Qualitative analysis of phytoconstituents from Azima tetracantha L. and evaluation of its antimicrobial activity

156 Thariq and Prince Int. J. Biosci. 202 5 RESEARCH PAPER RESEARCH PAPERRESEARCH PAPER RESEARCH PAPER OPEN ACCESS OPEN ACCESSOPEN ACCESS OPEN ACCESS Qualitative analysis of phytoconstituents from Azima tetracantha L. and evaluation of its antimicrobial activity J. Thaha Thariq * , L. Prince PG and Research Department of Microb iology, Maruthupandiyar College (Affliated to Bharathidasan University), Thanjavur, Tiruchirappalli, Tamil Nadu, India Key words: Phytomedicine, Medicinal plants, Phytocompounds, Azima tetracantha L., Antimicrobial activity http://dx.doi.org/10.12692/ijb/26.1.156-162 Article published on January 10, 2025 Abstract Azima tetracantha L., a medicinal plant from the Salvadoraceae family, is traditionally recognized for its diverse therapeutic properties, including antimicrobial, anti-inflammatory, hepatoprotective, and hypoglycemic effects. This study aimed to screen the phytochemical constituents and evaluate the antimicrobial potential of its leaf extracts. The phytochemical analysis revealed the presence of key bioactive compounds such as tannins, flavonoids, phenolic compounds, lignin, and terpenoids in ethyl acetate and ethanol extracts. The antimicrobial activity was assessed using the disc diffusion method against various bacterial (Escherichia coli, Pseudomonas aeruginosa, Streptococcus mutans, Salmonella typhi, and Staphylococcus epidermidis) and fungal (Aspergillus niger, Aspergillus flavus, and Candida sp.) strains. The ethanol extract demonstrated the highest inhibition against Escherichia coli (11 mm), Streptococcus mutans (11 mm), and Staphylococcus epidermidis (12 mm). The ethyl acetate extract exhibited notable inhibition against Escherichia coli (8 mm) and Staphylococcus epidermidis (6 mm). The diethyl ether extract showed strong inhibition against Escherichia coli (12 mm) but was less effective against other bacterial strains. Petroleum benzene extract exhibited limited activity, with inhibition observed only against Streptococcus mutans (7 mm). Mixed solvent extracts displayed the highest inhibition against Escherichia coli (20 mm), Streptococcus mutans (20 mm), Staphylococcus epidermidis (15 mm), and Candida sp. (16–20 mm). GC-MS analysis identified several bioactive metabolites, further highlighting the pharmaceutical potential of Azima tetracantha L. The findings support its traditional medicinal use and suggest its potential role in developing novel antimicrobial agents. * Corresponding Author: J. Thaha Thariq  [email protected] International Journal of Biosciences | IJB | ISSN: 2220-6655 (Print) 2222-5234 (Online) http://www.innspub.net Vol. 26, No. 1, p. 156-162, 2025 157 Thariq and Prince Int. J. Biosci. 202 5 Introduction Since ancient times, medicinal plants have been valued for their potent abilities to treat and manage a wide range of health issues (Begum et al., 2009). The World Health Organization reports that 80% of the world's population relies on medicinal plants, which are incorporated into over 30% of pharmaceutical products. Medicinal plants owe their diverse range of effects to the many phytochemicals they contain, including alkaloids, anthraquinones, flavonoids, glycosides, phenols, saponins, steroids, sterols, tannins, terpenoids, triterpenoids, phytosterols, hydrocarbons, monoand sesquiterpenes, phlobatannins, and other secondary metabolites (Bennett et al., 2004). Various studies, including in vitro and in vivo experiments, have investigated the antioxidant properties of spices, their role in promoting digestion, reducing lipid levels, and their possible antibacterial, anti-inflammatory, antiviral, and anticancer effects. These plant components or their extracts are usually taken orally, either alone or in combination with various ingredients like water, honey, milk, juices, or black pepper (Nandgude et al., 2007; Rall et al., 1967; Rao and Prasada Rao, 1978). In modern times, plant-derived secondary metabolites are employed in numerous sectors, including