GCMS-based phytochemical profiling and in vitro pharmacological activities of plant Alangium salviifolium (L.f ) Wang

Background There is an urge for traditional herbal remedies as an alternative to modern medicine in treating several diseases. A significant number of modern pharmaceutical drugs are based on or derived from medicinal plants or their extracts. These drugs derived from the plant origin have various antimicrobial, antioxidant, anticancer, anti-inflammatory activities. Alangium salviifolium belongs to Cornaceae family and is well known for its medicinal properties. The present study was carried out to evaluate the antibacterial, antioxidant effect and possible bioactive components present in the chloroform, acetone, ethanol, methanol and aqueous extract of Alangium salviifolium leaves. Methodology Dried leaves of Alangium salviifolium were subjected to serial solvent extraction using increasing polarity of solvents, i.e., chloroform, acetone, methanol, ethanol, and distilled water. Crude extracts were further tested for qualitative analysis of phytochemicals using standard procedure, while GCMS analysis was performed to identify the probable phytocompounds. Antibacterial activity was performed against bacterial pathogens using agar well method, whereas antioxidant activity was performed using in vitro PM, DPPH and FRAP assays. Results Phytochemical analysis of the extracts revealed the presence of key phytochemical classes. Using gas chromatography-mass spectrometry, several high and low molecular weight chemical compound kinds were discovered. These chemical substances are regarded as having significant biological and pharmacological effects. All crude extracts had considerable and comparable in vitro antioxidant and antibacterial properties. Conclusions According to the findings of this study, Alangium salviifolium leaves are a rich source of phytoconstitu-ents that are crucial in stopping the advancement of numerous disorders.


Background
Numerous metabolites that are more useful in the field of medicine are naturally produced by plants.More than 80% of the world's population, according to the World Health Organization (WHO), relies on traditional medicine for their primary healthcare requirements [1].21,000 plants are known to be used as medicines worldwide, according to the WHO [2].As plants are the primary source of medicines in Siddha, Unani, and Ayurveda systems of medicine, India has a rich cultural history of ancient medicines [3].Many of the chemicals produced by these medicinal plants have been shown to have therapeutic benefits [4].More research works on ethnopharmacognosy were increased due to the result of the emergence of negative effects and microbial resistance to the chemically synthesized medications [5].
Secondary metabolites including tannins, steroids, phenolic compounds, and alkaloids, sometimes known as phytochemicals or phytocompounds, are what give plants their therapeutic potential.Some plant secondary metabolites with medicinal promise include morphine, vincristine, vinblastine, taxol and quinine [6].Due to fewer side effects as compared to synthetic pharmaceutical chemicals, these phytochemicals have recently gained importance throughout the world and are used in the pharmaceutical industry for the drug development and treatment of major diseases like asthma, arthritis, cancer, and diabetes [7,8].These medicines have different antimicrobial, antioxidant, anticancer, and anti-inflammatory effects because they are derived from plants [9].Antioxidants are substances that slow down or stop oxidative reactions that catalyze free radicals.The presence of phenolic substances such as flavonoids, phenolic acids, tannins, and phenolic diterpenes [10,11] is primarily responsible for the antioxidant activity of plant products.Plants are protected from oxidative assault by antioxidants like BHT (Butylated Hydroxy-Toluene) and BHA (Butylated Hydroxy-Anisol), which bind oxidative damage to metallic ions, break down peroxides, or destroy free radicals [12].According to Lobo et al. [13] and the rise in pathogen antimicrobial resistance, infectious diseases account for almost 50% of all deaths [14].As a need of the hour researchers have created new, efficient antibacterial medications from natural phytochemicals [15,16] demonstrated the potential of numerous herbs as sources of pharmaceuticals with lower toxicity.By screening a wide range of plant groups, the search for new antibacterial compounds continues.Higher plants are a possible source of novel antibiotic prototypes, according to research on the antibacterial activity of plant extracts and plant products [17,18].There is a need for more research into traditional health systems because certain traditional medicines have already developed substances that are effective against bacteria strains that are resistant to antibiotics [19,20].
Alangium salviifolium, also known as sage leaved alangium or ankola, was chosen for the current study.It is a member of the Cornaceae family, which is indigenous to China, India, Bangladesh, and the Philippines [21].This family of plants often grows in arid or hotter climates.It is used to treat a variety of illnesses.According to numerous studies [22][23][24][25], it is widely used as a treatment for skin conditions, leprosy, asthma, epilepsy, hepatitis, scabies, and as an antidote for snake and dog bites.According to numerous studies [26][27][28], roots can be used to cure diarrhea, paralysis, piles, and vomiting.Although Alangium salviifolium leaves are a storehouse of many nutrients and bioactive chemicals, the systematic analysis of these leaves is still not sufficient in terms of the specific biological activity of their chemical constituents.The potential benefits of A. salviifolium for treating different medical issues should be further investigated.Thus, the objective of the current study was to identify the phytochemical components, antioxidant, and antibacterial properties of various solvent extracts of A. salviifolium leaves.Additionally, utilizing GC-MS and FTIR analysis, the bioactive elements of the extracts and the functional groups of the compounds were also found.

