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Pharmacognostical and phytochemical analysis and identification of bioactive compounds using GC-MS Techniques of Ruellia tuberosa

Dr. K. L. Senthil kumar1 , Dr. P. D. Gokulan2 , Mr. S. Venkateshwaran3, G. Praveen Kumar4, S. Priyadharshini5, M. Punith Kumar6

1 Principal, Sri Vijay Vidyalaya College of Pharmacy, Dharmapuri, Tamilnadu, India

2 Head of the Department of Pharma ceutical Chemistry, Sri Vijay Vidyalaya College of Pharmacy, Dharmapuri, Tamil nadu, India)

3* Associate professor, Sri Vijay Vidyalaya College of Pharmacy, Dharmapuri, Tamil Nadu (India)

*4, 5, 6 B. Pharm Students, Sri Vijay Vidyalaya College of Pharmacy, Dharmapuri, Tamil Nadu (India)

Abstract- Aim: The aim of this study was to analyze the different bioactive compounds found in the leaves of Ruellia tuberosa collected from the Dharmapuri district. The study focused on identifying the bioactive constituents present in the chloroform extract and ethanol extracts of the Ruellia tuberosa leaves using the GC-MS technique along with phytochemical screening.

Materials and methods: The collected leaves of Ruellia tuberosa were thoroughly washed, air-dried, and ground into a coarse powder. The leaf extract was prepared by using the Soxhlet extraction method. The selection of solvents is based on their polarity.

Result: The phytochemical screening and GC-MS analysis confirmed the presence of various bioactive compounds in Ruellia tuberosa leaves. The study revealed several active chemical constituents, such as alkaloid, glycosides, flavonoids, proteins, tannins, terpenoids, etc.

Conclusion: The present study shows that Ruellia tuberosa leaves contain a rich source of secondary metabolites.

Key words: Ruellia tuberosa, extraction, pharma cogenetic studies, phytochemical analysis, and GC-MS technique.

Introduction

Ruellia tuberosa is a medicinal plant that has been traditionally used since ancient times. It is an erect, perennial herb belongingtothefamilyAcantharean.Theplantisknownbyvarious names,suchasMinnieroot,feverroot,snapdragon root, andsheeppotato.Itiscommonlyreferredtoasthe“crackerplant.”Theplantgrowsneartheroadsideingrasslands,cultivated fields, and even in a dry(xerophilic)area It is a short-lived perennial plant with striking funnel-shaped violet bracteates flowersondichotomouscymes.Thefruitisasub-cylindricalpuberulentcapsulehavingmoreorless 20seedsperlocule.The plant also has thick, fusiform tuberous roots that grow in clusters. Ruellia tuberosa L. is a tropical plant that is commonly found in many parts of Asia, including Indonesia, Malaysia, and India. The plant flowers and produces fruits from June to February. It can be easily recognized by its attractive, trumpet-shaped, violet-colored flowers with a five-lobed corolla. The plantis mainlygrowninmoistand shady environments.Theplant attracts manypollinators,especiallybutterfliesandother insects.Theflowersappearlightbluish-purpleduetothepresenceofanthocyanin, a flavonoidpigmentthatisalsousedasa natural dye in industries. The plant contains several important phytochemical constituents, such as alkaloids, glycosides, flavonoids, tannins, proteins, terpenoids, diterpenes, sterols, etc. This study aims to investigate the extraction of bioactive compoundsfromRuelliatuberosausingdifferentsolventsandevaluatethephytochemicalcontentusingGaschromatographymassspectrometryanalysis

International Research Journal of Engineering and Technology (IRJET) e-ISSN: 2395-0056

Volume: 13 Issue: 01 | Jan 2026 www.irjet.net p-ISSN: 2395-0072

Traditionally, the plant is used for anti-diabetic, anti-inflammatory, ant nociceptive, antipyretic, analgesic, antioxidant, antihypertensive,insecticidal,anticancer,andantidotaltoxicagents.

TheleafextractofRuelliatuberosaL.hasanti-diuretic,anti-cancer,andanti-oxidantactivity.

Ruelliatuberosarootextractisusedforanti-diureticactivity,reducingbloodsugarlevelsandkidneydisease.