food production (for flavor enhancement, nutritional improvement, and spoilage prevention), medicine (for the prevention and treatment of diseases, chemoprevention, and as natural antimicrobials), as well as in pharmacology and cosmetology (e.g., cosmetics and dietary supplements without preservatives). Ayurveda, Siddha, and Unani are traditional forms of medicine that have been practiced for hundreds of years. Drugs developed from Ayurvedic practices to treat current health conditions are now accessible in the market (Williams and Nagarajan, 1988). The World Health Organization reports that about 80% of individuals in developing countries use traditional medicine as their main source of primary health care. Azima tetracantha L. (belonging to the Salvadoraceae family) is valued for its significant medicinal properties (Nandagude et al., 2007). Its roots, leaves, fruits, and stems have been traditionally used to address various health conditions and are noted for their stimulant, expectorant, antispasmodic, analgesic, antiinflammatory, anti-ulcer, anti-diarrheal, antimicrobial, hepatoprotective, nephroprotective, hypoglycemic, and hyperlipidemic effects (Bennett et al., 2004; Kekuda and Raghavendra, 2017). The plant Azima tetracantha L. is recognized by a variety of synonyms, such as Monetia barlerioides L’Herit., Azima nova J. F. Gmel., Kandena spinosa Rafin., Monetia angustifolia Boj. Ex A. DC., and Monetia tetracantha (Lam.) Salisb. The plant is called "Kundali" in Ayurvedic medicine, whereas in Siddha, it goes by the name "Mulchangan." Azima tetracantha is a perennial shrub that grows to about 3 meters in height, often thriving in hot, dry riverine scrub regions, especially in soils that are alluvial or saline (Tiwari et al., 2011). The leaves are noted for their rigid texture and elliptical shape, along with a pale green color. These unisexual flowers, which are small and can be greenishwhite or yellow, cluster together in the axils. The fruits of this plant are spherical and shiny white, containing seeds that are flat and circular in shape (Oman et al., 2013; Prabu et al., 2013; Saxena et al., 2013; Mottaleb and Sarker, 2012). A. tetracantha L. is recognized for its diuretic properties, which help manage rheumatism, dropsy, dyspepsia, and chronic diarrhea, and it is additionally used as a tonic stimulant post-childbirth. Beneficial for addressing cough, phthisis, asthma, smallpox, and diarrhea, this plant's leaves, root, and root bark are particularly noted for their effectiveness in treating rheumatism. The root has been traditionally used as a diuretic and is applied in Siddha medicine to treat dropsy and rheumatism. In herbal practices, the leaves serve as a stimulant, expectorant, and antispasmodic. Moreover, the bark is utilized for its antiperiodic, astringent, and expectorant properties, particularly in managing cough and asthma (Raaman, 2006). In this study we screened and evaluate the phytoconstuents from Azima tetracantha L. plant extract. Materials and methods Gathering and classification of plants The garden-fresh plant materials were collected by using sterilized knife into clean polyethylene bags and finally it brought to the laboratory. Leaf of the plant was washed and identified by botanically, then cut into tiny pieces 158 Thariq and Prince Int. J. Biosci. 202 5 and allowed to dry for approximately two weeks in shade dry. The shaded dried plant leaf was ground into a coarse powder put through a filter and at last extracted by using various solvents of increased polarity. Preparation of phyto-extraction by using soxhlet The grainy powder was filled into the soxhlet apparatus and passed through two exacting solvents known as ethyl acetate and ethanol distinctly by continuous hot percolation method. After collecting the filtered extraction the solvents were eliminated through distillation under reduced pressure and to the end the coloured filtrate was attained (Rall et al., 1967). Phytoconstituent test The phytochemical analysis of the plant leaf was examined for carbohydrate, fixed oil and lipids, amino