Introduction

Medicinal plants have been used in treating various ailments and are natural sources for medicinally important secondary metabolites. Plant based medicines are reliable, cost effective with less side effects compared to chemical drugs (Zimila et al. 2020). About 80% of population of the world uses plant drugs as the primary treatment for various disorders. Many extracts from medicinal plants are used in traditional practices (Enerijiof et al. 2021). The World Health Organization (WHO) promotes the use of plant based medicine in developing countries, as secondary metabolites present in plants are having different biological activities like antioxidant, antimicrobial, anxiolytic and anti-inflammatory (Nagannawar and Jayaraj 2020). Analysis of secondary metabolites is very important to know the responses of the plants to environmental conditions. GC–MS analysis is used as one of the primary tools for secondary metabolite profiling of plant extracts, especially in the analysis of a single sample and in determining important metabolites like sugars, amino acids and organic acids (Abadie et al. 2022).

Calycopteris floribunda Lam. of Combretaceae is a large woody climber endemic to tropical forests of south east Asia and it is found in the central and southern parts of India. It is used in Asian medicinal practices like Ayurveda, folklore and Unani. The leaves have various medicinal properties and are used in the treatment of intestinal worms, colic, leprosy, malaria, dysentery, ulcers, vomiting (Bharat 2019), snake bites (Gomes et al. 2010), burned wounds (Shivanna and Rajakumar 2011). The stem and leaf extracts are used to prepare roborans tonic (Liu et al. 2009), and leaves are mixed with leaves of Jasminum malabaricum to calm the Asthmatic attack (Bhandary et al.1995). The aqueous extract of the leaves is reported to have antifungal activity (Vinayaka et al. 2009). Leaves also have anthelminthic activity (Ratnagiriswaran et al. 1934). Calycopterone, isocalycopterone and 4-demethylcalycopterone isolated from leaves showed activity against tumor cell lines (Mayer 1999). Calycopterin a flavonoid isolated from leaves showed anthelmintic, antiviral and anticancerous activity (Liu et al. 2009). Pachypodol is also flavonoid isolated from leaves showed anticancerous activity (Ali et al. 2008).

Though the leaves of C. floribunda have many medicinal properties, only phytochemical studies of the essential oils of leaf extracts and selected compounds from the leaf extracts have been reported (Mayer 1999; Ali et al. 2008; Liu et al. 2009). However, there are no reports on the overall phytochemical profiling of crude extracts of the leaves of C. floribunda. Hence, the present work is undertaken to study the physico-chemical parameters and to identify phytochemicals by GC–MS.

Material and methods

Plant material

The leaves of Calycopteris floribunda Lam. (Voucher specimen no: KUD/BOT/SY/MJ/02) were collected during the month of June 2021 from the plants grown in Karnatak University campus, Dharwad (15° 43ʹ 74.98″ N, 74° 98ʹ 36.86″ E), Karnataka, India (Fig. 1).

Fig. 1
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Branch with leaves of Calycopteris floribunda Lam.

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Extraction

The healthy leaves were collected and shade dried for 25 to 30 days to remove the moisture. Further, it is ground into powder by using electronic grinder and stored in an air tight zipper bag for further use (Fig. 2). Finely powdered sample (10 g) was used to extract the different organic solvents (the increasing polarity) such as petroleum ether, chloroform, acetone and ethanol for 18 h by using Soxhlet apparatus (Nagannawar and Jayaraj 2020).

Fig. 2
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Leaf powder of Calycopteris floribunda Lam.

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Physico-chemical study

Physico-chemical parameters like ash value, moisture content, fat content, foaming index, pH and extractive values of leaf was studied by following standard procedures (Nissar et al. 2021).

Preliminary phytochemical analysis

Leaf extracts in different organic solvents (petroleum ether, chloroform, acetone and ethanol) are used for preliminary analysis of secondary metabolites by using the standard procedures. Tests are conducted for the analysis of carbohydrates (Molisch’s test), reducing sugar (Fehling’s test and Benedict’s test), protein (Million’s test and Biuret’s test), amino acids (ninhydrin test), glycosides (anthrone test), phenols (ferric chloride test), tannins (gelatin test), flavonoids (Shinoda’s test) and alkaloids (Dragendorff’s test, Wagner’s test and Mayer’s test) (Banni and Jayaraj 2022).