Collection of plant material
The plant Alangium salviifolium (L.f ) Wang's leaves were collected in the month of February 2022 in Ankola, Uttar Kannada District, Karnataka, India.Dr. K. Kotresha, Professor, Department of Botany, Karnatak Science College, Dharwad, Fresh plant leaves were gathered, cleaned under running water, dried in the shade, and then blended to a coarse powder.For future use, the powder was kept in sealed containers.

Solvent extraction
Alangium salviifolium dry leaves were coarsely pulverized, and then, serial solvent extractions were performed using a Soxhlet apparatus.In increasing order of polarity, the following solvents were used for the extraction: chloroform, acetone, methanol, ethanol, and distilled water.Using a Rota-evaporator, the extracts were further concentrated.The airtight containers used to hold the concentrated extracts were chilled until use.

Phytochemical analysis
The presence of various phytochemical components, including alkaloids, tannins, phenols, sterols, terpenoids, glycosides, saponins, flavonoids, and carbohydrates, were screened for in the crude extracts of Alangium salviifolium plant leaves using a standard procedure [29].

Total phenol content (TPC) estimation
Utilizing 1.5 ml of the Folin-Ciocalteu (FC) reagent and 7.5% sodium carbonate (Na 2 CO 3 ) solution, the plant extract of known concentration, i.e., 1 mg/1 ml of respective solvent, was subjected to oxidation.The absorbance reading at 750 nm was obtained during an hour of incubation at room temperature.The experiment was performed in triplicate, and results were expressed as mean ± standard deviation.The quantity was determined using the calibration curve for gallic acid.Gallic acid equivalent (GAE) mg/100 ml of sample was used to express the results [30].

Total flavonoid content (TFC) estimation
The 10% aluminum chloride (AlCl 3 ) and 1 M sodium acetate were combined with the known quantity of plant extract, i.e., 1 mg/1 ml of respective solvent.Following a 45-min incubation in the dark, the absorbance was measured at 415 nm.Using the Quercetin calibration curve, the quantity was determined.Quercetin equivalent (QE) mg/100 ml of sample was used to express the results [31].The experiment was performed in triplicate, and results were expressed as mean ± standard deviation.

FTIR analysis
Utilizing a Perkin Elmer Spectrophotometer system, an FTIR study of Alangium salviifolium was carried out in order to identify the distinctive peaks between 400 and 4000 cm −1 and their functional groups.The FTIR's peak values were noted.

