*Corresponding Author:
Indiraleka Muthiah
Department of Biotechnology
Mepco Schlenk Engineering College (Autonomous)
Sivakasi, Tamil Nadu 626005, India
Date of Received 10 May 2022
Date of Revision 02 February 2023
Date of Acceptance 25 October 2023
Indian J Pharm Sci 2023;85(5):1408-1421

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Ruellia patula belongs to the family Acanthaceae. The leaves of this plant possess tremendous medicinal values like treating wounds, eyesores, gonorrhea, syphilis and renal infections. Phytochemicals extracted using four different solvents namely hexane, acetone, methanol and water by maceration technique were qualitatively analysed by chemical tests. Total phenolic content was estimated as 142.94±1.01, 104.41±7.06, 14.37 mg Gallic Acid Equivalent/g of extract in acetone, methanol, and aqueous extracts respectively. Total flavonoid content was estimated as 37.49±1.83, 26.73±10.65 mg Quercetin Equivalent/g of extract in acetone and methanol extracts respectively. The antimicrobial activity of plant extracts was determined by agar well diffusion assay and minimum inhibitory concentration test, acetone extract showed better results for both assays. The minimum inhibitory concentration of acetone extract against five different organisms were determined as, Bacillus subtilis-202 mg/ml, Escherichia coli-208 mg/ml, Staphylococcus aureus-208 mg/ml, Pseudomonas aeruginosa-203 mg/ml and for Proteus mirabilis-200 mg/ml. Antioxidant activity of extracts was evaluated by 2,2-diphenyl-1-picryl-hydrazyl-hydrate assay, and IC50 values of methanol and acetone extracts were estimated as 146.45 and 153.81 μg/ml respectively. Anti-inflammatory activity of extracts estimated using protein denaturation inhibition assay resulted in better anti-inflammatory property associated with acetone extract. Based on the results, the acetone and methanol extracts showed better activities at a lower concentration, further gas chromatography-mass spectrometry analysis of these extracts was carried out and biological activity of components were determined from literature survey.


Ruelliapatula, phytochemical compounds, gas chromatography-mass spectrometry, antimicrobial and anti-inflammatory activity, 2,5-diphenyl-2H-tetrazolium bromide assay

Phytochemicals are produced by plants for their growth, reproduction and protection against microorganisms or predators like insects and animals[1]. Phytochemicals show drug-likeness (antimicrobial, antioxidant, anticancer, etc.), biological friendliness and as alternatives to eradicate antibiotic-resistant pathogens[2-4]. Initially it is essential to screen and study the plants which are used as folklore medicines[5] to commercialize natural compounds as drug. The present study focuses on Dipteracanthus patulus (Jacq.) Nees. Synonym Ruellia patula (Vernacular name: Vedichedi, Kiranthinayagam) belongs to the family Acanthaceae[6]. When 5-6 leaves of this plant are chewed, the phytochemicals of these leaves act as an antidote to snakebite. Leaves are also used to treat cuts, wounds, an eyesore, gonorrhea, syphilis and renal infections[7]. Ruellia patula is an erect, pubescent, taproot, 50 cm tall, much-branched shrublet. Leaves are elliptic ovate. The fruit capsule is glabrous, 1.4-1.8 cm, 8-10 seeded. Fruits have flat and orbicular seeds. The plant is widely distributed in Arabia, India, Pakistan, Africa and Sri Lanka. In India, these plants are found in Western Ghats, Tamil Nadu, Rajasthan, Andhra Pradesh, and Haryana[8,9].