Determination of total phenolic content

Phenolic content of leaf extract was determined by using Singleton et al. (1999) method. About 0.1 mL of leaf extract was diluted using distilled water (3 mL) and mixed with Folin-Ciocalteu reagent (0.5 mL) and incubated for 3 min. Further, 20% sodium carbonate (2 mL) was added and the absorbance was recorded at 760 nm after 30 min of incubation using UV–visible spectroscopy (Hitachi U-3000). The results obtained were expressed in mg of gallic acid equivalent to per gram of extract (mg GAE/g) using calibration curve for gallic acid standard solutions.

Determination of total flavonoid content

Total flavonoid content of the leaf extract was determined using the aluminium chloride colorimetric method. In this method, 0.1 mL of leaf extract, 2% (w/v) AlCl3 (1 mL), 5% (w/v) and sodium acetate (3 mL) were mixed thoroughly. After incubation for 120 min, the absorbance was read at 440 nm. Results were expressed as mg of quercetin equivalent to per gram of extract (mg QE/g). Quercetin was used to make a standard calibration curve (Safari and Ahmady-Asbchin 2019).

FTIR spectroscopic analysis

1.0 mg of leaf powder was compressed into KBr pellets and scanned using FTIR within the range of 400 to 4000 cm−1 with resolution of 4.0 cm–1. The functional groups were identified using sigma Aldrich IR spectrum table (Silva da Costa et al. 2022).

GC‑MS analysis

GC–MS analysis of different leaf extracts of C. floribunda was carried out using Shimadzus (QP2010S) GC–MS equipped with ELITE-5MS column which is 30 m long, having 0.25 mm inner diameter and a 0.25 μm thickness. Helium was used as a carrier gas with the flow rate of 1 mL/min and an injection temperature of 280 °C. The initial temperature of the analysis was kept at 80 °C for 5 min and then the temperature was gradually increased at a rate of 10 °C/min till it reached 280 °C and the same temperature was maintained for 5 min. Samples were kept for analysis for 50 min in GC–MS column. The samples were injected in split mode with a pressure of 65.0 kPa of the carrier gas with the split ratio of 1:20. All data obtained are within the scan range of 50–500 m/z. The NIST 11 and WILEY 8 libraries were used to identify the compounds by comparing their retention values (Nagannawar and Jayaraj 2020; Banni and Jayaraj 2022).

Statistical analysis

Results were obtained in triplicate and expressed as mean ± standard deviation (SD). GC–MS results obtained in this study were analyzed by principal component analysis (PCA) to determine correlations between the biological activities of secondary metabolites of leaf extracts using IBM SPSS ver. 20 software.

Results and discussion

Physico-chemical study of plants gives the standard parameters for the purity and quality of the drugs. The ash content of the drugs helps to determine the inorganic chemicals like the oxides of Ca, Mg, K, Na, Si, P and Fe. These can be used as the parameters for the purity and authentication of drugs (Papiya, et al. 2014). In the present study the total ash value, acid insoluble ash value and water insoluble ash value of the leaves of C. floribunda were calculated, the results obtained show that the water soluble ash value is higher than to the acid insoluble ash value which indicated that leaf powder of the plant has more organic contents (Table 1).

Table 1 Physico-chemical parameters of leaf of Calycopteris floribunda Lam.
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Loss of moisture on drying is one of the basic parameters used to determine the quality of the drug. Thus the minimum value of the moisture content of the crude drug should be less than 14% to avoid any microbial contamination. The loss of moisture on drying of the leaf powder is below 14%. The formation of foam by crude drugs when dissolved in water is due to the presence of saponins, this foam formation is measured as the foaming index and used as a parameter for standardizing the quality of the drug (Nissar et al. 2021). In the present study, the foaming index of leaf powder is 100. The pH of the drug gives the ionic nature of the drug, in the present study on the leaf powder pH value is slightly acidic in nature (Table 1).

Extractive yield of the plant extracts is considered one of the important physico-chemical parameters for standardizing for quality of the plant drugs, the extractive yield gives information regarding the phyto-constituents of the extracts and in selection of the better suitable solvents for the extraction of the crude drugs (Aslam and Afridi 2018). In the present work three types of extraction were carried out. The results obtained showed the presence of more polar compounds in the leaves of C. floribunda have (Table 2).