Itisusedinancientdaysformanytraditionalpurposes.

Vernacular name:

 Assamese:Chapatti

 Bangladesh:Potpati

 Marathi :Ruwel

 SriLankan:Heenamukkara

 Tamil:Kiranthinayakam,Pattaskai

 Telugu:Jurbulagadda

Taxonomic Hierarchy:

 Kingdom:Plantae

 Subkingdom:Viridiplantae

 Infrakingdom: Streptophyta

 Superdivision:Embryophyta

 Class : Magnoliopsida

 Superorder:Asternae

 Order : Lamiales

 Family : Acanthaceae

 Genus : RuelliaL.

 Species:RuelliatuberosaL.

Pharmacognostical Studies:

Methodology

1. Macro soppy(SOP No. PCOG-004-SOP): The external feature of the test sample was documented using a Nikon D-5600 digitalcamera.

2. Micro scopy(SOP No. PCOG-005-SOP): Thesample was preserved in fixative FAA for more than 48 h. The preserved specimens were cut into thin transverse sections using a sharp blade, and the sections were stained with 0.8% safranin and 0.5% Astra blue. Transverse sections were photographed using an Axiolab5 trinocular microscope attached to a Zeiss Axiocam208colordigitalcameraunderbrightfieldlight.Magnificationswereindicatedbyascalebar.

FIG no : 01 Ruellia tuberosa

Result:

International Research Journal of Engineering and Technology (IRJET) e-ISSN: 2395-0056

Volume: 13 Issue: 01 | Jan 2026 www.irjet.net p-ISSN: 2395-0072

Microscopy:

Petiole

The transverse section of the petiole is urn-shaped. The lower surface is convex, while the upper surface is slightly concave and bears lateral wings. The outermost layer is a single-layered epidermis covered with a cuticle. Beneath it, 4-5 layers of collenchymatous tissue are present, which continue with the parenchymatous ground tissue. At the center, an arc-shaped vascularbundleisseen.Thevascularbundlesareconjoint,collateral,closed,andcomposedofnormalvasculartissues.Xylem isorientedtowardstheuppersideand Phloemtowardsthelowerside.Twotracebundlescanbeobservedoneachuppersidebelowthewingregion.Cystolithsare foundinepidermalcells,andstarchgrainsaredistributedintheinnerparenchymacells.

TS of petiole

Enlarged view of wing region

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Upper portion enlarged

Figure no: 02 Micro scopy of Ruellia tuberosa petiole

Enlarged view of vascular region

Enlarged view of lower portion

Col -collenchyma; Cys -cystolith; E -epidermis; Pa -parenchyma; Ph -phloem; SG -starchgrain; TB -tracebundle; V -vessel; VB -vascularbun

Leaf

TheTSoftheleafisdorsiventralwithaflatlowersurfaceandaslightlyelevateduppersurfaceandshowsribbon-likelateral laminarextensions.

Midrib

TS of the midrib shows an outer single-layered upper and lower epidermis covered by cuticle and bears several covering trichomes; 3 to 4 layers of collenchymatous hypodermal layer are present, followed by parenchymatous ground tissue embedded with a crescent-shaped conjoint, collateral, and closed vascular bundle at the center; xylem is placed towards the uppersideandphloemtowardsthelowerside;xylemisformedofvessels,tracheids,fibers,andparenchyma,whereasphloem is made up of sieve elements and phloem parenchyma; two or three trace bundles can be seen on the upper side facing towardsthelamina.

Lamina

The transverse section of the lamina shows that both the upper and lower epidermis are single-layered and covered with a cuticle. These layers also bear covering trichomes. The mesophyll tissue is well differentiated into an upper, tightly packed single layer of palisade cells,followed by four to five layers of loosely arrangedspongy parenchyma cells.Veins run through themesophylltissue.Cystolithsarefoundinspecializedepidermalcellsonbothsurfacesofthelamina,whileoilglobulesare scatteredthroughoutthelamina.Afewcollenchymacellsarealsopresentneartheleafmargin.