acid as well as protein, alkaloids, tannins, phenolic compounds, lignin, saponins, flavonoids, phytosterols, gums and mucilage carried out by standard (Rao and Prasada Rao, 1978). Microorganisms The microbe species were collected from MTCC- (Microbial Type Culture Collection) IMTECH, Chandigarh. The plant leaf extracts were examined for activity of antimicrobial in disc diffusion method for six important strains of bacteria including Escherichia coli, Pseudomonas aeruginosa, Streptococcus mutans, Salmonella typhi, Staphylococcus epidermidis and bacterial cultures were maintained at 4 °C nutrient agar. Slants 0.1 ml of bacterial cultures (18 hrs) was spread over suitable sterile Muller Hinton agar plates. The fungal strains, Aspergillus niger and Aspergillus flavus were maintained on Czapek Dox Agar (CDA) and the cultures were sub-cultured at regular intervals of one month. The Candida sp. was maintained and sub cultured in the Sauboraud’s Dextrose Agar. Preparation of inoculum The experimentally used bacterial cultures were subcultured in nutrient broth at 37  C for 18 hours. Fungal cultures were sub-cultured in Czapek Dox Agar and Sauboraud’s Dextrose Agar (Candida sp.) plates at 25 C for 24 hours and used for the subsequent tests. Disc preparation Different concentrations of Azima tetracantha L. with different solvents used extract were 50 mg, 60 mg; 90 mg and 100 mg were taken. 6 mm in diameter of Whatman No. 1 filter paper discs were imbibed in the extracts solutions and were allowed to dry. Each dried disc contained different concentrations of each extract such as 6.25 mg, 7.5 mg, 8.75 mg, 10 mg, 11.25 mg, 12.5 mg/disc. Antimicrobial assay Disc diffusion method was used to examine the antimicrobial activity (Williams and Nagarajan, 1988). Various concentration impregnated discs were placed on the selected bacterial, fungal and Candida sp. swabbed plates. Each plate contains 6 different concentrated discs. Bacterial and Candida sp. plates were incubated at 37 C for 24 hrs and fungal plates were incubated at 28 C for 72 hrs. The results were observed after incubation. Results and discussion The present study has screened the different organic solvents (Ethanol, Diethyl acetate and Petroleum benzene used extracts of the leaves of Azima tetracantha L. against Grams-positive organisms (Streptococcus mutans, Staphylococcus epidermidis), Gram-negative organism (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi) and fungal species (Aspergillus flavus, Aspergillus niger and Candida sp.). The phytoconstituent study of the plant leaf extract showed that the components present in the ethyl acetate and ethanol extracts were found to be carbohydrates, tannins and phenolic compounds, flavonoids and lignin. However, alkaloids, phytosterols, fixed oil and fats, proteins and aminoacids, gums and mucilage and saponins were not detected in the materials used (Table 1). Therefore, we can concluded that the antifungal and antibacterial of the ethanol and ethyl acetate exacts might be due to the above phytochemical components. The antimicrobial activity may be due to the presence of carbohydrates, tannins and phenolic compounds, flavonoids and lignin etc. the same report is also given by (Nandagude et al., 2007) for the leaves of Alstonia macrophylla. Highly susceptible organisms namely 159 Thariq and Prince Int. J. Biosci. 202 5 Streptococcus mutans and Escherichia coli were selected to analyse minimal inhibitory concentration of the test plant extract on that bacterial strains. Table 1. Phytochemical screening of Azima tetracantha L. Phytochemical compounds Observation Control Aqueous Acetone Methanol Tannins - - + + Flavonaids - + + + Terpenoids - - + + Saponins - + + + Phlobatannins - - - + Steroids - - - + Carbohydrates - - - - Glycosides - - - + Cournarins - - - - Proteins - - - - Emodins - - - - Anthraquinones - - - - Anthocyanins - - - - Leucoantho cyaninsturns - - - - The effect of ethanolic extract of leaves of Azima tetracantha L. incomycin (15mcg) was tabulated. The ethanol extract was found highly effective against