S. Murugesan / Journal of Pharmacy Research 2017,11(1),23-28 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Phytochemical Screening, GC-MS Analysis of Bioactive Compounds and Antibacterial activity from gendarussa Burm.F. Stem S. Murugesan Assistant Professor, Department of Botany, Periyar University, Salem, Tamil Nadu – 636 011,India.

Received on:27-11-2016; Revised on: 22-12-2016; Accepted on: 04-01-2017

ABSTRACT Background: The aim of the present study was to analyzed the phytochemical screening of methanol, chloroform and petroleum ether extract of the Justicia gendarussa stem and GC-MS technique to study the major and minor phytoconstituents and their antibacterial activity. Methods: In the present study, preliminary phytochemical analysis and GC–MS was carried out on the methanol, chloroform and petroleum ether extract of stem of Justicia gendarussa for identification of phytocompounds in the stem. The disc diffusion Method (Bauer et al. 1996) was used to screen the anti bacterial activity. Result : The preliminary phytochemical analysis of methanol, chloroform and petroleum ether extract of Justicia gendarussa stem showed the presence of bioactive components like carbohydrates, steroids, alkaloids, glycosides, flavonoids, tannins and saponins. Pharmacognosy studies In Stem Physico – chemical values were analysed such as foreign organic matter, Moisture content, Total ash. Florescence analysed in stem on Justicia gendarussa various solvents for Visible light condition under the UV rays (254nm, 366nm). The different extract showed varying degree of antibacterial activity against the P. vulgaris and P. pneumonia tested. Conclusion: Results confirmed the presence of therapeutically potent compounds in the stem of various extract. This study will also helps to predict the formula of biomolecules which can be used as drugs and further investigation may lead to the development of drug formulation and this plant extract in developing a novel broad spectrum antibacterial agent.

KEYWORDS: Phytochemical, GC-Ms analysis, Pharmacognosy, Justicia gendarussa and Antimicrobial activity.

1. INTRODUCTION The World Health Organization (WHO) has reported that about 80% family . The family Acanthaceae is a taxon of of the world’s population mainly depends on traditional medicine dicotyledonous, flowering containing almost 250 genera and and the use of plant extracts is mainly involved in the traditional 2500 species. Most are tropical herbs, shrubs or twining vines.[7] treatment.[1] Since long throughout the world man has relied on Justicia gendarussa Burm. F is well known for its medicinal properties traditional medicine and has since been on the developing his such as parricidal, antioxidant, immunosuppressive, antinociceptire, knowledge of medicinal plants.[2] Medicinal and aromatic plants and amidic, anti-arthritic and anti-inflammatory activities.[8,9] The increase their derivatives to the tune of nearly Rs 200 crores are produced in prevalence of multiple drugs resistance has slowed down the annually in the country.[3] The drugs were isolated either from the development of new synthetic antimicrobial drugs and has whole plant or from different parts of the plant like leaves, stem, bark, necessitated the search for new antimicrobials from alternative root, flower and seed. Some drugs are prepared from excretory plant sources.[10] In general, bacteria have the genetic ability to transmit products such as gums, resins and latex.[4] Plants have formed on and acquire resistance to drugs used as therapeutic agents.[11] There the basis of sophisticated traditional medicine systems among which is growing interest in correlating the phytochemical constituents of are Ayurvedic, Unani and Chinese.[5] Natural products derived from a medicinal plant with its pharmacological activity. They concluded plants have historically played an essential role in the discovery of that the observed antimicrobial activity was due to other active novel new pharmaceuticals.[6] Justicia gendarussa belongs to the principles present in the extracts that were used in the investigation.

2. Justicia gendarussa Burm. F. Description *Corresponding author. Dr. S. Murugesan Ph.D.,, Justicia gendarussa Burm. f. Syn: Gendarussa vulgaris (figure.1) is Asst.Prof; an erect undershrub, 0.6 to 1.2 m in height with subterete branches. Department of Botany, Leaves are simple, lanceolate or linear – lanceolate, 7.5 to 12.5 cm Periyar University, Salem, long, glabrous, short- petioled, pale green beneath and dark violet green above, 8 pairs of main nerves, mid rib and main nerves prominent Tamil Nadu – 636 011,India.