As Ruellia patula is traditionally used more frequently as a wound healer, wound healing properties of the extracts from leaves are studied. Wounds are either acute or chronic types. Acute wounds heal within the expected time, chronic wound types, take more time to heal and difficult to predict the healing time[10]. Acute wounds undergo four phases in the following order hemostasis, inflammation, proliferation, and remodelling[11]. Chronic wounds on the other hand show an extended inflammatory phase, proliferative phase, or remodelling phase leading to wound ulcers[12]. Hypoxic condition is created in the wound region due to two main reasons such as vascular disruption which leads to diminished oxygen delivery[13] and the exposure of subcutaneous tissue (due to loss of skin integrity in the wound region) to microorganisms causes existing oxygen to be utilized by the aerobic microorganisms. This hypoxic condition becomes an appropriate region for the anaerobic microorganism survival[10]. Hence the wound is not only occupied by aerobic microorganisms like Staphylococcus aureus, Pseudomonas aeruginosa, etc but also by the anaerobic microorganisms during chronic stage. So, there is a need for antimicrobial activity of the compound against those microorganisms to decrease the healing time. Further the healing time also delayed by production of excess Reactive Oxygen Species (ROS) by phagocytes. ROS being oxidizing molecules when present at a low-level help in getting rid of the infection and stimulate wound healing by producing cell survival signals[14,15], but when present in excess causes oxidative stress, cell damage and pro-inflammatory condition[16]. Antioxidant molecules on the other hand help to overcome oxidative stress by donating electron or hydrogen ions to ROS and prevent ROS from taking away electrons from macromolecules such as DNA or protein[17], thus preventing host cell damage. The present study focuses on the phytochemical analysis, evaluation of antimicrobial, antioxidant, and anti-inflammatory properties[18] of four different solvent extracts of Ruellia patula leaves and determining the more effective extract among the four. Identification of phytochemicals present in effective extract using gas chromatography-mass spectrometry analysis.

Materials and Methods

Collection of plant materials:

Ruellia patula plant was collected from different regions of Virudhunagar district in Tamil Nadu, India. Plant materials were taxonomically identified and authenticated by the Botanical Survey of India, Southern Regional Centre, Coimbatore, Tamil Nadu, India.


Leaves of collected plants were washed, shade dried and powdered using an electric blender. One nonpolar solvent hexane, one mid polar solvent acetone, and two polar solvents methanol and water were chosen. The maceration technique was carried out to avoid the loss of thermolabile compounds and to ensure a longer exposure time of leaf powder to the solvent[19]. The leaf powder was added to solvents in the ratio of 1:20 (W/V). Maceration was carried out for 24 h. Then the contents were filtered using Whatman filter paper No.1 obtained filtrate was air-dried[20].

Phytochemical analysis:

For phytochemical analysis, crude extracts were dissolved in their respective solvents. Phlobatannins were detected by the formation of a red precipitate when a few drops of extract boiled in 1 % HCl. Terpenoids or sterols were detected by the formation of a reddish-brown ring when 1 ml of the extract was mixed with 0.5 ml of chloroform followed by the addition of a few drops of concentrated sulphuric acid. Tannins or phenolics were detected by brownish-green colour formation when 2 ml of the extract was added with a few drops of 10 % ferric chloride[21]. Alkaloids were detected by adding a few ml of extract and a few ml of diluted HCl, the mixture was then filtered, and filtrate obtained was added with drops of saturated picric acid (Hager's reagent), and the formation of a yellow precipitate indicates the presence of alkaloids. Flavonoids were detected by an alkaline reagent test where 2 ml of the extract was added with a few drops of 1 N NaOH, the formation of a yellow solution indicates the presence of flavonoids. Glycosides detection was done by following the Keller-Killiani test, in which 2 ml of extract was added with 2 ml of water, 0.5 ml of lead acetate and shaken well, mixture obtained was then filtered, filtrate then added with an equal volume of chloroform, evaporated, the obtained residue dissolved in glacial acetic acid and few drops of ferric chloride was added, the obtained mixture was then transferred to test tube containing 2 ml of concentrated sulphuric acid[19]. The formation of a reddish-brown layer at the interface which on standing turns to bluish green indicates the presence of glycosides.

