Extraction of Bioactive Compounds for Antioxidant, Antimicrobial, and Antidiabetic Applications

This study was designed to check the potential of secondary metabolites of the selected plants; Citrullus colocynthis, Solanum nigrum, Solanum surattense, Calotropis procera, Agave americana, and Anagallis arvensis for antioxidant, antibacterial, antifungal, and antidiabetic agents. Plant material was soaked in ethanol/methanol to get the crude extract, which was further partitioned via solvent extraction technique. GCMS and FTIR analytical techniques were applied to check the compounds responsible for causing antioxidant, antimicrobial, and antidiabetic activities. It was concluded that about 80% of studied extracts/fractions were active against α-amylase, ranging from 43 to 96%. The highest activity (96.63%) was exhibited by butanol fractions of A. arvensis while the least response (43.65%) was shown by the aqueous fraction of C. colocynthis and the methanol fraction of fruit of S. surattense. The highest antioxidant activity was shown by the ethyl acetate fraction of Anagallis arvensis (78.1%), while aqueous as well as n-hexane fractions are the least active throughout the assay. Results showed that all tested plants can be an excellent source of natural products with potential antimicrobial, antioxidant, and antidiabetic potential. The biological response of these species is depicted as a good therapeutic agent, and, in the future, it can be encapsulated for drug discovery.


Introduction
The utility and discovery of plants having bioactive ingredients are as ancient as their uses as medicines, food, and nutraceuticals [1]. A huge diversity of plants is being used by native people for the curing of many diseases throughout the whole world. It is documented that about eighty percent of the community of developing countries is reliant on medicinal plants for health maintenance along with food purposes for humans as well as animals [2]. Currently, in the food industry, preservation of food, quality maintenance, and safety are considered major growing concerns [3,4]. The bad quality of food can cause several infectious diseases, due to food contamination and spoilage which is highly increased due to the presence of bacteria and various fungi. The chief rewards of attaining drugs from a natural source are easy availability and relatively fewer side properties [5]. Due to the revolution of traditional culture, aboriginal knowledge of herbal plants is being decreased [1]. Citrullus colocynthis fruits produce the colocynthin alkaloid, and roots, as well as the fruit of this plant, are used to treat snake bites [6]. Bryonia alba and Ecballium elaterium have been used as homeopathic therapeutic agents [7]. The fruit and leaves of Capsicum annuum L. are being used for the reduction of pain due to dog bites and other skin problems [8]. The

DPPH Radical Scavenging Assay
The antioxidant activity of crude extracts/fraction were measured in terms of the radical scavenging ability by the DPPH method [26]. Solution (1 mL) of all extracts/fraction at 100 mg/mL concentration was added to 2 mL methanolic solution of DPPH (2 mg/mL). The absorbance was noted at 517 nm after 30 min of incubation. The results were evaluated as the percentage scavenging of radicals using the reported formulae mentioned below. The results were compared with the standard (Gallic acid). Inhibition (%) = 1 − Absorbance of sample Absorbance of control × 100

Antimicrobial Activity
To evaluate the antimicrobial activity of the plant extracts, antibacterial and antifungal activities were performed. For this purpose, 10 bacteria (Klebsiella pneumonia, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Halomonas salina, Neisseria gonorrhoeae, Shigella sonnei, Halomonas halophila, and Chromohalobacter israelensis) and 2 fungi strains (Aspergillus flavus and Aspergillus niger) were selected on the availability of resources. All the plant extracts and fractions were screened in vitro for antimicrobial activities against bacterial and fungal strains. The medium was prepared, autoclaved, and poured into petri plates. Each petri plate was seeded with respective bacterial and fungal strains. A total of 20 µL of the sample was introduced on each disc with a micropipette. Streptomycin (5 mg/mL) was used as a positive reference for bacterial strains and Fluconazole (5 mg/mL) was used as a positive reference for fungal strains. After loading samples, cultures were allowed to grow for 24 h in an incubator at 37 • C for bacterial strains, and, for fungal strains, plates were incubated at 25 • C for 48 h. After reported time, the activity of the microbes was measured in millimeters and average activity and zone of inhibition were taken [25,27].