Journal of Pharmacy Research Vol.11 Issue 1 January 2017 23-28 S. Murugesan / Journal of Pharmacy Research 2017,11(1),23-28 on the under surface. Stems and branches are dark violet. Flowers 4.3. Preliminary Phytochemical Screening are 5-12.5 cm long from the uppermost leaf -axils; white coloured, Preliminary Phytochemical analysis was carried out for all the extracts spotted with purple and clustered in the interrupted spikes. Fruits as per standard methods described by Brain and Turner 1975 and glabrous capsules. Calyx 3.8-5mm; with nearly glabrous linear Evans 1966. segments. The leaves and roots are acrid, febrifuge, thermogenic, emetic, anodyne, emmenagogue, diaphoretic, insecticidal and Detection of alkaloids antipyretic.[12,13] Extracts were dissolved individually in dilute hydrochloric acid and filtered. The filtrate was used to test the presence of alkaloids. 3. Scientific Classification a) Mayer’s test: Filtrates were treated with Mayer’s reagent. Formation Kingdom : Plantae of a yellow cream precipitate indicates the presence of alkaloids. Division : Tracheophyta Class : Magnoliopsida b) Wagner’s test: Filtrates were treated with wagner’s reagent. Order : Formation of brown/ reddish brown precipitate indicates the presence Family : Acanthaceae of alkaloids. Genus : Justicia Species : gendarussa Detection of Flavonoids

a) Lead acetate test: Extracts were treated with few drops of lead acetate solution. Formation of yellow color precipitate indicates the presence of flavonoids.

b) H2SO4 test: Extracts were treated with few drops of H2SO4. Formation of orange colour indicates the presence of flavonoids.

Detection of Steroids

Liebermann- Burchard test: 2ml of acetic anhydride was added to

0.5g of the extracts, each with 2ml of H2SO4. The color changed from violet to blue or green in some samples indicate the presence of steroids.