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no: 03 Microscopy of Ruellia tuberosa leaf passing through midrib

TS of leaf

Figure

Midrib

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Enlarged wing portion

Ts of lamina

TS of leaf margin

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withcystolith Underapolarizedfield

Col - collenchyma; Cu - cuticle; Cys - cystolith; LE - lower epidermis; MCr- micro sphenoidal crystals; OG - oil globule; Paparenchyma; Pal - palisade; Ph - phloem’s - spongy parenchyma - trichome; TB - trace bundle; UE - upper epidermis; Vvessel; VB -vascularbundle; Ve –vein

PHYTOCHEMICAL STUDIES:

The obtained extracts were subjected to phytochemical analysis. This qualitative study was carried out to identify the presenceofsecondarymetabolitessuchasalkaloids,glycosides,flavonoids,terpenoids,diterpenes,proteins,tannins, sterols, etc.

1. Test for Alkaloids

Dragendorff’s test

To 1 ml of extract, add 1 ml of Dragendorff’s reagent (potassium bismuth iodide solution). An orange-red precipitate indicatesthepresenceofalkaloids.

Lamina

Microsphenoidalcrystals

Crystalsunderpolarizedfield

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Mayers Test

To 1 ml of extract, add 1 ml of Mayer’s reagent (potassium mercuric iodide solution). Whitish yellow or cream-colored precipitateindicatesthepresenceofalkaloids.

2. Test for Glycosides

Test solution: Itwaspreparedbydissolvingthesampleinalcohol.

Keller-Killiani Test

To a test solution, a few dropsof ferricchloridesolutionand concentrated sulphuricacid wereadded; the formation of two layersoccurred,alowerlayerofreddish-browncolorandanupperlayerofbluish-greencolorsimultaneously.

3. Test for Triterpenoids

Test solution: Itwaspreparedbydissolvingtheextractsinchloroform.

Salkowski test

Afewdropsofconcentratedsulfuricacidwereaddedtothesolutionandallowedtostandforsometime.Thelackofformation ofyellowcolorinthelowerlayerindicatestheabsenceoftriterpenoids.

4. Test for Carbohydrate

The Molisch testto 2-3ml of the test solution, added few drop Molish reagent solution and was shaken. Concentrated sulphuricacidwasaddedfromthesideofthetesttube.Avioletringwasformedatthejunctionoftwolipids.

5. Test for Protein

Test solution: Itwaspreparedbydissolvingextractinwaterandmakingaqueousextract.

Biuret test

To about 3 ml of the extract,40% sodium hydroxide solution and a few drops of 1% copper solution were added.White producesbluecolor.

6. Test for Anthraquinone

Borntrager’s Test:Add10mlof10%ammoniasolutionwithafewmlof3mlofAq.Theextractisshakenwithmlofbenzene and filtered (shaken vigorously 30 mins) appears as a pink, violet, or red-colored solution, it shows the presence of anthraquinones.

7. Test for Flavonoids

Test solution:A small amount of extract and 2M HCL was added and heated for about 30-40 mins. The extract was cooled downandagainextractedwithethylacetate,whichwasfurtherconcentratedtodrynessandreadytobeusedasatestsample.

Lead acetate test: Toasmallquantityofextract,leadacetatewasadded.Formationofyellow-colored precipitateshowsthe presenceofflavonoids

8. Test for Quinone

Conc. HCl test

Plantextractwithconc.HClappearsgreenincolor;itshowsthepresenceofquinones.

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9. Test for Tannins

Test solution: Thetestsolutionwaspreparedbydissolvingtheextractinwaterandalcohol.

Ferric chloride solution test

To 1 ml of the extract, ferric chloride was added. The formation of a dark blur or greenish-blackcolor shown in the presenceindicatesthetannins.

10. Test for Cholesterol

Add2mlofchloroformwith10dropsofacetic anhydrideandadd2-3dropsofconc.H₂SO₄. Theappearanceof aredrosecolorshowstheabsenceofcholesterol.

11. Test for Diterpenes

Plant extract is dissolved in distilled water, and 3-4 drops of copper acetate solution are added. It appears emerald green incolor;itshowsthepresenceofditerpenes.