Total phenolic content:

Assay carried out based on Ainsworth et al.[22] with slight modifications, 0.25 ml of each gallic acid standard aliquots of following concentrations, 10, 20, 40, 60, 80, 100 µg/ml prepared in 10 % (v/v) Dimethylsulfoxide (DMSO) was added with 1 ml of 10 % (v/v) Folin-Ciocalteu reagent and 2 ml of 7.5 % (v/v) sodium carbonate. Shaken well and kept in dark condition for 30 min. Spectrophotometric reading of absorbance taken at 765 nm. Blank was prepared by replacing gallic acid aliquot with 10 % (v/v) DMSO. Whereas extracts at two different concentrations were prepared as 0.1 and 1 mg/ml in 10 % (v/v) DMSO, 0.25 ml of extracts were added in place of gallic acid aliquot. The standard curve of gallic acid concentration versus absorbance plotted was used to determine the total phenolic content in extract and expressed as mg of Gallic Acid Equivalent (GAE) per g of extract.

Total flavonoid content:

The assay was carried out based on Correa et al.[23] with slight modifications. 0.25 ml of each Quercetin standard aliquots of concentrations, 20, 40, 60, 80, 100 µg/ml prepared in 80 % ethanol was added with 0.65 ml of 80 % ethanol, 0.05 ml of 1 M sodium acetate, and 0.05 ml of aluminium chloride (10 %) and placed in dark condition for 40 min. Absorbance was read in the spectrophotometer at 415 nm. Blank was prepared by mixing 0.95 ml of 80 % ethanol and 0.05 ml of 1 M sodium acetate. Extracts of 0.5 mg/ml (in 10 % DMSO) were prepared and 0.25 ml of extract was used in place of Quercetin. Negative control samples were prepared by replacing 0.05 ml of aluminium chloride with 0.05 ml of distilled water. The standard curve of quercetin concentration vs. absorbance plotted was used to determine the total flavonoid content in the extracts and expressed results as mg of Quercetin Equivalent (QE) per g of extract.

Antimicrobial assay:

Preparation of Inoculum: The antimicrobial activity of the extracts was tested against bacteria such as Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Staphylococcus aureus, and Proteus mirabilis. The bacterial strains were procured from Microbial Type Culture Collection, India. Microorganisms were cultured overnight in nutrient broth (Peptone 5 g/l, NaCl 5 g/l, Meat Extract 1.5 g/l, Yeast Extract 1.5 g/l) at 37° in a rotary shaker. Later, each strain was adjusted to a concentration of 108 cells/ml using 0.5 McFarland standard using a spectrophotometer at a wavelength of 600 nm[24].

Agar well diffusion assay: The antimicrobial activity of extracts was tested by Agar well diffusion method. The surface of the agar plate was inoculated by spreading a 100 µl volume of the microbial culture over the entire surface of the agar plate. A well was punched aseptically using a sterile cork well borer, and 100 µl of plant extract was introduced into the well. Then the agar plates were incubated at 37° in an incubator. For the diffusion of the extracts into agar, the plates were refrigerated for 30 min[25]. After 24 h of incubation, the zone of inhibition (diameter) was measured in mm. This assay was repeated twice to minimize the errors.

Minimum Inhibitory concentration (MIC): MIC was identified by the microtiter broth dilution method. The procedure involved preparing different concentrations of plant extracts in nutrient broth in a 96 well microtitration plate. Different serial dilutions were made for different organisms for each extract. Then each well was inoculated with a microbial culture which was adjusted to a 0.5 McFarland scale. After mixing, the microtiter plates were incubated at 37° for 24 h[25]. The concentration of extract at which no visible growth was observed was taken as MIC value. This experiment was repeated twice to minimize errors and to confirm the activity.

Antioxidant activity:

2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) free radical scavenging assay: Different concentrations of 20, 40, 60, 80 and 100 µg/ml of ascorbic acid and extracts were prepared in absolute ethanol and 10 % DMSO respectively. 0.25 ml of each standard aliquots and samples was added with 1 ml of 0.1 mM DPPH reagent freshly prepared in absolute ethanol. The reaction mixture was incubated at room temperature in dark conditions for 30 min. Spectrophotometric reading of absorbance at 517 nm for all standard and sample aliquots was determined[26]. Percentage of inhibition estimated using the formula as follows:


Asample=Absorbance of sample/standard Control is 0.25 ml of ethanol or 10 % DMSO and 1 ml of 0.1 mM DPPH for standards or samples respectively. Blank is absolute ethanol or mixture of 0.25 ml of 10 % DMSO and 1 ml of ethanol for standards or samples respectively.

