Plant material collection and sample preparation

Fresh M. calabura leaves and roots were collected from the greenhouse at (18°01'35.1"N 79°33'31.7"E). The collected plant materials were thoroughly washed under running tap water to remove impurities. These were dried for 12 days in the shade at room temperature (27 ± 2 °C) and ground into a fine powder. Five gram of plant materials were mixed with 50 mL of methanol and incubated in an orbital shaker for 48 hours at 130 rpm. Whatman No. 1 filter paper was used to filter the supernatant. The supernatants were evaporated at room temperature to obtain a crude extract, then subjected to GC-MS analysis and anti-microbial activity testing.

GC-MS instrumentation

M. calabura leaf and root methanol extracts were GC-MS analysed using a Perkin-Elmer GC system coupled with a silica capillary column 30m x 0.25mm ID x 0.25m df equipped with Elite-5MS (5 % diphenyl and 95 % dimethyl polysiloxane). Helium was used as a gaseous carrier in a 1:1 mg/minute ratio, with an injector volume of 2μl and a temperature of 250 °C and ionization at 70-eV. The oven temperature began at 110 °C and ended at 280 °C. The extraction time was 29 minutes for M. calabura leaf extracts and 13 minutes for root methanolic extracts. The total peak area of M. calabura was used to calculate the percentage of each bioactive compound in leaf and root extract. The Sophisticated Analytical Instrument Facility, IIT Bombay, performed the GC-MS analysis.

Microbial cultures and culture conditions

Muntingia calabura methanolic leaf and root extracts were tested against gram-negative and gram-positive bacteria. Escherichia coli, Proteus vulgaris, Bacillus sphericus, and Pseudomonas fluroscens were used as test organisms. Preparation of 24 hr old bacterial strains: inoculate nutrient broth with original cultures and incubate overnight at 37 °C. The antibacterial activity was tested using the agar well diffusion method with streptomycin (10 µg/mL) as the standard. The media was prepared and poured 15 mL into a petri dish for 5 minutes to solidify. The inoculums were swabbed uniformly over the media and dried. The standard (10 µg/mL) and three different leaf and root extract concentrations (45, 60, and 75 µg/mL) were incubated overnight at 37 °C in each well. After incubation, zones emerged, and the proportion of inhibition was scaled in millimetres.

GC-MS analysis of leaf methanolic extract

The bioactive molecules found in methanolic leaf extracts were thirty-eight compounds. Among the thirty-eight compounds, the first compound identified with less retention time (4.85 min) was 1,3-dihydroxypropan-2-one and the peak area (0.24%), whereas the last compound with the longest retention time (33.68 min) was 3-Hydropregn-5-en-20-one hydrazone and the peak area (0.33%). Many of these compounds possess various pharmacological activities. The remaining phytochemical compounds are as follows: 1-(6-oxabicyclo[3.1.0]hexan-1-yl)ethan-1-one (or) 6-oxabicyclo(3.1.0)hexan-3-one, a phytoconstituent of methanolic leaf and root extracts of pili and safedshatavar (Asparagus racemosus), known to possess antioxidant activity (Banakar & Jayaraj, 2017). The 3,5-Dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4-one reported antimicrobial, automatic nerve, and antioxidant properties in ethanolic stem extracts of an important medicinal plant Waltheria indica L (Ashwathanarayana & Naika, 2018). 2,3-dihydro-1-benzofuran, a bioactive compound, possesses anti-inflammatory activity in crude extracts of leaf and flower of Pavetta crassicaulis Bremek (Correa et al., 2017). Decan-3-yl acetate, reported to possess activities like asthma, anticancer, anti-inflammatory activities and Histamine H3 and H4 receptors, have been discovered drugs for Parkinson’s and Alzheimer’s disease (Tatipamula et al., 2019). The 1,2,3-benzenetriol (Pyrogallol), identified as a phytochemical compound that possesses anticancer activity in ethanolic extract of Clathria baltica (Sukprasert, Pansuksan, & Sriyakul, 2020). The compound (1R,2S,3R,4S)-cyclopentane1,2,3,4-tetrol, known to possess antiviral activity in Lysiphyllum strychnifolium plant extracts that treat against influenza virus (Rubaye, Hameed, & Kamal, 2018). The compound, 4-[(1E)-3-hydroxyprop-1-en-1-yl]-2-methoxyphenol have multiple biological properties like anti-hyperglycemic, hepatoprotective, anti-obesity and antibiotic activities in root extracts of Eclipta alba (L) (Naik, Gurushanthaiah, Kavimani, & Mahesh, 2019). More recently, it was reported that the compound (2E)-3,7,11,15-tetramethyl-2-hexadecen-1-ol has antifungal and antibacterial activity in leaf extracts Rhizophora apiculata (Lakshmanan et al., 2019).