12. Test for Terpenoids

Add2mlofchloroformwith5mlofplantextractandadd3mlofconc.H₂SO₄(boiledinawaterbath).Donotappear inagrey-coloredsolutionintheabsenceofterpenoids.

13. Test for Saponins

Test solution: Itwaspreparedbydissolvingextractinwaterandmakingaqueousextract.

Foam Test

The drug extract was vigorously shaken with water Persistent foam formation does not indicate the absence of saponins.

14. Test for Fat

Solubility test

Fatsareinsolubleinwaterandethanol.Butsolubleinchloroform,benzene,andether.ItshowsthepresenceofFat.

15. Test for sterols

Liberman-Burchard Test

1gmofthetestsubstancewasdissolvedinafew dropsofchloroform,3mlofaceticanhydrideand3mlofglacialaceticacid wereadded,anditwaswarmedandcooledunderthetap,anddropsofconc.H₂SO₄wereaddedalongthesideofthetesttube. Thenon-appearanceofabluish-greencolorshowstheabsenceofsterols.

Table 1: Phytochemical analysis of Ruellia tuberosa

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PHYTOCHEMICAL SCREENING: (+) Presence (-)/Absence

TheextractofRuelliatuberosaL.wassubjectedtophytochemicalanalysis.Theselectionofsolventisbasedontheirpolarity. The solvents used for the extraction were chloroform, ethanol, and water. The phytochemical screening of secondary metabolites such as alkaloids, glycosides, flavonoids, triterpenoids, diterpenes, carbohydrates,proteins, tannins, cholesterol, andsterolswasdetermined.

GAS CHROMATOGRAPHY AND MASS SPECTROPHOTOMETRY

TheRuelliatuberosaleavescontainarichsourceofsecondarymetabolites.Theextractionandcharacterizationofbiologically activecomponentsfrommedicinalplantshaveresultedinthetreatmentforvariousdiseases.Thebioactivecompoundpresent intheRuelliatuberosawasseparatedbyusinggaschromatographyandmassspectrometry.

GC-MS analysis of ruellia tuberosa

PROCEDURE

Sample preparation:

Weigh1gofhomogenizedsample(chloroformandethanol)intoa15mlplastictube.Add1mlofethylacetateandvortex for5minutes.

Thentransfertoa1mlvialforinjectionofµlofthesampleintoGC-MS

INSTRUMENT CONDITIONS:

C–MS analysis was performed using a Shimadzu QP2020 gas chromatograph–mass spectrometer equipped with a split/splitlessinjectorandanelectronimpactionizationsourceoperatinginEI⁺mode.SeparationwasachievedonanHP-5MS capillary column (30 m × 0.32 mm, film thickness 0.25 µm). An injection volume of 1.0 µL was employed, with the injector temperaturemaintainedat285°C.

Helium (99.999% purity) wasused asthe carrier gasat a constant flow rate of1.0 mL/min. The oven temperatureprogram was set with an initial temperature of 50°C, which was gradually increased to 290°C and held for 10 minutes to ensure complete elution of all components. The split valve was opened 3 minutes after injection to purge the injector. All injections werecarriedoutusinga10µLmicrosyringe.

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Volume: 13 Issue: 01 | Jan 2026 www.irjet.net

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Table 2: Phytochemical screening by using chloroform extract

Conclusion:

The extraction of Ruellia tuberosa using solvents of varying polarity resulted in different percentage yields. Phytochemical screening was carried out to identify the presence of bioactive constituents in each extract. The detection of these phytochemicalsthroughpreliminaryanalysissubstantiatesthemedicinalsignificanceoftheplant.

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The plant extract was subjected to gas chromatography–mass spectrometry (GC–MS) analysis under standardized experimentalconditions.Theseparationofcompoundswasachievedbasedontheirretentiontimeandrelativepeakarea,and detection was performed using a suitable detector such as a flame ionization detector (FID). The resulting chromatogram exhibited multiple peaks corresponding to diverse bioactive constituents. GC analysis further confirmed the chemical heterogeneity of the extract by effectively resolving volatile compounds. The identified constituents may contribute to the therapeuticpotentialof Ruellia tuberosa

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