Anti-inflammatory activity:

The anti-inflammatory activity of Ruellia patula extracts was studied according to the protocol of Padmanabhan et al.[27], Gunathilake et al.[28]. The activity of the extracts was evaluated using the "Albumin Denaturation Inhibition Assay”. The reaction mixture was prepared by mixing 50 μl of egg albumin (from fresh hen’s egg), 700 μl of phosphate- buffered saline of pH 7.4, and 0.5 ml of plant extract (0.6 to 1.6 mg/ml). Positive controls were prepared using 0.5 ml of diclofenac sodium (0.6 to 1.6 mg/ ml) in the place of plant extract. Negative control was prepared by mixing 50 µl of Albumin and 750 µl of phosphate-buffered saline. Controls and test samples were incubated at 37° for 15 min and again incubated at 75° for 10 min. Then the absorbance was read at 660 nm (To nullify the absorbance due to extracts at different concentrations, mixture of 0.5 ml of extract (0.6 to1.6 mg/ml) and 750 μl of 10 % DMSO was prepared and spectrophotometric reading of absorbance taken at 660 nm). The percentage of inhibition was calculated using the formula


Gas Chromatography-Mass Spectroscopy (GC- MS) analysis of Ruellia patula leaf extracts:

Plant extracts were subjected to GC-MS. The analysis was performed using Agilent GC 7890A/ MS 5975C, an inert mass spectrometer fused with the capillary column of 30 m×0.25 mm with a film thickness of 0.25 µm. Pure nitrogen gas was used as the carrier gas at a flow rate of 1 ml/min and the pressure was maintained at 7.6522 psi. The oven temperature was increased from 50° to 300° at a rate of 50°/min. A 1 µl aliquot of the crude extract of acetone and methanol leaf extracts was injected in split mode. The runtime for analysis was 23.833 min. The peak retention time, peak area (%), and mass spectral fragmentation patterns to that of the known compounds described by the National Institute of Standards and Technology (NIST) library[29].

Results and Discussion

According to some investigators, it is best to carry out initially the phytochemical analysis before evaluating the biological activity of plant extracts. Determining the phytoconstituents helps to forecast the pharmacological function of the plant extracts[30,31]. The presence of phytochemicals such as flavonoids, tannins or phenols, alkaloids, glycosides, phlobatannins, terpenoids, or sterols of each extract wasdetectedbychemicaltests. Flavonoids, glycosides, and phlobatannins were detected in acetone and methanol extracts, tannins or phenols detected in acetone, methanol and aqueous extracts, terpenoids detected in hexane and methanol extracts (Table 1).

Solvent Hexane Acetone Methanol Aqueous
Flavonoids - + + -
Tannins or phenolic - + + +
Alkaloids - - - -
Glycoside - + + -
Phlobatannins - + + -
Terpenoids or sterols + - + -

Table 1: Phytochemical Analysis of the Extracts by Chemical Tests

Under basic conditions (sodium carbonate) phenolic compounds lose their proton and form phenolate anions which can reduce Folin-Ciocalteu's Reagent (FCR) which results in blue color formation due to the reduction of molybdate to molybdenum oxide in the FCR. Formed molybdenum oxide shows maximum absorbance at 765 nm. Hence the intensity of blue coloration is directly proportional to the concentration of phenolics[32]. The standard curve for total phenolic content was obtained (fig. 1), and the total phenolic content of extracts was interpolated from the standard curve. Total phenolic content in extracts decreases as follows, acetone extract>methanol extract>aqueous extract, and no phenolic content was detected in hexane extract (Table 2).