Antidiabetic Activity
α-Amylase inhibitory activity was carried out according to the reported method [28] with some modifications. A total of 200 µL of sample solution, 200 µL amylase (0.5 mg/mL), and 100 µL phosphate buffer (20 mM) of pH 6.9 were incubated at room temperature for 10 min. After 10 min, 100 µL of starch solution was added and again incubated at 25 • C for 20 min. After incubation, 100 µL of dinitrosalicylic acid (DNS) and 100 µL NaOH were added to the above solution mixture and test tubes were put into boiling water for about 20 min and then cooled at room temperature. A total of 1.0 mL of distilled water was added to the reaction mixture and the absorbance was measured at 540 nm. Each assay for all samples was carried out in triplicate. Percentage inhibition of all samples was calculated using the below-mentioned formula.
Absorbance of sample Absorbance of control × 100

GC-MS and FTIR Analysis
GC-MS analysis was performed with a gas chromatogram (Shamzadu GC-2010) along with mass spectrometer 9 (Shamazdu AOC-201). Samples for GC-MS were prepared in nhexane. The sample was introduced in non-polar capillary Colum SH-Rxi™-1MS by a splitless injection system. Helium gas of ultra-high purity was used with the 1 mL/min flow rate [29]. FTIR spectra of plant extracts were recorded at room temperature through Nicolet iS5 FTIR spectrometer having ATR/iD3 with argon horizontal cell (Thermo Scientific ® , Waltham, MA, USA).

Statistical Analysis
All experiments were performed in triplicate formation and standard deviation (±SD) was calculated against each assay using the MS Excel program.

Results and Discussion
The various parts of different plant species were collected and extracted in different solvents. The crude extracts were further partitioned with different solvents based on polarity with the solvent extraction technique ( Table 1). The crude extracts and their respective fractions were subjected to phytochemical analysis using reported protocols which suggested that these plant materials are a rich source of secondary metabolites.

FT-IR Analysis of Plant Extracts
Identification of functional groups present in the studies of plant extracts is necessarily for the structure elucidation of the bioactive compounds. To find the functionality of the compounds in the crude extract and fraction, the FTIR technique was used, and functional moieties were identified based on different bands respective to their functional groups. The absorption band at 3550-3450 cm −1 showed the characteristic band of aromatic OH group in chelate while OH of primary and tertiary alcohol absorbed at 1350-1260 cm −1 and 1410 cm −1, respectively. Fundamental stretching frequencies of the C-N bond appeared in the range of 1200-1000 and 1350 cm −1 to 1280 cm −1 , and N-H absorbed at 3400-3380 and 3510-3460 cm −1 for aliphatic and aromatic amines, respectively. The bands at 1150-1060 cm −1 , 1270-1230 cm −1 , and 1250-1150 cm −1 are representing the presence of C-O (stretch) of dialkyl, alkyl, aryl, and diaryl ethers, respectively. Oximes can be confirmed by comparing band frequency at 1690-1590 cm −1 representing the presence of C=N and also broad OH stretch at 3300-3150 cm −1 . The

FT-IR Analysis of Plant Extracts
Identification of functional groups present in the studies of plant extracts is nec sarily for the structure elucidation of the bioactive compounds. To find the functional of the compounds in the crude extract and fraction, the FTIR technique was used, a functional moieties were identified based on different bands respective to their functio groups. The absorption band at 3550-3450 cm −1 showed the characteristic band of aroma OH group in chelate while OH of primary and tertiary alcohol absorbed at 1350-1260 c and 1410 cm −1, respectively.

FT-IR Analysis of Plant Extracts
Identification of functional groups present in the studies of plant extracts is necessarily for the structure elucidation of the bioactive compounds. To find the functionality of the compounds in the crude extract and fraction, the FTIR technique was used, and functional moieties were identified based on different bands respective to their functional groups. The absorption band at 3550-3450 cm −1 showed the characteristic band of aromatic OH group in chelate while OH of primary and tertiary alcohol absorbed at 1350-1260 cm . The CH 2 of R 2 C-CH 2 vibrates at the frequency range from 3090 to 3075 cm −1 , for in-plane CH 2 vibrates at 1420-1410 cm −1 , and for out-plane, CH 2 vibration occurs at 895-885 cm −1 (Figures S1-S11). The IR bands at 3463, 3439, 3442, 3424, and 3423 cm −1 are due to OH/NH, while bands at 1647, 1649, and 1652 cm −1 are due to carbonyl functionality in the compounds. The bands near 2900 cm −1 are indicated the presence of an aromatic C-H bond in the molecules. Similarly, bands ranged 1052-1062 cm −1 because of C-O-C (ether) linkage. Based on the FT-IR analysis results, it was depicted that these compounds have various functional moieties such as C=O, -OH, C-O, and C=C. From the results, it is analyzed that Oxime, ester, amide, carboxylic acid, and ether functional groups are present in most plant extracts (Tables 2-4).