GC-MS profiling
The different solvent extracts of A. salviifolium were subjected to a GC-MS analysis with instrument model GCMS-QP 2010 Plus, Shimadzu).At an ionization voltage of 70 eV, injector temperature of 250 °C and injector mode was split with linear velocity 36.5 cm/s and pressure was 57.5 kPa.The instrument was run in electron impact mode.Approximately 1 µL of the sample was injected into mobile phase consisting of helium (99.9% purity) at a flow rate of 1 mL/min.The oven's temperature was first set to 60 °C for 2 min of isothermal operation before being raised to 100 °C at a rate of 10 °C per minute and then to 280 °C at a rate of 5 °C per minute for 9 min.The GC ran for 34 min in total.Comparing each component's average peak area to the total areas allowed us to determine the proportional percentage amount of each component.With the help of The National Institute of Standard and Technology-5 (NIST-5), a comparison was made between the spectra of the unknown component and the spectrum of the known components including the compound's name, chemical formula, molecular weight, and structure, which were identified.

Phosphomolybdenum assay
Using a standardized process, the phosphomolybdenum method was used to assess the antioxidant activity of the plant extracts [32] and ascorbic acid was used as reference standard.Alangium salviifolium extracts of concentration 1 mg/ml were added to each test tube separately along with 3 ml of distilled water and 1 ml of the phosphomolybdate (PM) reagent in varying concentrations ranging from 100 to 500µL.For 90 min, the tubes were incubated in a water bath at 95 °C.Following incubation, these tubes were cooled to room temperature, and the reaction mixture's absorbance was assessed at 695 nm.

2, 2 Diphenyl-2-picryl hydrazyl (DPPH) radical scavenging assay
A. salviifolium plant extracts were subjected to the Rice-Evans et al. [33] method for the DPPH radical scavenging assay.The known concentration of test samples (100 µg) was combined in various concentrations with 100 µl of a DPPH solution made in methanol (40 mM).The combination was incubated for 30 min at room temperature and in the dark to produce the desired color, which was measured at 517 nm along with ascorbic acid as a standard.
Each sample's DPPH scavenging activity was estimated using the formula below: where At is the absorbance of the test sample and Ac is the absorbance of the control reaction (100 µL of methanol plus 100 µL of DPPH solution).

Ferric ion reducing power (FRAP) assay
With a few minor modifications, the Oyaizu [34] method was used to assess the reducing power of ferric ions.The extracts of A. salviifolium (1 mg/ml) were pipetted into mixtures containing 2.5 ml of 0.2 M phosphate buffer, 2.5 ml (1% w/v) potassium ferricyanide, and various concentrations ranging from 100 to 500 µL.The mixture was then incubated at 50 °C for 20 min.After cooling the mixture, 2.5 ml of 10% w/v trichloroacetic acid and 0.5 ml of 0.1% w/v ferric chloride were added, and the combination was left at room temperature for 10 min to form a complex that was green in color.The absorbance was calculated using a spectrophotometer at 700 nm.The reference standard used was ascorbic acid.

Antibacterial activity
The antibacterial activity was screened using the agar well diffusion method [35] using Pseudomonas aeruginosa and Staphylococcus aureus cultures as the test organisms.100µL of the uniformly diluted saline suspension were swabbed over the sterile agar plates.Along with the control drug Ciprofloxacin (30 µg), various concentrations of the extract (1 mg/ml) (30, 60, 90, and 120 µL/well) were added to the medium.The plates were incubated for 24 h at 37 °C.The diameter of the inhibition zones that formed around the wells after the incubation time was measured in millimeters.

Statistical analysis
The experiments were performed in the triplicate, and the results were expressed as mean ± standard deviation using IBM SPSS Statistics 20.0.

Plant chemical analysis
The qualitative phytochemical analysis of five different solvent extracts of Alangium salviifolium revealed the presence of flavonoids, reducing sugar, and carbohydrates in chloroform, acetone, methanol, ethanol, and distilled water; in addition, glycosides, saponins, and tannins were present in aqueous extract.All five extracts contained alkaloids and phenols.The other four extracts, with the exception of methanol, all included lignin The results of phytochemical analysis was shown in Table 1.

