also for treating nasal polypus (Umoren et al., 2016). For the cure of liver respiratory disorders, E. debile is used in the form of juice. For treatment of bone dislocations its root paste is helpful. It is also used for the removal of urinary track and kidney stones; and for treatment of rheumatism, hemorrhoids, bloody urine, swelling, enteritis influenza, fever and treatment of eye (Ali et al., 2017). Other Equisetum species have revealed to present antioxidant, antinociceptive and anti-inflammatory activities (Kloucek et al., 2005).E. giganteum contains phytochemicals which include flavonoids, styryl pyrones glycosides, hydroxycinnamic acid derivatives (Francescato et al., 2013), metals, silica (Ovalles et al., 1996) and some polar compound (oleoresin, as alkanes, fatty acids, methyl esters and steroid triterpenes) (Michielin et al., 2005). From different parts of E. arevenas following phytochemicals have been reported; phenolic compounds, tannins, flavonoids, triterpenoids, and alkaloids(Četojević-Simin et al., 2010; Mojab et al., 2010). However currently no data is available regarding the phytochemical constituents present in Equisetumdebile Roxb. collected from Pakistan. The present study was carried out with the aim to find out phytochemicals present in different extracts of Equisetumdebile Roxb. plant. Material and Methodology Plants collection and identification E. debile Roxb. (local name: satgandibooti) was collected from the hilly area of Kokarai (a local area of Swat valley, Pakistan) and identified by Prof.Dr. ShaukatAli (Department of Chemistry) and Assistant Professor

Wisal Sahib (Department of Botany) of Islamia College Peshawar, Pakistan. Extract preparation The plant was first washed with distilled water and then the stem and roots were separated. Then they were shade dried for few days. After drying they was grinded via Willy Mill to 60 mesh size. Powdered of plant root and stem was soaked in 150 ml of water (polar) and n-hexane (non-polar) separately for up to 3 days in order to get the extract. Filtration of extract was then done via Whatman No. 1 filter paper. The filtrate was concentrated using rotary evaporator (Panchun Scientific Co. Kaoshsiung, Taiwan) and stored in air tight bottles at 4 ℃ for further phytochemical screening. Phytochemical screening Phytochemical screening of aqueous and n-hexane extract was carried out by following procedure as followed by Ali et al. (2014) and Mallik and Nayak (2011) with some modification in them. Test for carbohydrate Molish’sTest: To extractofE. debile Roxbwasadded few drops of alcoholic solution of alcoholic solution of alpha- naphthol. The mixture was shaken welland from walls of test tube conc. H2SO4waspoured. Presence of carbohydrates will be confirmed by formation of violetringatthe intersection of two liquors. Test for reducingsugar Benedict’s Test: In the test tube Benedict’s test solution and extract in equal amount. That mixture is heated for 5 minutes in the boiling water bath. On the concentration of reducing sugar present in the extracts red, yellow or green will appear in the solution. Fehling’s Test: 2 mL of extract was added to Fehling solution (FehlingAandFehlingBsolution in equal amount boiled for 1oneminute) and for 5-10 minutes boiled in hot water bath. Reducing sugar presence in the extract will be indicted by formation of first ayellow thenabrick red ppt. Test for monosaccharide Barfoed’s Test: Extract and Barfoed’s reagent are taken in equal amount and boiled for few minutes in hot water bath and then allowed cool at room temperature. The formation of brick red precipitates was taken as symbol for the monosaccharide’s presence. Test for hexose sugar Cobalt chloride Test: Three milliliters of extract and two milliliters of cobalt chloride solution was taken boiled and then allowed to cool down.AfterthatfewdropsofNaOH solution wereadded. As a sign for glucose, fructose or both presence following colours; greenish blue, purplish or lower layer purplish, respectively, will be observed in solution. Seliwanoff's Test: Three milliliters ofSeliwanoff’s reagent is added to one milliliter of extract and boiled for few minutes in boiling water bath and waited forred colour to appear which is an indicator for the presence of monosaccharides.