Detection of Terpenoids Figure 1: Justicia gendarussa Salkowski’s test: 0.2g of the extract of the whole sample was mixed 4. MATERIALS AND METHODS with 2ml of chloroform and concentrated H2SO4 (3ml) was carefully added to form a layer. A reddish brown coloration of the inner face 4.1. Collection of plant samples was indicates the presence of terpenoids. Plant was collected locally from Irulappatti (Vill) in Pappireddipatti (Po), Dharmapuri (Dt), Tamil Nadu, India, and got identified by Detection of Anthroquinones Assistant Prof. Dr. S.Murugesan, Department of Botany, Periyar University, Salem, Tamil Nadu and India. Borntrager’s test: About 0.2g of the extract was boiled with 10% HCl for few minutes in a water bath. It was filtered and allowed to 4.2 . Extraction of Plant Material cool. Equal volume of CHCl3 was added to the filtrate. Few drops of The fresh plant samples (Plant leaves) were collected and washed 10% NH3 were added to the mixture and heated. Formation of pink under running tap water and dried an oven at 400c for 3 days. The color indicates the presence anthraquinones. dried plant materials were ground into powder. About 10 g of dry powered plant material was extracted by soxhlate apparatus using Detection of Phenols methanol, Chloroform and Petroleum Ether solvent. Extracts was then concentrated using a rotary evaporator and the concentrated residual a) Ferric chloride test: Extracts were treated with few drops of 5% extracts were stored at 40C in a dry airtight container until further ferric chloride solution. Formation of bluish black color indicates the use. presence of phenol. Journal of Pharmacy Research Vol.11 Issue 1 January 2017 23-28 S. Murugesan / Journal of Pharmacy Research 2017,11(1),23-28 b) Lead acetate test: Extract was treated with few drops of lead acetate on the surface of medium and the extract was allowed to diffuse for solution. Formation of yellow color precipitate indicates the presence 5 minutes and the plates were kept for incubation at 370C for 24 hrs. of phenol. At the end of incubation, inhibition zones formed around the disc were measured with transparent ruler in millimeter. Detection of Saponins 5. RESULTS AND DISCUSSION Froth test: About 0.2g of the extract was shaken with 5ml of distilled water. Formation of frothing (appearance of creamy stable persistent 5.1. Phytochemical Investigation of small bubbles) shows the presence of saponins. Preliminary phytochemical screening of stem extract of Justicia gendarussa Burm. f. was performed and results were compared in Detection of Tannins the Table 1. The methanol extract showed better result for preliminary phytochemical screening than Chloroform extract and Petroleum ether Ferric chloride test: A small quantity of extract was mixed with water extract extract. The phytochemical groups like alkaloids, flavonoids, and heated on water bath. The mixture was filtered and 0.1% ferric reducing sugars, tannins, amino acids and protein are present in chloride was added to the filtrate. A dark green color formation both leaf and stem parts of the investigated plant.[14] indicates the presence of tannins. Table: 1. Preliminary Phytochemical Screening of Justicia gendarussa Burm.F. Stem Detection of Carbohydrates S.No. Class of Methanol Chloroform Petroleum Fehling’s test: 0.2gm filtrate is boiled on water bath with 0.2ml each Chemical extract extract ether extract Compounds of Fehling solutions A and B. A red precipitate indicates the presence of sugar. 1 Alkaloids Mayer’s test + - - Detection of Oils and Resins Wagner’s test + - - 2 Flavonoids Test solution was applied on filter paper. It develops a transparent Lead acetate test + - + appearance on the filter paper. It indicates the presence of oils and H2SO4 test + - + resins. 3 Steroids Liebermann- - + - Bur chard test 4.4. Gas Chromatographic–Mass Spectrophotometric (GC-MS) 4 Terpenoids Analysis Salkowski’s test + - - GC-MS analysis was carried out for all the three extracts (Methanol, 5 Anthraquinones Borntrager’s test - - - chloroform, petroleum ether) of the leaves and stem of Justicia 6 Phe no l’s gendarussa Burm.F. Alpha Omega Hi-tech Bioresearch Centre, 16, Ferric chloride test + - - Anbu Nagar, Gorimedu, Salem – 636 008, Tamil Nadu, India. Lead acetate test + - - Pharmacognosy Analysis of Physico chemical constant soft the 7 Saponin - + - 8 Tannin + - - powder bark has been done to evaluate the quality and purity of the 9 Carbohydrates + + + drug. Various physicochemical parameters like Moisture contents, 1 0 Oils & Resins - + + foreign organic matters and Ash values were calculated as per WHO + = Present; = Absent guidelines. Many herbs show fluorescence when the cut surface or powder is exposed to UV light and this can be useful in their 5.2. GC-MS Analysis identification. The present study was undertaken to find out the bioactive compounds present in the methanol, Chloroform and Petroleum 4.5. Antibacterial activity Ether extract of Justicia gendarussa by using Gas chromatography The disc diffusion Method (Bauer et al. 1996) was used to screen the and Mass spectroscopy. The active principles with their molecular antimicrobial activity. In vitro antimicrobial avitivity was screened formula, retention time, molecular Weight, peak area. Which shows by using Muller Hinton Agar (MHA) obtained from Hi-media the presence of 12 bioactive phytochemical compounds in the (Mumbai). The MHA plates were prepared by pouring 15 ml of molten Chloroform extract of Justicia gendarussa. More than bioactive media in to sterile petriplates. The plates were allowed to solidify for phytochemical compounds are identified in Chloroform extract 5 minutes and 0.1% inoculums suspension was swabbed uniformly compared than Methanol and Petroleum Ether extract (Tables 2.1 – and the inoculums were allowed to dry for 5 minutes. The 2.3; Fig - 2.1 – 2.3). It was found that the secondary metabolites and concentration of extracts (Control, 25µl, 50µl, 75µl and 100 µl) are 40 bioactive phyto-constituents identified by GC/MS in different mg/disc was loaded on 6 mm sterile disc. The loaded disc was placed plants.[15]