all the test organisms at a concentration of 12.5 mg/disc (Table 2). When the zone formation for each organism was compared to that of concentration there was an increase in zone formation for every increased concentration and the zone of inhibition is 11 mm as the highest, against Streptococcus mutans. The negative control (Disc having only the solvent ethanol) had no effect on both the Gram-negative and Gram-positive organisms used in this study. Bennett et al. (2004) reported about the ethanolic extract of Gymnema sylvestre leaves exhibited antimicrobial activity against Pseudomonas aeruginosa and inactivity against Escherichia coli. Similarly ethanolic extracts appeared to exert more inhibitory action against the bacteria (Escherichia coli). This was reported by Kekuda and Raghavendra (2017) in the same way, the ethanolic extracts of leaves of Azima tetracantha L were also exhibited good inhibitory activity on the Escherichia coli. The results showed that the diethyl ether extract had good effect on Ampicillin resistant Escherichia coli (12mm) than Staphylococcus epidermidis (5 mm). They didn’t have any activity against Streptococcus mutans, Salmonella typhi and Pseudomonas aeruginosa. The variation in the zone formation for every increased concentration is only by 10 mm for Ampicillin resistant Escherichia coli. The negative control (Disc containing the solvent diethyl ether alone) showed no activity against all the organisms. The ethanolic, diethyl ether and ethyl acetate extract of leaves of P. reticulates had good inhibitory effect on Streptococcus mutans growth (Bennett et al., 2004). But, the ethanolic and petroleium benzene extract of leaves of Azima tetracantha L. also exhibited the same effect on Streptococcus mutans. A similar work was done by (Tiwari et al., 2011) on aerial parts of Drosera peltata who showed that the ethanol, diethyl ether, ethyl acetate and petroleium ether exhibited antimicrobial activity against Streptococcus mutans. The effect of petroleum benzene extract of Azima tetracantha L. against five organisms are indicated. Petroleum benzene was active only against Streptococcus mutans as 7 mm and no activity was observed for other tested organisms. The negative control (disc containing the solvent petroleum benzene alone) had no effect on all the tested organisms. It was seen that ethyl acetate extracts showed high activity against Escherichia coli (8 mm). At higher concentration the ethyl acetate extract showed activity against Staphylococcus epidermidis (6 mm). They did not exhibit any activity against Streptococcus mutans, Pseudomonas aeruginosa and Salmonella typhi. When the zone formation for Escherichia coli was compared to that of concentration, there was an increase of 1.0 mm for every concentration increased. The negative control (disc contained the solvent ethyl acetate) has no activity on bacterial cultures used in the study. The Euphorbia thymifolia ethyl acetate extract inhibited the Escherichia coli growth. Ethyl acetate extract appeared to be more effective. This was given by (Oman et al., 2013), But, the ethyl acetate extracts of Azima tetracantha L. leaves has no effect on Escherichia coli and diethyl ether extract and ethanolic used extracts found to be more potent than ethyl acetate. 160 Thariq and Prince Int. J. Biosci. 202 5 Table 2. Antimicrobial activity (Zone of inhibition) Microorganisms Ethanol (mm) Ethyl acetate (mm) Diethyl ether (mm) Petroleum benzene (mm) Mixed solvent (mm) Escherichia coli 11 8 12 0 20 Pseudomonas aeruginosa 0 0 0 0 5 Streptococcus mutans 11 0 0 7 20 Salmonella typhi 0 0 0 0 0 Staphylococcus epidermidis 12 6 5 0 15 Aspergillus niger - - - - - Aspergillus flavus - - - - - Candida sp. - - - - 16 - 20 Table 3. GC-MS analysis of Azima tetracantha L. extract Sl RT Compound name Area % Height % 1 4.117 N,N - DIMETHYL - 1,3 - BUTADIEN - 1 - AMINE 18.9 14.72 2 8.469 3 - PYRIDINECARBOXYLIC ACID, 1,2,5,6 - TETRAHYDRO - 1 - METHYL - , METHYL ESTER 0.75 1.08 3 10.656 Methanamine, N - [3 - methyl - 2 - butenylidene] 0.85 0.86 4 10.954 Neophytadiene 0.74 1.12 5 