Total crude extraction
The phytochemical examination of A. salviifolium leaves revealed a significant amount of the plant secondary metabolites.Using the solvents chloroform, acetone, ethanol, methanol, and distilled water, the total yield of crude extracts from A. salviifolium leaves was determined to be 1.8%, 2.1%, 3.8%, 2.4%, and 2.8% (w/w), respectively.

Fourier transform infrared spectroscopy analysis
The FTIR spectra of the all extracts revealed the presence of halogen and nitrogen compounds in common.The chloroform, acetone, and ethanol extracts revealed the presence of alkane and alcohol functional groups; the chloroform, acetone, methanol, and ethanol extracts revealed carbon dioxide and alkene groups; the chloroform extracts revealed the presence of aldehyde and ketone groups; the chloroform, methanol, and acetone extracts revealed amine groups; the acetone and ethanol extracts revealed esters groups; acetone, ethanol,

GC-MS analysis
The compounds from the GC-MS analysis of the chloroform, acetone, methanol, ethanol, and aqueous extracts of A. salviifolium leaves are shown in Tables 8,  9, 10, 11, and 12.In case of the chloroform extract, the compound Androst-5-ene-3, 17-diol, 17-methyl-, dipropan was found to be major compound, in case of acetone extract the compound Phytol was found to be major compound, for methanol extract the compound ethyl (dimethyl) isopropoxysilane seems to be major compound, in case of ethanol extract diethyl phthalate was found to be major compound, and for aqueous extract benzofuran, 2, 3-dihydro compound was found to be major compound.Overall, the GC-MS results of chloroform, acetone, ethanol, methanol, and aqueous extract showed the presence of 24, 53, 23, 31, and 6 compounds, respectively, and the results are shown in Tables 8, 9, 10, 11, and 12.Additional file 1: Figs.

A. salviifolium's antioxidant activity in vitro Phosphomolybdenum (PM) assay
On comparison between the all extracts, i.e., for acetone, chloroform, ethanol, methanol, and aqueous extracts in the phosphomolybdenum (PM) assay, aqueous extract has shown the highest activity with higher absorbance, i.e., 0.991 ± 0.004 for 500 µg concentration, whereas standard ascorbic acid has shown 1.078 ± 0.003.The results of all test samples are shown in Table 13 and Additional file 1: Fig. S12.

Radical scavenging assay using 2, 2 diphenyl-2-picryl hydrazyl (DPPH)
In case of DPPH assay among the selected chloroform, acetone, ethanol, methanol, and aqueous extracts, aqueous extract has exhibited prominent activity with a higher percentage of inhibition, i.e., 84.892% and standard ascorbic acid shown around 86.271%.The study found that when aqueous extract was compared to regular ascorbic acid, it had the comparable antioxidant activity than the remaining extracts (Table 14).

Ferric ion reducing power (FRAP) assay
Using standard ascorbic acid, the ferric ion reducing power (FRAP) assay was carried out on extracts of acetone, chloroform, ethanol, methanol, and distilled water.
According to the study's findings (Table 15, Additional file 1: Fig. S13), aqueous extract had the highest antioxidant activity with higher absorbance OD value when compared to the other four extracts.

Antibacterial activity of Alangium salviifolium leaves extract
Aqueous extract from the leaves of A. salviifolium had the greatest zone of inhibition against Pseudomonas aeruginosa and Staphylococcus aureus of all the leaf extracts tested for antibacterial activity.With a maximum inhibitory zone of 10 mm, leaf aqueous extract was found to be more effective to Pseudomonas aeruginosa than ethanol, acetone, and chloroform (5 mm) or methanol extract (3 mm).With a maximum inhibitory zone of 11 mm, Staphylococcus aureus was found to be more sensitive to the aqueous extract than to acetone, chloroform, ethanol, or methanol.