Journal of Pharmacy Research Vol.11 Issue 1 January 2017 23-28 S. Murugesan / Journal of Pharmacy Research 2017,11(1),23-28 Fig 2.1. GC-MS Analysis of Justicia gendarussa Burm.f. of Stem Methanol Extract

Table - 2.1 Identification of chemical constituents from Methanol extract of the stem by GC-MS.

S.No Retention Peak Name Molecular Molecular % peak time Formula weight area

1 13.25 1H-Indole, 1-methyl- C9H9N 131 0.25

2 19.78 3-Methylene-5- methylhepten-5-ol C9H16O 140 0.18

3 23.25 Phytol Acetate C22H42O2 338 0.69

4 25.23 Eicosanoic acid, methyl ester C21H42O2 326 2.40

5 29.02 2-Hexadecen-1-ol, 3,7,11,15-tetramethyl- C20H40O 296 5.57

6 35.1 2,3-epoxypropylcyclooctane C11H20O 168 1.03

7 37.46 1,2- Benzenedicarboxylic acid, bis(2- methylpropyl) ester C16H22O4 278 0.27

8 40.37 3-Chloro-2- methylidenepropylisobutyrate C8H13ClO2 176 1.16

9 42.62 4,5-Diacetoxy-3- butylpentanal C13H22O5 258 2.76 Fig – 2.2 GC-MS Analysis of Justicia gendarussa Burm.f. of Stem chloroform Extract

Table – 2.2 Identification of chemical constituents from Chloroform extract of the stem by GC-MS. S.No Retention Peak Name Molecular Molecular % peak time Formula weight area

1 4.16 Benzene, 1,4-dichloro- C6H4Cl2 146 2.92 2 6.28 4,6-Octadienoic acid C8H12O2 140 0.80 3 8.74 Eugenol C10H12O2 164 2.86 4 9.20 Germacrene D C15H24 204 0.67 5 9.82 Caryophyllene C15H24 204 0.53 6 10.47 1,3-Cyclohexadiene, 5-(1,5- dimethyl-4-hexenyl)-2-methyl,C15H24 204 3.23 7 10.85 1,6,10-Dodecatriene, 7,11- dimethyl-3-methylene-, (Z)- C15H24 204 0.35 8 11.80 3-Pyridinecarboxylic acid, 6-amino- C6H6N2O2 138 1.76 9 13.69 Tetradecanoic acid C14H28O2 228 1.47 10 16.41 1,2-Benzenedicarboxylic acid, butyl octyl ester C20H30O4 334 1.26 11 19.30 9,12-Octadecadienoic acid (Z,Z)- C18H32O2 280 23.41 12 19.61 Octadecanoic acid C18H36O2 284 6.08 Journal of Pharmacy Research Vol.11 Issue 1 January 2017 23-28 S. Murugesan / Journal of Pharmacy Research 2017,11(1),23-28 Fig – 2.3 GC-MS Analysis of Justicia gendarussa Burm.f. of Stem petroleum ether Extract

Table - 2.3. Identification of chemical constituents from petroleum ether extract of the stem by GC-MS. S.No Retention Peak Name Molecular Molecular % peak area time Formula weight

1 10.29 Benzene, 1-(1,5-dimethyl-4- hexenyl)-4-methyl- [Ar-Curcumene] C15H22 202 3.07

2 11.17 Dodecanoic acid C12H24O2 200 0.73

3 18.38 Hexadecanoic acid, ethyl ester C18H36O2 284 1.69

4 19.30 9,12-Octadecadienoic acid (Z,Z) C18H32O2 280 23.41

5 26.07 Docosanoic acid C22H44O2 340 4.96

6 28.83 1,1-Dideutero- ,6,10,14-tetra methylpentadeca- 9,13-diene-1,3,5-triol C19H32D2O2 294 2.07