11.18 1 - Methyl - pyrrolidine - 2 - carboxylic acid 3.49 1.01 6 11.357 2 - METHYL - 2 - NONENE 4.83 3.02 7 11.529 Dodecane 0.56 0.6 8 12.43 4,6,7 - TRIMETHYL - 2H - AZEPINE - 2,5(6H) - DIONE 2.92 2.55 9 17.779 2 - CYCLOPENTEN - 1 - OL, 5 - (1,1,3 - TRIMETHYL - 2 - BUTENYL) - 0.35 0.56 10 18.087 2 - CYCLOPENTEN - 1 - OL, 5 - (1,1,3 - TRIMETHYL - 2 - BUTENYL) - 0.99 1.39 11 19.979 1,2 - BENZENEDICARBOXYLIC ACID, DIETHYL ESTER 0.36 0.47 12 22.005 Oxalic acid, cyclohexylmethyl tridecyl ester 4.73 7.18 13 23.5 2(4H) - BENZOFURANONE, 5,6,7,7A - TETRAHYDRO - 6 - HYDROXY - 4,4,7A - TRIMETHYL-, (6S-CIS)- 0.46 0.44 14 23.83 PLUCHIDIOL 1.29 1.01 15 24.534 Neophytadiene 1.09 1.51 16 24.629 2 - PENTADECANON, 6,10,14 - TRIMETHYL - 0.79 1.09 17 24.954 Phytol, acetate 0.5 0.69 18 25.17 9 - Heptadecanone 0.29 0.48 19 25.271 Neophytadiene 0.39 0.6 20 25.807 5,9,13 - PENTADECATRIEN - 2 - ONE, 6,10,14 - TRIMETHYL - , (E,E) - 0.68 0.96 21 26.037 Hexadecanoic acid, methyl ester 1.91 2.5 22 26.113 Benzenepropanoic acid, 3,5 - bis(1,1 - dimethylethyl) - 4 - hydroxy - , methyl ester 1.04 1.51 23 26.693 DECANE, 5,6 - BIS(2, 2 - DIMETHYLPROPYLIDENE) - , (E,Z) - 4.44 5.49 24 26.829 Sulfurous aci d, cyclohexylmethyl hexyl ester 2.57 3.24 25 27.148 HEXADECANOIC ACID, ETHYL ESTER 0.54 0.71 26 27.434 Decane, 5,6 - bis(2, 2 - dimethylpropylidene) - , (E,Z) - 7.39 1 0.99 27 28.714 9,12 - OCTADECADI ENOIC ACID (Z,Z) - , METHYL ESTER 0.24 0.43 28 28.811 8,11,14 - Ei cosatrienoic acid, methyl ester 0.71 0.86 29 28.983 Phytol 5.62 8.04 30 29.228 Methyl stearate 0.54 0.81 31 29.379 gamma - Sitosterol 0.46 0.49 32 29.899 beta - Sitosterol 0.49 0.55 33 31.818 Octadecan oic acid, 10 - oxo - , methyl ester 1.42 1.4 34 32.451 4,8,12,1 6 - Tetramethylheptadecan - 4 - olide 1.35 1.14 35 32.584 Friedelan - 3 - one 1.45 0.73 36 32.736 Octadecanoic acid, 10 - ox o - , methyl ester 0.67 0.77 37 34.903 Tetradecanal 0.58 0.52 38 37.975 13 - Docosenamide, (Z) - 21.56 16.59 39 38.957 alpha - Tocospiro A 0.95 0.67 40 40.047 delta - Tocopherol 1.09 1.24 Prabu et al. (2013) reported the alkaloid fraction of LWF from the leaves of P. discoideus inhibited the growth of Escherichia coli and Pseudomonas aeruginosa. The same results were obtained in the Azima tetracantha L. incomycin (15mcg) leaf extract. It was found that the ethanol and ethyl acetate 161 Thariq and Prince Int. J. Biosci. 202 5 extracts was found to be effective against most of the organisms. For interest, the four dissolvable concentrate of leaves of Azima tetracantha L. were combined or joined as one and tried against eight microorganisms at different fixation. The results indicated that the mixed solvent extract of Azima tetracantha L. leaves had much more effect on Escherichia coli, Streptococcus mutans, and Staphylococcus epidermidis. They displayed less action as 5 mm against Pseudomonas aeruginosa and no impact on lay on different organisms utilized in the study. To the extent as susceptibility of the organisms concerned, Staphylococcus epidermidis and Escherichia coli was known to be the most susceptible and Salmonella typhi and Pseudomonas aeruginosa were the most resistant towards the four solvent extracts. The examination was done with the fungus Candida sp. from the ongoing study results, the ethanol extract of Azima tetracantha L. have antifungal activities. The zone of inhibition denotes the growth inhibition effect. The zone ranges from 7 to 9 mm and 11 to 20 mm in diameter as connected with their concentration. Whereas the inhibition of Candida species to the Phyllan this reticulates extract is a side spectrum and the results denotes a strong antifungal activity with the inhibition zone of 16-20 mm in diameter as related to concentration of lower to higher level. Validation of the antimicrobial properties and GC-MS analysis of Azima tetracantha L. has been achieved (Table 3). Conclusion This study highlights the significant antimicrobial potential of Azima tetracantha L., confirming its traditional medicinal use. The phytochemical analysis revealed the presence of key bioactive compounds, including