Discussion
Numerous chemicals found in plants that are known to be biologically active and to have a variety of pharmacological effects [36,37].These plant secondary metabolites include several important natural antioxidant sources that are safer and more efficient than synthetic antioxidants [38].The most prevalent phenolic molecules that act as natural antioxidants in plants include ascorbic acid, carotenoids, and flavonoids [39].The phytochemicals found in the plant A. salviifolium were extracted in the current study utilizing a variety of increasing polarity solvents, including chloroform, acetone, ethanol, methanol, and distilled water.Different phytochemical tests were used to identify the phytochemicals, which included the presence of reducing sugar, alkaloids, flavonoids, phenols, lignin, glycosides, and tannins.Plants use alkaloids, the majority of which have a severe bitter taste and are very toxic, to protect themselves from herbivory, pathogenic microbial attack, and invertebrate pests.Numerous studies on phenolic compounds have demonstrated the significance of these compounds in demonstrating biologically active properties like anti-inflammatory, antidiabetic, antioxidant, antibacterial, anticancer, etc. [38].Because of this, the total phenolic and total flavonoid contents of various extracts of A. salviifolium leaves were determined, as well as their antioxidant potential by in vitro phosphomolybdenum, DPPH, and ferric ion reducing power (FRAP) assay methods.In the current investigation, chloroform extract (71.86 mg/g QE) and ethanol extract (82.86 mg/g GAE) were shown to have the greatest concentrations of total flavonoid and phenol, respectively.It can be expected that the biological activity of the leaves of A.    salviifolium may be caused by the presence of flavonoids and phenolic compounds in it based on the measurement of the total phenol and flavonoid content in them.
According to Neha et al. [52], an antioxidant is a chemical that can inhibit or block the oxidation of lipids or other molecules by avoiding the onset of oxidative chain reactions.As a result, it can stop or undo the harm that oxygen does to the body's cells.Natural antioxidants are more popular these days because of their potential to improve health and fend off diseases.In the current work, three assay methods-the Phosphomolybdenum (PM), DPPH, and ferric ion reducing power (FRAP) assay methods-were used to determine the antioxidant activity of Alangium salviifolium leaves extract.Different crude extracts of Alangium salviifolium leaves underwent PM, DPPH, and FRAP assays, and the results were compared to ascorbic acid standard.The phosphate-Mo (V) complex is a bluish green color, and its synthesis results in the reduction of molybdate ions, which is evaluated spectrophotometrically in the phosphomolybdenum test [32].It is a method that is often used in laboratories to evaluate  the overall antioxidant activity of plant extracts.Aqueous extract had the highest activity in the current investigation (0.991 ± 0.004) (Table 13, Additional file 1: Fig. S12).
The assessed antioxidant's potential to scavenge free radicals is revealed by the decrease in DPPH solution absorbance during the reaction.Alangium salviifolium plant secondary metabolites such as alkaloids, flavonoids, tannins, phenols, and glycosides are abundant in the plant's crude extracts.By contributing a hydrogen molecule, each of these bioactive compounds has the ability to oxidize the DPPH solution [53].Using chloroform, acetone, ethanol, methanol, and aqueous extract, the antioxidant activity of Alangium salviifolium was assessed in the current study and compared to that of conventional ascorbic acid.According to the results (IC 50 value: 58.89 µg/ml) (Table 14), aqueous extract had the highest level of scavenging activity compared to all other extracts.
The ferric ion reducing power (FRAP) assay is a method that examines how antioxidants in an acidic media reduce ferric ion (Fe3+)-ligand complex to the strikingly blue ferrous (Fe2+) complex.According to this approach, absorption is inversely related to reducing potential; the greater the absorbance, the greater the antioxidants' capacity to reduce [54].In the current analysis, aqueous extract was proven to be significant than chloroform, acetone, ethanol and methanol extracts in terms of antioxidant activity (0.997 ± 0.002) (Table 15, Additional file 1: Fig. S13).The investigated extracts' antioxidant activity varied greatly among the different solvent extracts, and the results concluded that on comparison among the tested extracts aqueous extract demonstrated a greater overall antioxidant capacity with noticeably superior outcomes.According to research by Shravya et al. [1], the antioxidant activity of Alangium salviifolium leaves was found to be superior than that of the plant's roots.However, Alangium salviifolium leaf extract demonstrated significant antioxidant activity against the DPPH radical and was comparable to earlier findings for this plant, but there was no relationship between antioxidant activity and TPC for Alangium salviifolium leaves [55].
Over the past three decades, pharmaceutical companies have created a variety of innovative antibiotic treatments, but bacteria have grown more resistant to these medications.Plant extracts are a fantastic source of pathogen-fighting antibacterial compounds.They can therefore be utilized to treat a variety of infectious disorders brought on by virulent microorganisms.Staphylococcus aureus and Pseudomonas aeruginosa were used in this work to test the plant extract from Alangium salviifolium for antibacterial activity.In both test organisms (Zone of Inhibition-19 mm and 22 mm), aqueous extract stood out among the extracts for its strong antibacterial activity (Table 16, Additional file 1: Figs.S14 and S15).However, Alangium salviifolium stem bark and flower extract have been found in the past to have strong antibacterial activity against a variety of bacteria [56,57].Additionally, there are not many reports on the antibacterial properties of Alangium salviifolium leaf extract.The results of the current investigation make it abundantly evident that water extract proven to be have significant antibacterial activity, whereas acetone, chloroform, ethanol, and methanol have noticeable antibacterial properties.Our study's findings indicate that Alangium salviifolium leaves can act as a natural antioxidant to stop the onset and spread of a variety of ailments.To isolate and purify the plant chemicals for this antioxidant and antibacterial properties, more research is required.