7 36.84 1-Propanamine, 3-dibenz[b,e]oxepin-11(6H)-ylidene-N,N-dimethyl- C19H21NO 279 3.36

8 36.96 9-Octadecenoic acid, methyl ester C19H36O2 296 1.23

5.3. Physico – Chemical Values Table - 3.2. Florescence analysis of stem on Justicia gendarussa The results of Physico – Chemical values and Florescence analysis Burm.F. various solvents. of Justicia gendarussa stem recorded were given. In stem Physico – chemical values such as Foreign organic matter recorded for as S. Solvent Visible light Short UV Long UV No treatment (254nm) (366nm) 0.37, Moisture content recorded for as 05.16, Total ash recorded for as 10.50, Acid Insoluble ash recorded 7.21, Water soluble ash recorded 1 Drug + Distilled Greenish Yellow green Blackish green for as 7.03, Water insoluble recorded for as 6.58 and Sulphated ash water colour 2 Drug + H SO Dark green Light green Light green recorded for as 4.25 respectively. (Tables – 3.1). Florescence analysed 2 4 3 Drug + NaOH in Dark brown Light green Dark green by different solvents such as Distilled water, H2SO4, NaOH in water, water NaOH in methanol and HCl. Justicia gendarussa stem of florescence 4 Drug + NaOH Yellowish Greenish black Ceramic yellow showed in short UV (254nm) and long UV (366nm) (Table 3.2). in methanol brown 5 Drug + HCl Dark brown Brownish black Dark brown Table – 3.1. Physico - Chemical Parameters of Justicia gendarussa 5.4. Antibacterial activity Burm.f. stem The antimicrobial study was also carried out against two micro S.No Parameters Stem powder (%) organisms. The maximum zone of inhibition was observed against P. pneumonia and it was followed by P.vulgaris. Methanol extract of 1 Foreign organic matter 0.37 ± 0.23 stem of Justicia gendarussa exhibited the highest inhibition zone 2 Moisture content 05.16 ± 0.51 3 Total ash 10.50 ± 0.35 was observed against P. pneumonia. Chloroform and Petroleum Ether 4 Acid Insoluble ash 7.21 ± 0.96 Extracts were showed highest antibacterial activity against 5 Water soluble ash 7.03 ± 0.02 P. vulgaris. The present findings corroborate with the report of 6 Water insoluble ash 6.58 ± 0.37 antibacterial activity by J. gendarussa aqueous stem extract shown 7 Sulphated ash 4.25 ± 0.19