Solvent Total phenolic content (mg GAE/g of extract)
Acetone 142.94±1.01
Methanol 104.41±7.06
Aqueous 14.37

Table 2: Estimation of Total Phenolic Content of Four Extracts


Fig. 1: Total phenolic content-Gallic acid standard plot

Flavonoids are estimated by the aluminium chloride colorimetric method where aluminium chloride forms the complex with either C-5 or C-3 hydroxyl, the C-4 group of flavonols, and flavones. Those mentioned complexes are said to be acid stable complexes. Additionally, aluminium chloride forms ortho-dihydroxyl groups in the B- or A- ring of flavonoids which are acid labile complexes[33]. Standard plot for total flavonoid content obtained, it was estimated that only the acetone and methanol extracts possess the flavonoid content which is correlating to the phytochemical analysis data of four different extracts. Among acetone and methanol extracts, acetone extract contains a higher content of flavonoids than methanol extract (fig. 2 and Table 3).

Solvent Total flavonoid content (mg QE/g of extract)
Acetone 37.49±1.83
Methanol 26.73±10.65

Table 3: Estimation of Total Flavonoid Content of Four Extracts


Fig. 2: Total flavonoid content-Quercetin standard plot

Antibiotic resistance is a challenge that continues to affect the healthcare industry in both developing and developed countries all over the world. The rise and spread of multidrug-resistant organisms have put conventional antibiotic therapy in jeopardy. This has forced a search for new antimicrobial substances, such as plants, which produce a wide range of bioactive chemicals with recognized medicinal characteristics. In this study, the antimicrobial property of extracts was studied against five microorganisms Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis.

Other than the aqueous extract of leaves, all other extracts showed a significant zone of inhibition. Zone of inhibition decreased as positive control>acetone extract>methanol extract>hexane extract. Ampicillin was used as positive control against Escherichia coli, Bacillus subtilis and Proteus mirabilis. Levofloxacin and penicillin were used as positive control against Staphylococcus aureus and Pseudomonas aeruginosa respectively. Ampicillin was used in three different concentrations against Escherichia coli and Bacillus subtilis (fig. 3 and fig. 4). Disc containing 2 µg/disc of ampicillin developed zone of inhibition of 18.55 mm against Proteus mirabilis. Disc containing 2 units/disc of penicillin developed zone of inhibition of 15.2 mm against Pseudomonas aeruginosa and levofloxacin disc (5 µg/disc) developed zone of inhibition of 22.2 mm against Staphylococcus aureus. Acetone showed a better zone of inhibition from 100-300 mg/ml. Zone of inhibition of acetone extract at 300 mg/ml against microbes such as Escherichia coli-18.75 mm, Bacillus subtilis-20.5 mm, Staphylococcus aureus-20.5 mm, Pseudomonas aeruginosa-22.3 mm and Proteus mirabilis-18.5 mm (fig. 3-fig. 7).


Fig. 3: Antimicrobial activity-zone of inhibition developed by antibiotic (Ampicillin) and extracts in the concentration range of 100-300 mg/ml against Escherichia coli
Note: Image Methanol


Fig. 4: Antimicrobial activity-zone of inhibition developed by antibiotic (Ampicillin) and extracts in the concentration range of 100-300 mg/ml against Bacillus subtilis
Note: Image Methanol


Fig. 5: Antimicrobial activity-zone of inhibition (mm) developed by extracts in the concentration range 100-300 mg/ml against Pseudomonas aeruginosa
Note: Image Methanol


Fig. 6: Antimicrobial activity-zone of inhibition (mm) developed by extracts in the concentration range of 100-300 mg/ml against Staphylococcus aureus
Note: Image Methanol


Fig. 7: Antimicrobial activity-zone of inhibition (mm) developed by extracts in the concentration range of 100-300 mg/ml against Proteus mirabilis
Note: Image Methanol

MIC is defined as the lowest concentration of the antimicrobial agent that prevents visible microbial growth after 24 h of incubation. Extracts exhibited antimicrobial activity against all the 5 microorganisms such as Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis. MIC of acetone extract was lower than other extracts, Escherichia coli-208 mg/ml, Bacillus subtilis-202 mg/ml, Staphylococcus aureus-208 mg/ml, Pseudomonas aeruginosa-204 mg/ml, Proteus mirabilis-204 mg/ ml (Table 4). The aqueous extract didn't inhibit the growth of microorganisms.