Table 2 Extractive yield (%) of leaf of Calycopteris floribunda Lam.
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Preliminary phytochemical analysis

The preliminary phytochemical analysis of leaf extracts showed the presence of carbohydrates, steroids, alkaloids, phenols and triterpenoids in all the solvents used in the present study (Table 3). Reducing sugars were present in chloroform, acetone and ethanol extracts, protein and amino acids are present only in petroleum ether. Saponins are present in petroleum ether, chloroform and aqueous extracts. Tanin is present in petroleum ether and aqueous extracts. Organic acids are present in ethanol and aqueous extract (Table 3). Results obtained were compared with the preliminary phytochemical study carried out by Glory et al. (2016) on the leaves of C. floribunda. The petroleum extract showed presence of alkaloid, flavonoid, steroid, saponin and tannin in the present study is similar to study of Glory et al. (2016). However, the chloroform extract of the present study showed presence of more phytochemicals.

Table 3 Preliminary phytochemical analysis of leaf extracts of Calycopteris floribunda Lam.
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Total phenolics and flavonoids

Total phenolics and total flavonoids are considered as the indicators of antioxidant properties of plant extracts. Earlier studies reported that C. floribunda has biologically important phytochemicals belonging to flavonoids (Liu et al. 2009). The ethanolic leaf extract showed 472.66 ± 2.51 mg GAE/ g dw of phenolics and 303.00 ± 3.60 mg QE/g dw of flovonoids (Table 4). The results obtained showed better results compared to previous study (Vantamuri et al. 2018) in methanolic and aqueous root extracts (343.77 ± 2.02 and 87.87 ± 6.06 gallic acid equivalent/mg), the study of whole plant extracts of C. floribunda by Santharam et al. (2015) showed that the presence of total flavonoids is 0.038 ± 0.076 mg/g in petroleum ether extract, 3.545 ± 0.023 mg/g in ethyl acetate extract and 1.489 ± 0.043 mg/g in methanolic extract.

Table 4 Total Phenolics content,and flavonoids content of ethanolic leaf extract of Calycopteris floribunda. Lam.
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FTIR spectroscopic analysis

FTIR analysis is used conventionally to identify the components of phytochemicals in medicinal plants. The characteristic absorption peaks of FTIR spectrum show specific functional groups present in the chemical components. The IR fingerprint is used to analyze the integrity and universality of chemical components (Bai et al. 2021). The IR spectrum of C. floribunda leaf powder was analyzed for the identification of functional groups. FTIR spectrum was obtained in between the range of 4000–400 cm − 1. The peak observed at 3351.74 cm − 1 was due to strong O–H bond stretching (alcohol group). Band at 2919.23 cm − 1 (strong broad) was due to O–H bond stretching (carboxylicacid), and the band at 1619.91 cm − 1 (strong broad) is due to C = C stretching (α, β unsaturated ketone). Bands at 1519.20 and 1445.02 (strong) were denoted as nitro compound (N–O) stretch. The bands observed at 1317.32 and 1061.41 cm − 1 (strong) were due to S = O (sulfone) and C-O (primary alcohol) stretching, the medium sharp bands at 823.04 cm − 1 and 781.16 cm − 1 indicated the stretching of C = C bending of alkene trisubstuted group. These functional groups infer the possible presence of several bioactive metabolites in the leaves of C. floribunda (Fig. 3). The FTIR study which showed strong O–H, C = C, N–O, S = O (sulfone), C-O and C = C bonds stretching similar to the study (Selvaraju et al. 2021) on Ocimum gratissimum.

Fig. 3
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FTIR spectrum of Calycopteris floribunda Lam. leaf powder

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GC‑MS analysis

GC–MS study is used to detect the potential phytochemicals in C. floribunda leaf extracts by comparing their mass spectra and retention time with the NIST 11 and WILEY 8 libraries. The results obtained from leaf extracts of C. floribunda showed the presence of various phytochemicals of the different classes viz. long chain alkanes, phytosterols, phenols, diterpenes and triterpenoids contributing to the medicinal properties of the plant. The identified compounds with their peak number, retention time (RT), peak area (%), molecular formula and molecular weight in each extract presented (Tables 5, 6, 7, 8) (Fig. 4). Comparative GC–MS analysis of different solvent extracts of leaf showed the presence of 18 compounds in the present study (Table 9). However, GC − MS study by Liu et al. (2009) on volatile oils of leaf of C. floribunda showed 52 compounds in which only phytol and dibutyl phthalate are recorded for the leaf extracts of C. floribunda of the present study is similar to the earlier work.