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Tollen’s phloroglucinol Test: 4 mL of phloroglucinol solution (0.5 %) and 2.5 mL of conc. HCl was taken in test tube. To that solution 1 mL of extract was added. Appearance of cherry-red colour in the solution indicates the presence of ribose and galactose whereas this test does not give any indication for the presence of glucose because it does not form any coloured furfural derivative upon reaction with phloroglucinol. Test for non-reducingsugars When the tests such as Fehling’sand Benedict’s test gives negative results then it will be indication for the presence of non-reducing sugar because they have no aldehyde or ketone group. Test for non-reducingpolysaccharides(Starch) IodineTest: When addition of few drops Iodine solution (dilute) to three milliliters of extract (aqueous or n-Hexane)results in appearance of bluecolour, taken as sign for starch presence, which upon boiling disappears and on cooling appears again. Tannic acid Test: 20% of tannic acid was added to test solution. Formation of ppt is an indication for tannic acid presence in the test solution. Test for proteins Biuret Test: To 3 mL o f testsample4 % NaOHwasadded.Afterthatfewdropsof1% CuSO4 solution werepoured into it.Violet orpink colour appearance is a sign for protein presence. Million’s Test: Five milliliters of Million’s reagent was added to three milliliters of extract, gives white precipitates which upon heating dissolves to give solution of green colour or turn into brick red precipitates. Testsfor aminoacids Ninhydrin Test: To heated three milliliters of extract, 5% of ninhydrin solution was added followed by heating for 10 minutes in water bath. Purple or bluish colouration will confirm the existence of amino acids. Tyrosine Test: Few drops of Million’s reagent was added to heated extract (3 mL). Red colour observation is an indication for amino acid existence. Xanthoprotein Test: 1 mL of conc. H2SO4 was added to 3 mL of test solution. The protein containing tyrosine phytophan presence will be indicated by formation of white precipitates. Which upon boiling gives yellow ppts and on addition of 1 mL of ammonium hydroxides gives orange colour precipitates. Tests for fats & oils Acrolein Test: 2 drops of sample were heated with KHSO4 powder, pungent smell of acrolein will show the presence fats and oils. Bromine water Test: Bromine water was added to alcoholic solution of extract to detect presence of fats and oils. The presence of unsaturated acid will be detected by decolouration of bromine water. Test for steroids Salkowski Test: Five milliliters of extract were mixed with two milliliters ofaceticanhydride. To that mixture

two milliliters of H2SO4 was added. Steroids presence will be indicated by change in colour from violet to blueorgreenin somesamples. Test for cardiacglycosides Keller-Killani Test: Few drops of ferric chloride solution were added to two milliliters of glacial acetic acid. Then that solution was mixed with five milliliters of extract followed by the addition of one milliliters of conc. H2SO4 was added to that mixture. Brown ring formation occur atinterference is a sign for adeoxy-sugar (characteristicofcardenolides). Legal Test: Two milliliters of extract was mixed with one milliliter of sodium nitroprusside and one milliliter of pyridine. For glycosides presence the appearance of the pink or deep red colour will be taken as a sign. Test for anthraquinoneglycosides Modified Borntrager’s Test: Equal volume of dil. hydrochloric acid and extract were mixed and heated in boiling water bath for one hour and then allowed to cool followed by the addition of benzene. Mixture was shaken well. Benzene layer was recovered and to that equal amount of dilute ammonia solution was added. Appearance of pinkish red colour in ammoniacal layer was an indication for anthraquinone glycosides presence. Test for saponinglycosides FoamTest: Vigorous shaking of equal volume of extract and water was done. Formation of persistent foam is a positive indicator for presence of saponins glycosides. Hemolysis Test:To one drop of blood extract was added. The appearance of hemolytic zone will confirm the presence of saponins glycosides. Test for alkaloids Hager’s Test: Three milliliters of saturated solution of picric acid (Hager’s reagent) was mixed with one milliliter of extract. The alkaloids presence will be indicated by the formation of precipitates of yellow colour. Wagner’s Test: Some drops of Wagner’s reagent were added to few milliliters of extract. The appearance of precipitates of reddish brown colour is an indication for alkaloids presence. Dragendorff’s Test: 1 mL of Dragendorff’s reagent was added to 3 mL of extract. The presence of brick red/brown or orange colour indicates the presence of alkaloids. Test for tannins and phenolic compounds Lead acetate Test: Additionof3 mL of lead acetate to 3 mL of extract gives white precipitate which is an indicator for phenolic compounds presence. Iodine solution Test: Transient red colour appearance on addition of dil. iodine solution to extract is a sign for phenolic compounds and tannins presence. Dil. HNO3 solution Test: To three milliliters of extract addition of some drops of dilute HNO3 gives reddish to yellow colour as a sign for indication of phenolic compounds presence.