Solvent Escherichia coli Bacillus subtilis Staphylococcus aureus Pseudomonas aeruginosa Proteus mirabilis
Hexane (mg/ml) 288 264 289 313 315
Acetone (mg/ml) 208 202 208 204 204
Methanol (mg/ml) 215 210 210 208 207

Table 4: Minimum Inhibition Concentration of Extracts against Five Microorganisms

DPPH assay works on the principle of free radical scavenging. The highly sensitive and simple nature of the assay made the assay popular nowadays. DPPH free radical is purple which turns to yellow color DPPH when antioxidant provides hydrogen atom to the free radical. As the concentration of antioxidants increases, there occurs a decrease in the intensity of the purple color. DPPH free radical shows maximum absorbance at 517 nm[34]. Absorbance at 517 nm vs. concentration of extract (µg/ml) was plotted (fig. 5) and analysed that percentage of inhibition was directly proportional to the concentration of extract. The IC50 value of methanol extract in DPPH free radical scavenging was interpolated as 146.4483 µg/ml which is less than the IC50 value of acetone extract of 153.8131 µg/ml (Table 5). While other extracts did not show radical scavenging activity within a concentration range of 10-200 (µg/ml).

Solvent IC50 (µg/ml)
Methanol 146.4483
Acetone 153.8131

Table 5: Determination of IC50 Value of Four Extracts in Scavenging DPPH Free Radicals

The egg albumin (protein) denaturation inhibition assay provides a cheap alternative method for testing the anti-inflammatory activity of herbal extracts. As the concentration of extract had increased, absorbance decreased, indicating that denaturation of egg albumin by the applied heat was inhibited by leaf extract. Percentage of inhibition by the extracts at 1.6 mg/ml decreased as diclofenac (90 %)>acetone extract (89 %)>aqueous extract (86 %)>methanol (79 %)>hexane (45 %) (fig. 6). The result was compared with Ruellia tuberose. Hexane extract of Ruellia tuberosa was not showed antimicrobial activity on Escherichia coli, Pseudomonas aeruginosa and Protease sp at any concentration[78]. Anti-inflammatory activity of Ruellia tuberose was studied by in vivo experiment and it showed the activity at maximum concentration 300 mg/kg[79].

As acetone and methanol extracts showed better activities than other two extracts, acetone and methanol extracts were further analysed by GC-MS method. In the case of acetone extract, compounds with greater percentage area are phytol (14.99) and diethyl phthalate (10.05) (fig. 8 to fig. 11). Phytol possesses both anti-inflammatory and antimicrobial activities. Diethyl phthalate possesses antimicrobial activity (Table 6)[35-77]. In the case of methanol extract, compounds with greater percentage area are beta-sitosterol (17.01), dimethyl sulfone (7.53), beta-amyrin (6.51) and alpha-amyrin (6.51). Antimicrobial activity associated with dimethyl sulfone, antibacterial and antioxidant activities associated with beta-sitosterol, anti-inflammatory activity associated with beta-sitosterol, dimethyl sulfone, beta- amyrin, alpha-amyrin (Table 7)[78,79].