Table 5 GC–MS analysis of petroleum ether leaf extract of Calycopteris floribunda Lam.
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Table 6 GC–MS analysis of chloroform leaf extract of Calycopteris floribunda Lam.
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Table 7 GC–MS analysis of acetone leaf extract of Calycopteris floribunda Lam.
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Table 8 GC–MS analysis of ethanol leaf extract of Calycopteris floribunda Lam.
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Fig. 4
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Chromatograms of leaf extracts in different solvents

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Table 9 Comparative GC–MS analysis of the bioactive Components of Leaf extracts of Calycopteris floribunda Lam.
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1, 2-benzenedicarboxylic acid is one of the major peaks in petroleum ether, chloroform and acetone extracts. 1, 2-benzenedicarboxylic acid and its derivatives have anticancerous, antimicrobial and antiarthritic activities (Reddy et al. 2020). Squalene and phytol are present in petroleum ether, acetone and alcohol extracts. Tulika et al. (2017) in their study on GC–MS analysis of bioactive constituents in the extract of Pistia stratiotes and extracts of Eichhornia crassipes are also reported to have antibacterial, antioxidant, antitumor, antiinflammatory, antinociceptive, potential antiplatelet components, hypoglycemic, and hypolipidemic activities of squalene. Antinociceptive, antioxidant, anti-inflammatory, antiallergic, hypolipidemic, anticancer, antimicrobial, cytotoxic, anti-teratogenic, antidiabetic, antispasmodic, anticonvulsant, disinfectant and antidiuretic activities of phytol were studied (Chirumamilla et al. 2022). Neophytadiene is present in chloroform, acetone and alcohol extracts. Ahmad et al. (2022) reported the presence of neophytadiene in aerial parts of Leucophyllum frutescens and mentioned its analgesic, antipyretic, anti-inflammatory, antimicrobial and antioxidant activities.

Tetratetracontane is identified in petroleum ether and ethanol leaf extracts in the present study. Varsha Jayakar et al. (2020) reported its anti-inflammatory, hypocholesterolaemia, cancer preventive, hepatoprotective and antioxidant activities in their study on leaf extracts of Garcinia cambogia and Garcinia indica. Gamma.-sitosterol is a steroid identified in both petroleum ether and ethanol extracts. Vats and Gupta (2017) reported gamma-sitosterol in the hydroethanolic extract of Moringa oleifera and documented its anti-diabetic, antiangeogenic, anticancerous, anti-inflammatory and antimicrobial activities. Dibutyl phthalate is a phthalate ester present in chloroform and ethanol extracts which is reported to have antifungal, antimicrobial and antimalarial activities (Akpuaka et al. 2013).

GC–MS analysis of leaf extracts C. floribunda of present study showed phytochemicals which were present in one of the extracts, pentacosane in petroleum ether, 2, 4-ditert-butylphenol, 1-nonadecene, E-15-heptadecenal, 10-heneicosene, heptadecyl trifluoroacetate, beta-sitosterol and methyl commate c in chloroform extract and tricosane in ethanol extract were indentified. These phytochemicals are reported to have antibacterial, anti inflammatory, antifungal, antioxidant and anticancerous activites (Table 10).

Table 10 Biological activities of the compounds of leaf extracts of Calycopteris floribunda Lam.
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Principle component analysis (PCA)

Phytochemicals are identified by GC–MS have biological activities and were subjected to a principal component analysis (PCA) (Fig. 5). The resulted data showed the relationship between the secondary metabolites having different bioactivities like antioxidant, anticancerous, anti inflammatory and antimicrobial with the solvents used for extraction (Fig. 5). The PCA showed 95.31% of the overall data variation and the PC 1 showed 68.29% of data variation and composed of antioxidant, antifungal, antibacterial and anticancerous activities. The PC 2 inferred anti-inflammatory and antimicrobial activities with 27.01% of the data variation.

Fig. 5
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PCA map showing the relationship among different solvent leaf extracts of Calycopteris floribunda Lam. and biological activities of secondary metabolites. AF, antifungal; AB, antibacterial; AM, antimicrobial; AML, antimalarial; AO, antioxidant; AI, anti-inflammatory; AC, anticancerous

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The principal component analysis (PCA) of the bioactivities of secondary metabolites of each extract showed that the leaf of Calycopteris floribunda Lam. is rich in secondary metabolites having antioxidant, antifungal and antibacterial activities.

Conclusion

GC–MS analysis of leaf extracts of Calycopteris floribunda Lam. showed the presence of 31 major bioactive compounds from the four different solvent extracts that are having greater number of biological activities. Present study concludes that the leaves of Calycopteris floribunda Lam. are rich in phytochemicals having antioxidant, antibacterial and antifungal activities. These phytochemicals can be extracted individually and they can be used to develop new classes of drugs to treat various disorders with respect to their biological activities by pharmaceutical industry.