 Hexadecanoic acid, a phytochemical compound, has properties like strong antimicrobial, anti-inflammatory activities and acts against arthritis in medicinal plants like Albizia adianthifolia, Pterocarpus angolensis and in some plants against food-borne pathogens (Abubakar & Majinda, 2016; Preethi, Devanathan, & Loganathan, 2010). In Cycas revolute, a medicinal plant is known to have antioxidant properties in the 1–hentetracontanol bioactive molecule (Olabisi & Olubunmi, 2019). The (9Z,12E)-octadeca-9,12-dienoic acid and (9Z)-octadec-9-enoic acid have phytochemical properties like hypercholesterolemia, anti-acne, anti-cancer, anti-bacterial, and diabetes mellitus in methanolic leaf extract Crateva adansonii DC and root extract of Jatropha pelargoniifolia Courb (Christiana et al., 2014). Another bioactive molecule, 3-[(4-ter-butylcyclohexyl)oxy]-3-phenyl-2-benzofuran-1(3H)-one is identified to synthesize green synthetic particles using ferrous and nickel nanoparticles by leaf extract of eucalyptus (Weng, Guo, Luo, & Chen, 2017).

5-hydroxy2-phenyl-4–chromanone is one compound that possesses Helicobacter pylori and Gastric antisecretory activity (Das et al., 2010). 1-tetradecene,2–decyl reported to have phytochemical properties like an anti-inflammatory in leaf extracts of Azima tetracantha Lam (Jose & Panneerselvam, 2019) and antioxidant activity in leaf and stem of Strobilanthes species (Fernandes et al., 2019). Recently, the anti-inflammatory activity of (2E)-1,3-diphenylprop-2-en-1-one has been reported by (Landim et al., 2019) in Lonchocarpus cultratus.

The compound, 5-hydroxy-7-methxy-2-phenyl-4H-1-benzopyran-4-one, possesses anti-cancer, anti-prostate and anti-inflammatory activities (Marques, Silva, Marques, & Braga, 2019; Wang et al., 2019). Similarly, a compound is known as (Naringenin) (2S)-5,7-dihydroxy-2-(4-hydroxyphenyl)-2,3-dihydro-4H-1- benzopyran-4-one is observed to have two biological properties like anti-tuberculosis and anti-cancer activities (Sahu et al., 2020). The major compound identified in the M. calabura leaf extract was found to be 2-{3-[(E)-2-(1H-indol-3-yl)ethenyl]-1,2,4-oxadiazol-5-yl}phenol 5.78% peak area, and have antioxidant and antitumor activity (Figure 1). These bioactive compounds are synthetic aromatic C-nucleoside derivatives developed by (Sadek et al., 2014). 2,2'-((1E,1'E)-(1,4- phenylenebis(azanylylidene))bis(methanylylidene))diphenol (Nitrilo methyl dyne) has been reported to have an anti-tumor activity (Kopoytkoba et al., 2019).The compound 7-hydroxy-3-methoxy-2-(4- methoxyphenyl)-4H-1-benzopyran-4-one that is identified to possess antioxidant activity from the bran of Thai black rice (Suttiarporn et al., 2016) and 5,7-dihydroxy-2-(4-hydroxyphenyl)-6-methoxy-4H-chromen-4-one reported to have strong antioxidant activity (Karker et al., 2019; Mu et al., 2009; Yausheva et al., 2019). 2,3,7,8-tetramethoxy-5,11-dihydro10H-dibenzo[a,d][7]annulen-10-one has anti-inflammatory activity (Fernandes et al., 2019). The compound, Anthraquinone, 1-(4-methylphenyl)anthracene-9,10-dione is identified to act against the cancer cell and has antibacterial activities (Chu et al., 2019). 8,9-dihydro-aflatoxin B1 has the mycotoxin activity (Aparna et al., 2012; Pok et al., 2020), and 1-Docosene has the anticancer activity of Xestospongia testudinaria. Some compounds have been identified, but their biological activity has not yet been discovered (Table 1).