Name RT % Area MW Molecular formula Biological activity
Dimethyl sulfone 5.964 2.1 94.14 C2H6O2S Antimicrobial and anti-inflammatory[35]
Diethyl Phthalate 13.1 10.05 222.24 C12H14O4 Antimicrobial[36]
Tetradecanoic acid 14.62 0.51 228.37 C14H28O2 Antimicrobial[37]
Tridecanoic acid 14.62 0.51 214.34 C13H26O2 Antimicrobial, anti-inflammatory[38]
2-Hexadecene, 3,7,11,15-tetramethyl 15.4 0.55 280.5 C20H40 Antimicrobial and antioxidant[39]
5-Nonadecen-1-ol 15.55 0.59 282.5 C19H38O Antimicrobial and anti-inflammatory[40]
1,4-Eicosadiene 15.55 0.59 278.5 C20H38 Antimicrobial[41]
Cyclohexanol, 1-ethynyl 15.72 0.69 124.18 C8H12O Anticancer and antioxidant[42]
Dodeca-1,6-dien-12-ol, 6,10-dimethyl 15.72 0.69 210.36 C14H26O Antimicrobial, antioxidant, and anti-inflammatory[43]
Squalene 16.04 1.08 410.7 C30H50 Antioxidant, antibacterial[44]
Hexadecanoic acid, methyl ester 16.1 2.23 270.451 C17H34O2 Antibacterial and antifungal[45]
Pentadecanoic acid, 14-methyl-, methyl ester 16.1 2.23 270.5 C17H34O2 Antimicrobial, antifungal[46]
n-Hexadecanoic acid 16.39 3.3 256.42 C16H32O2 Antioxidant[47]
Nerolidol 1 16.94 0.55 222.37 C15H26O Antimicrobial, anti-biofilm, antioxidant, anti-inflammatory[48]
Methyl 10-trans,12-cis-octadecadienoate 17.64 1.18 294 C19H34O2 Antioxidant and antimicrobial[47]
9,12,15-Octadecatrienal 17.52 4.13 262.43 C18H30O Antimicrobial[49]
Phytol 17.61 14.99 296.5 C20H40O Anti-inflammatory and antimicrobial[50]
Octadecanoic acid, methyl ester 17.7 0.44 294.47 C19H34O2 Antiviral[51]
Octadecanoic acid 17.95 0.59 284.48 C18H36O2 Anti-inflammatory and antioxidant[47]
Tetradecanoic acid 17.95 0.59 228.37 C14H28O2 Antimicrobial[52]
3-Hydroxymyristic acid 18.64 0.61 244.37 C14H28O3 Antifungal and antimicrobial[53]
7-Tetradecyne 19.19 0.78 194.36 C14H26 Antimicrobial[54]
1,2-Benzenedicarboxylic acid, mono(2-Ethylhexyl) ester 20.57 0.5 278.34 C16H22O4 Antimicrobial[46]
Di-n-octyl phthalate 20.57 0.5 390.6 C24H38O4 Antimicrobial[55]
Phthalic acid, isohexylisoporpyl ester 20.57 0.5 292.4 C17H24O4 Antimicrobial[56]
Nonanoic acid, 9-(3-hexenylidenecyclopropylidene)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester 21.64 1.79 52 C29H36O4 Antioxidant, antimicrobial[57]
i-Propyl 9,12,15-octadecatrienoate 21.64 1.79 320.5 C29H36O2 Anti-inflammatory[58]
2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl 22.33 1.5 410.72 C30H50 Antioxidant[59]