Conclusions
In the present study, the plant Alangium salviifolium was selected and using phytochemical and GCMS analysis the different solvent extracts of the plant have shown the presence of several metabolites such as phenols and alkaloids, GC-MS analysis has shown the presence of several compounds which are having industry and medicinal applications.Among the different solvent extracts, ethanol and chloroform extract have shown the presence of highest phenolic and flavonoid content, respectively.Further, all selected extracts were screened for biological activities such as antioxidant and antibacterial activity.The results concluded that aqueous extract of plant Alangium salviifolium proven to be having potent properties in all performed assay.Hence, in future the molecular level of studies and animal model can be studied to understand its pathway studies.

Table 2
Total phenol and total flavonoid content from A. salviifolium (leaf extract) , aqueous extract showed sulfoxide group; carboxylic acid group was found in methanol and aqueous extracts; vinyl ether was found in methanol and aqueous extract revealed sulfonyl chloride groups.The results of FTIR are shown in Tables3, 4, 5, 6, and 7 (Additional file 1: Figs.S1-S6). methanol

Table 3
FTIR Interpretation of compounds of leaf chloroform extract of A. salviifolium

Table 4
FTIR Interpretation of compounds of leaf acetone extract of A. salviifolium

Table 5
FTIR interpretation of compounds of leaf methanol extract of A. salviifolium

Table 6
FTIR Interpretation of compounds of leaf ethanol extract of A. salviifolium Table 16 and Additional file 1: Figs.S14 and S15 display the measured zone of inhibition for leaf extracts produced from various solvents.

Table 7
FTIR Interpretation of compounds of leaf aqueous extract of A. salviifolium

Table 8
Compound identified in the chloroform extract of A. salviifolium using GCMS Sl. no.

Compound name Molecular formula Molecular weight Retention time Peak area Compound nature
Sl. no.

Table 9
Compound identified in the acetone extract of A. salviifolium using GCMS

Table 10
Compound identified in the methanol extract of A. salviifolium using GCMS Sl.

Table 11
Compound identified in the ethanol extract of A. salviifolium using GCMS

Table 12
Compound identified in the aqueous extract of A. salviifolium using GCMS

Table 14
DPPH assay for A. salviifolium leaf extract

Table 15
FRAP assay for A. salviifolium leaf extractThe results are represented as mean ± standard deviation

Table 16
Zone of inhibition (in mm) for different solvent extracts of leaves of A. salviifolium