tannins, flavonoids, phenolic compounds, and lignin, which likely contribute to its antimicrobial properties. The antimicrobial activity assessment demonstrated that ethanol and ethyl acetate extracts exhibited strong inhibitory effects against Escherichia coli, Streptococcus mutans, and Staphylococcus epidermidis, with inhibition zones reaching up to 12 mm in ethanol extracts and 8 mm in ethyl acetate extracts. The mixed solvent extract exhibited the highest inhibition, particularly against Escherichia coli (20 mm), Streptococcus mutans (20 mm), Staphylococcus epidermidis (15 mm), and Candida sp. (16–20 mm). These findings suggest that Azima tetracantha L. possesses potent antimicrobial compounds that could be explored for pharmaceutical and therapeutic applications. Furthermore, GC-MS analysis identified multiple bioactive metabolites, reinforcing the plant’s potential as a natural source for novel antimicrobial agents. Future research should focus on isolating and characterizing these bioactive compounds and conducting in vivo studies to validate their efficacy and safety for clinical use. Acknowledgements The authors are thankful to PG and Research Department of Microbiology, Marudupandiyar College (Affiliated to Bharathidasan University), Thirucirappalli, Thanjavur, Tamil Nadu, India and Specialty Lab & Research, Thanjavur for offering facilities to carry out this study. References Begum TN, Muhammad Ilyas MH, Burkanudeen A, Kalavathy S, Vijaya Anand A, Sampathkumar P, Jaswanth. 2009. Hypoglycemic and antihyperlipidemic activity of ethanolic leaf extract of Azima tetracantha Lam. on alloxan-induced diabetic rats. J. Cell Tissue Res. 9(1), 1681–1685. Bennett RN, Mellon FA, Rosa EA, Perkins L, Kroon PA. 2004. Profiling glucosinolates, flavonoids, alkaloids, and other secondary metabolites in tissues of Azima tetracantha Lam. (Salvadoraceae). J. Agric. Food Chem. 52(19), 5856–5862. Bennett RN, Rosa EA, Perkins L, Kroon PA. 2004. Profiling glucosinolates, flavonoids, alkaloids, and other secondary metabolites in tissues of Azima tetracantha L. (Salvadoraceae). J. Agric. Food Chem. 52(19), 5856–5862. 162 Thariq and Prince Int. J. Biosci. 202 5 Kekuda PT, Raghavendra HL. 2017. Phytochemistry, traditional uses, and pharmacological activities of Azima tetracantha Lam. (Salvadoraceae) - An updated review. International Journal of Green Pharmacy 10, 217– 229. Mottaleb MA, Sarker SD. 2012. Accelerated solvent extraction for natural products isolation. Methods in Molecular Biology 864, 75–87. Nandagude TD, Bhojwani AP, Krishna Kinage. 2007. Analgesic activity of various extracts of leaves of Azima tetracantha Lam. Int. J. Green Pharm. 29, 37–38. Nandgude TD, Bhojwani AP, Krishna Kinage. 2007. Analgesic activity of various extracts of leaves of Azima tetracantha Lam. Int. J. Green Pharm. 1(1), 37–38. Oman M, Skerget M, Knez Z. 2013. Application of supercritical fluid extraction for the separation of nutraceuticals and other phytochemicals from the plant material. Macedonian Journal of Chemistry and Chemical Engineering 32(2), 183– 226. Prabu K, Samydurai P, Subbaiyan B, Thangapandian V. 2013. Phytochemical constituents and gas chromatography-mass spectrometry analysis of Caralluma diffusa (weight) N.E.BR. Aerial part. International Journal of Pharmacy and Pharmaceutical Sciences 5(3), 602–605. Raaman N. 2006. Phytochemical Techniques. 1st ed. New India Publishing Agency, New Delhi, p. 19–25. Rall GJH, Smalberger TM, De Waal HL, Arndt RR. 1967. Dimeric piperidine alkaloids from Azima tetracantha. Tetrahedron Lett. 3465–3469. Rao VE, Prasada Rao PRS. 1978. Occurrence of triterpenoids in Azima tetracantha. Curr. Sci. 47, 857. Saxena M, Saxena J, Nema R, Singh D, Gupta A. 2013. Phytochemistry of medicinal plants. Journal of Pharmacognosy and Phytochemistry 1(6), 168–182. Tiwari P, Kumar B, Kaur M, Kaur G, Kaur H. 2011. Phytochemical screening and extraction: A review. Internationale Pharmaceutica Sciencia 1(1), 98–106. Williams UV, Nagarajan S. 1988. Isorhamnetin-3O-rutinoside from leaves of Azima tetracantha Lam. Ind. J. Chem. 27, 387.