Table 6: Compounds Identified from GC-MS Analysis of Acetone Extract

Name RT % Area MW Molecular formula Biological activity
Dimethyl sulfone 6.453 7.53 94.13 C2H6O2S Anti-inflammatory and antimicrobial[35]
Diethyl Phthalate 13.075 0.79 222.24 C12H14O4 Antimicrobial[36]
2-Furanethanol, .beta.-methoxy-(S) 14.874 0.58 142.15 C7H10O3 Antimicrobial[40]
1-Methoxy-3-(2-hydroxyethyl) nonane 15.341 1.59 202.33 C12H26O2 Antimicrobial, antioxidant activity[60]
9-Nonadecyne 15.719 0.51 264.5 C19H36 Antifungal, antioxidant and antimicrobial[40]
Squalene 16.041 0.85 410.7 C30H50 Antioxidant[44]
n-Hexadecanoic acid 16.374 2.28 256.42 C16H32O2 Antioxidant[47]
5,9-Undecadien-2-one, 6,10-dimethyl 16.941 1.06 194.31 C13H22O Antimicrobial[61]
9,12,15-Octadecatrienoic acid, methyl ester 17.507 0.49 292.5 C19H32O ALA–omega 3 fatty acid
          Reduce blood clots[47]
7,10,13-Hexadecatrienoic acid, methyl ester 17.507 0.49 264.4 C17H28O2 Fatty acids that reduce blood clots[62]
Phytol 17.585 0.87 296.539 C20H40O Anti-inflammatory and antimicrobial[50]
Isophytol 17.585 0.87 296.5 C20H40O Antimicrobial and antifungal[63]
9,12,15-Octadecatrienoic acid 17.796 3.85 278.4 C18H30O2 ALA – omega 3 fatty acid
          Reduce blood clots[47]
2,6-Octadien-1-ol, 3,7-dimethyl 18.241 0.46 308.5 C20H36O2 Antimicrobial[64]
Tetradecanoic acid, 2-hydroxy- 18.64 0.46 244.37 C14H28O3 Antioxidant[65]
17-Pentatriacontene 18.696 1.97 490.9 C35H70 Antibacterial[66]
Vitamin E 19.663 0.93 430.71 C29H50O2 Antioxidant[67]
Phenol, 2,4-bis(1-phenylethyl)- 19.84 0.49 302.4 C24H26O Anti-inflammatory[68]
Xanthen-9-one, 1-hydroxy-3,5,8-trimethoxy- 19.84 0.49 302.28 C16H14O6 Antimicrobial, Antioxidant, Anti-inflammatory[69]
Methanone, [1,4-dimethyl-7-(1-methylethyl)-2-azulenyl]phenyl- 19.84 0.49 302.42 C22H22O Antimicrobial[70]
Phenol, 2,4-bis(1-phenylethyl)- 20.318 0.8 302.4 C22H22O Antioxidant[71]
Xanthen-9-one, 1-hydroxy-3,5,8-trimethoxy 20.318 0.8 302.28 C16H14O6 Antimicrobial, Antioxidant, Anti-inflammatory[69]
Ergosta-5,24-dien-3-ol, (3.beta.)- 21.351 0.58 398.7 C28H46O Moderate cytotoxicity against the human foreskin fibroblast cell line (Hs27 cells)[72]
Ergost-5,8(14)-dien-3-ol 21.351 0.58 398.7 C28H46O Immunosuppressive, anti-tumor[73]
Stigmasta-5,24(28)-dien-3-ol, (3.beta.,24Z) 21.351 0.58 412.7 C29H48O Antioxidant, anti-inflammatory[74]
Pregnenolone 21.985 0.5 316.47 C29H32O2 Anti-tumor[75]
2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl-, (all-E)- 22.329 0.57 410.71 C30H50 Antioxidant[59]
gamma.-Sitosterol 22.784 17.01 432.7 C29H52O2 Anti-cancer[76]
beta.-Sitosterol 22.784 17.01 414.71 C29H50O  
26,26-Dimethyl-5,24(28)-ergostadien-3.beta.-ol 22.973 1.94 426.7 C30H50O maintain cell membrane integrity[77]
beta.-Amyrin 23.44 6.51 426.7 C30H50O Antimicrobial[44]
Alpha.-Amyrin 23.44 6.51 426.7 C30H50O Antimicrobial[44]

Table 7: Compounds Analysed from GC-MS Analysis of Methanol Extract

In conclusion, the results of this study show the presence of phytochemicals such as flavonoids, tannins/phenolics, glycosides, and phlobatannins, higher phenolics and flavonoids content in both acetone and methanol extracts than in the other extracts. Higher antimicrobial, antioxidant and anti- inflammatory properties are associated with acetone and methanol extracts than the other extracts due to the presence of bioactive compounds illustrated in GC-MS analysis. Purification of most essential compound from the Ruellia patula leaves extract, investigating the wound healing properties by performing in vivo studies can be explored to bring natural product as a wound healing drug.


Fig. 8: Antioxidant assay-DPPH free radical scavenging assay
Note: Image Linear (Acetone)


Fig. 9: Anti-inflammatory assay-protein denaturation inhibition assay
Note: Image Aqueous


Fig. 10: GC-MS analysis of acetone extract


Fig. 11: GC-MS analysis of methanol extract


Authors thank to the Department of Biotechnology, Mepco Schlenk Engineering College, Sivakasi, Tamil Nadu, India for providing facilities to conduct our experimental work.

Conflict of interests:

The authors declare that they have no competing interest.