Journal of Pharmacy Research Vol.11 Issue 1 January 2017 23-28 S. Murugesan / Journal of Pharmacy Research 2017,11(1),23-28 significant antibacterial activity against S. flexneri (26.20mm), 4. Sridevi Sangeetha Kothandaraman Sivapraksam, Kavitha P. mirabilis (24.50mm), E. coli (21.40mm), B. subtilis (20.25mm), Karunakaran, Umamaheswari Subburaya, Sujatha S. paratyphi (19.50mm) and S. typhimurium (17.20mm) (Table 4).[16] Kuppusamy, Subashini TS. A Review on Phytochemical and Pharmacological Profile of Hedyotis corymbosa Linn. Table: 4. Antibacterial activity of Justicia gendarussa Stem in International Journal of Pharmaceutical Sciences Review Methanol, Chloroform and Petroleum Ether Extracts (The and Research. 2014; 26(1): 320-324. Concentration of control, 25µl, 50µl, 75µl, 100µl) 5. Kavitha K, Sridevi sangeetha KS, Sujatha K, Umamaheswari S. Phytochemical and Pharmacological Profile of Justicia Name of the Name of Zone of inhibitor (mm) gendarussa Burm f. – review. Journal of Pharmacy Research. Extract organism Control 25µl 50µl 75µl 100µl 2014; 8(7): 990-997. Methanol P. vulgaris 18 - 12 17 21 6. Mukherjee Pulok K. Quality control of herbal drugs. P. pneumonia 19 - 15 20 23 Business Horizons publication. New Delhi. 2002; 2: 2-24. Chloroform P. vulgaris 18 - 13 19 24 P. pneumonia 19 - 14 19 22 7. Mohamed farook NA. EJournal of Chemistry. 2009; 6(3): Petroleum Ether P. vulgaris 18 - 15 19 24 938-942. P. pneumonia 19 - 14 17 21 8. Senthilkumar N, Varma P, Gurusubramanian G. Larvicidal and 6. CONCLUTION adulticidal activities of some medicinal plants against the Fast dissolving drug delivery system have better patient compliance malarial vector Anopheles stephensi (Liston). Parasitology and may offer improved biopharmaceutical properties, improved Research. 2009; 104:237–244. efficacy and better safety compared with conventional oral dosage 9. Jothimanivannan C, Kumar RS, Subramanian N. Anti- forms. Fast dissolving tablets have gained considerable attention as inflammatory and analgesic activities of ethanol extract of a preferred alternative to conventional tablets and capsules due to aerial parts of Justicia gendarussa Burm. International better patient compliance. In Identification Chemical constituents Journal of Pharmacology. 2010; 6(3): 278- 283. and Physico Chemical characters which may be useful for 10. Amghalia E, Nagi AA, Shamsudin MN, Radu S, Rosli R, Pharmacognostical studies. In the present study, the plants contain Neela V, Rahim RA. Multiplex PCR Assays for the Detection potential antibacterial components that may be useful for evolution of Clinically Relevant Antibiotic Resistance Genes in of pharmaceutical for the therapy of ailments. It is concluded that Staphylococccusaureus Isolated from Malaysian Hospitals. the selected ethno medicinal plant Justicia gendarussa has the Research Journal of Biological Sciences. 2009; 4(4): 444- medicinal potential to develop new leads of therapeutic interest 448. against various diseases. 11. Cohen ML. Epidemiology of drug resistance: implications for a post-antimicrobial era. Science. 1992; 257: 1050-1055. Acknowledgement 12. Warrier PK, Nambiar VPK, Ramankutty C, Indian Medicinal I express my sincere thanks to the UGC to providing the financial Plants— A Compendium of 500 Species, vol. 3. Orient assistance to do the entire work. Longman Ltd., Chennai, 1995, p. 272–273. 13. Khare CP, Indian medicinal plants, springer science – REFERENCES business media LIC, 2007, p.350. 1. Naidoo KK, Coopoosamy RM A. Comparative analysis of 14. Fatima A, Singh PP, Agarwal P, Irchhaiya R, Alok S, Amita V. Treatment of various diseases by carissa spinarum L. - a two medicinal plants used to treat common skin conditions promising shrub. International journal of Pharmaceutical in South Africa. African Journal of Pharmacy and Science and Research. 2013; 4(7): 2489-2495. Pharmacology. 2011; 5(3): 393-397. 15. Kumar PP, Kumaravel S, Lalitha C. Screening of antioxidant 2. Diehl MS, Atindehou KK, Tere H, Betschart B. Prospect for activity, total phenolics and GCMS study of Vitex negundo. anthelminthic plants in the Ivory Coast using African Journal of Biochemistry Research. 2010; 47(7): 191- ethnobotanical criteria. Journal of Ethnopharmacology. 2004; 195. 95: 277-284. 16. Subramanian N, Jothimanivannan C, Moorthy K. 3. Selvarasuvasuki Manikandan. Ethnomedicinal Flora of Antimicrobial activity and preliminary phytochemical Ivanur Panchayat in Cuddalore District, Tamil Nadu, India. screening of Justicia gendarussa (Burm. f.) against human International Journal of Research in Plant Science. 2013; pathogens. Asian Journal of Pharmaceutical and Clinical 3(2): 39-46. Research. 2012; 5(3): 229-233.

Source of support: UGC, India, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.11 Issue 1 January 2017 23-28