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Acetic acid solution Test: To three milliliters of test sample addition of acetic acid solution (few drops) gives red colour (a sign for presence of phenolic components or tannins). Test for flavonoids Shinoda Test: To 2g of dry extract we added 5 mL of ethanol (95% v/v) followed by the addition of 5 drops of conc. HCl. After that 0.5g of magnesium turnings were added, appearance of pink colour indicates the presence of flavonoid. Results Initial screening test for phytochemicals was done for the presence of phenolic compounds, flavonoids, tannins, alkaloids, glycosides (cardiac, saponins, and anthraquinone), steroids, fats and oils, amino acids, proteins and carbohydrates for n-hexane and aqueous extract of stem and roots of E. debile Roxb. are shown in Table No. 1. Table No. 1: Preliminary screening of E. debile Roxb. stem and roots

S.No. Tests Aqueous Extracts n-Hexane Extract

Stem Root Stem Root

1. Test for carbohydrates

a. Molish’s Test + + + +

2. Test for reducing sugars

a. Benedict Test + + + +

b. Fehling’s Test + + + +

3. Test for monosaccharides

a. Barfoed’s Test + - + -

4. Test for hexose sugar

a. Seliwanoff’s Test - - - -

b. Tollen’s Test - - - -

c. Cobalt chloride Test - - - -

5. Test for non-reducing polysaccharides (Starch)

a. Iodine Test - - - -

b. Tannic acid Test - - - -

6. Test for proteins

a. Biuret test - - - -

b. Million’s Test - - - -

7. Test for amino acids

a. Tyrosine Test - - - -

b. Ninhydrin Test - - - -

c. Xanthoprotein Test - - - -

8. Test for fats and oils

a. Acrolein Test + + + +

b. Bromine water Test + + + +

9. Test for steroids

a. Salkowski Test - - - -

10. Test for cardiac glycosides

a. Keller-Killani Test - - - -

b. Legal Test - - - -

11. Test for anthraquinone glycosides

a. Modified Borntrager’s Test + + + +

12. Test for saponins glycosides

a. Foam Test + + + +

b. Hemolysis Test + + + +

13. Test for alkaloids

a. Hager’s Test + + + +

b. Wagner’s Test + + + +

c. Dragendorff’s Test + + + +

14. Test for tannins & phenolic compounds

a. Lead acetate Test + + + +

b. Iodine solution Test + + + +

c. Dil. HNO3 solution Test + + + +

15. Test for flavonoids

a. Shinoda Test - - - -

(+) = present; (-) = absent

Discussion Around the world for curing many diseases the medicinal plants are vital source (Bhatia et al., 2015). As the time going on the value of therapeutic plants are increasing because of the bioactive phytonutrients they have with the ability to treat different disease (Petrovska, 2012). Medicinal plants have number of phytonutrients possesses different charges, such compounds distribute themselves based on the rule “like dissolves like” when they are dissolved in various solvents with different polarities. So, in order to determine the phytochemical with bioactive potential within the plant screening of plant is an important step (Jones and Kinghorn, 2006; Starmans and Nijhuis, 1996). The phytochemical screening of E. debile Roxb. aqueous and n-hexane extract waspositive for carbohydrates, tannins, phenolic compounds, reducing sugar, fats and oils, anthraquinone glycosides, and alkaloids, whereas it was negative for hexosesugar, non-reducing sugars,amino acids, proteins, flavonoids,cardiacglycosides,steroids, starch, and non-reducing for both stem and roots. Phytochemical screening of aqueous extract of S. grandiflora Linn. reveals the presence of amino acids, carbohydrates, alkaloids, tannins, and glycosides which is line with our findings (Mallik and Nayak, 2011). The phytochemical screening of n-hexane and aqueous extract of Allium sativum indicates the presence of tannins, anthraquinone, phenolics while shown negative results for flavonoids and steroids (Divya et al., 2017). For saponins glycosides and monosaccharides screening test are positive for stem whereas negative in case of root for both of extracts. Aqueous extracts for stem of Malva silvestris L. shown positive tests for saponins and tannins which supports our findings (Zohra et al., 2012). Flavonoids and phenols are known to possess anti-oxidant and anti-inflammatory potential (Wu et al., 2008). Research has revealed that on

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Alzheimer disease good effect have been shown by alkaloids (Maelicke et al., 2001). Conclusion In the above work, phytochemical screening of E. debile Roxb. aqueous and n-hexane extract wascarried out which shows the presence of following phytochemicals in stem and roots extract of E. debile Roxb. carbohydrates, tannins, phenolic compounds, reducing sugar, fats and oils, anthraquinone glycosides, and alkaloids, while monosaccharides and saponin glycosides in case of stem only. The presence of above bioactive chemicals (tannins, alkaloids, phenolic compounds, saponins and anthraquinone glycosides) may be reason that E.debile Roxb. was used by folklore practioners due to their medicinal activities. References: Ali, S., Omer, M.O., Chaudhry, M.A., Ashraf, M.,

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