Figure 1

GC-MS chromatogram of leaf methanolic extracts of Muntingia calabura and potent bioactive compound 2-{3-[(E)-2-(1H-indol-3-yl)ethenyl]-1,2,4-oxadiazol-5-yl}phenol (antioxidant and antitumor 5.78%).

GC-MS analysis of root methanolic extract

Of all the bioactive compounds exhibited, cholest-4-en-6-on-3-ol has the highest 63.7% retention time (24.58 min) and has anti-obesity activity (Shaheed et al., 2019). At the same time, another compound, Lupeol, was observed during prolonged retention (35.9 min), and the maximum area percentage was 7.36% (Figure 2). Similarly, a compound is known, 17-octadecynoic acid has phytochemical properties like Hypocholesterolaemia, anti-inflammatory, anti-acne, and anti-coronary activities from various plant extracts (Benzidia et al., 2019; Mishra & Patnaik, 2020; Revathi et al., 2019). (2R)-2,5,8-trimethyl-2-[(4R,8R)-4,8,12-trimethyltridecyl]-3,4-dihydro-2H-1-benzopyran-6-ol (β-Tocopherol) is one of the compounds that have anti-spasmodic activity (Lanuzza et al., 2017). The compounds like cholestan-3-one-4,4-dimethyl-(5α); 9-octadecenoic acid(Z)-phenyl methyl ester; (9Z)-octadec-9-enoic acid (Oleic acid); androst1-en-3-one,4,4-dimethyl-(5α) identified to possess anti-cancer, anti-inflammatory, antioxidant, antiulcer genic, antipyretic activities in various plant extracts of medicinally important plants (Safwat, Hamed, & Moatamed, 2018; Shelke et al., 2019; Yamuna, Abirami, Vijayashalini, & Sharmila, 2017; Yue et al., 2020).

Figure 2

GC-MS chromatogram of root methanolic extracts of Muntingia calabura, potent anti-obesity compound cholest-4-en-6-on-3-ol(63.7%) and anti-cancerous compound Lupeol (7.3%).

Cholest-4-en-6-on-3-ol and Lupeol are the two identified compounds with highest percent of compounds among all the compounds in root methanolic extracts of M. calabura and reported to possess anticancer, antioxidant, anti-prostate, anti-inflammatory activities in different plant extracts of medicinal plants (Geetha & Varalakshmi, 2001; Shahaby et al., 2019; Shaheed et al., 2019; Yang et al., 2020). There are a few identified compounds, 1,12-Dibromododecane; 5-(2-bromopropan-2-yl)-2-methylcyclohexan-1-ol; 2H-1-benzopyran-6-ol,3,4-dihydro-2,8-dimethyl(-2-4,8,12-trimethyl tridecyl); A'-neogammacer-22(29)-en-3-one; 4,4,6a,6b,8a,11,11,14b-octamethyl-1,4,4a,5,6 yet to be identified (Figure 2 and Table 2).

Figure 3

Effect of Muntingia calabura leaf methanolic extract on various bacterial cultures.

Figure 4

Effect of Muntingia calabura root methanolic extract on various bacterial cultures.

Antibacterial activity for leaf and root methanolic extracts

The antibacterial activity of leaf methanolic extracts (45µg/mL, 60 µg/mL, 75 µg/mL) and the control Streptomycin (10 µg/mL) was evaluated. The maximum antibacterial activity was shown towards E. coli at 75µg/mL concentration, followed by B. sphericus, P. vulgaris and P. fluorescens. As the concentration of the plant extract increased, the inhibition zone was also found to increase. (Figure 4; Figure 3). The leaf methanolic extract of M. calabura was considered the most effective extract with high anti-bacterial activity (Zakaria et al., 2010). In M. calabura, the ethanolic leaf extract has shown strong antibacterial activity against E. coli (Gurning et al., 2021). The root extracts of Carica papaya have shown the highest antibacterial activity against Salmonella typhi among tested gram-negative and gram-positive bacterial strains (Doughari, Elmahmood, & Manzara, 2007).