GC-MS Analysis
GC-MS (gas chromatography-mass spectrometry) technique is an excellent technique used to identify unknown compounds present in plant extracts, drug metabolites, lipids, and environmental contaminants. Plant extract was completely dissolved in n-hexane, filtered through micro filter paper, and subjected to GC-MS analysis. Electronic voltage of −70 eV was given to the compound to fragmentize it [30]. The chromatogram of the n-Hexane fraction of A. americana, A. arvensis, and the chloroform fraction of C. colocynthis is presented in Figures 1-3

Antioxidant Activity
The medicinal plants are a reliable source of antioxidant moieties being used for a long time as a crude extract as well as their purified compounds. The community of developing countries and people living in rural areas are still dependent on such therapeutic agents for their health issues. In the present study, we have extracted different plants in ethanol/methanol which were further partitioned with various solvents. The extracts/fractions were screened for their antioxidant potential using DPPH as a free radical. It was depicted that most of the plant fusion has antioxidant potential as mentioned in Table 5. The maximum activity was exhibited by AAEA while the minimum potential was shown by AARH (15.3 ± 0.1%), which may be due to the presence of low molecular weight compounds like a hydrocarbon. The highest activity of the AAEA may be due to polar components in the fraction (78.1 ± 0.3%). It was found that aqueous fractions in most of the cases remained less effective against a tested activity as depicted in Figure 4. The polar solvent has polar compounds such as phenolic acid or flavonoids which have great potential to quench the free radicals. Our findings about the antioxidant potential of understudy compounds are lined with already reported literature that medicinal plants have secondary metabolites that are responsible for antioxidant activities [31][32][33][34].

Antioxidant Activity
The medicinal plants are a reliable source of antioxidant moieties being used for a long time as a crude extract as well as their purified compounds. The community of developing countries and people living in rural areas are still dependent on such therapeutic agents for their health issues. In the present study, we have extracted different plants in ethanol/methanol which were further partitioned with various solvents. The extracts/fractions were screened for their antioxidant potential using DPPH as a free radical. It was depicted that most of the plant fusion has antioxidant potential as mentioned in Table 5. The maximum activity was exhibited by AAEA while the minimum potential was shown by AARH (15.3 ± 0.1%), which may be due to the presence of low molecular weight compounds like a hydrocarbon. The highest activity of the AAEA may be due to polar components in the fraction (78.1 ± 0.3%). It was found that aqueous fractions in most of the cases remained less effective against a tested activity as depicted in Figure 4. The polar solvent has polar compounds such as phenolic acid or flavonoids which have great potential to quench the free radicals. Our findings about the antioxidant potential of understudy compounds are lined with already reported literature that medicinal plants have secondary metabolites that are responsible for antioxidant activities [31][32][33][34].

Antibacterial Activity
The crude extracts along their fractions (solvent extraction) were subjected to antibacterial assessments. The extracts of C. colocynthis, S. nigrum, S. surattense, C. procera, A. americana, A. arvensis, and B. hispida were evaluated against 10 bacterial strains as mentioned in the experimental section. The ethanol fraction of C. colocynthis, the chloroform fraction of fruit of S. surattense, the methanol fraction of A. americana, and the n-hexane fraction of A. americana inhibited most of the bacteria. The standard (STD) used against these bacteria was Streptomycin. Ethanol, methanol, n-hexane, and chloroform fractions of plant extracts were mostly active against these bacteria. The variations in the antibacterial potential of plant extracts are due to the different chemical compositions of different extracts.

Antibacterial Activity
The crude extracts along their fractions (solvent extraction) were subjected to antibacterial assessments. The extracts of C. colocynthis, S. nigrum, S. surattense, C. procera, A. americana, A. arvensis, and B. hispida were evaluated against 10 bacterial strains as mentioned in the experimental section. The ethanol fraction of C. colocynthis, the chloroform fraction of fruit of S. surattense, the methanol fraction of A. americana, and the n-hexane fraction of A. americana inhibited most of the bacteria. The standard (STD) used against these bacteria was Streptomycin. Ethanol, methanol, n-hexane, and chloroform fractions of plant extracts were mostly active against these bacteria. The variations in the antibacterial potential of plant extracts are due to the different chemical compositions of different extracts. The highest value exhibited against E. coli by AAH was 21 mm; against K. pneumonia by SSFC it was 30 mm; against B. subtilis by CCE it was 15 mm; against S. aureus by CCE it was 32.5 mm; against S. typhimurium by AAH it was 20 mm; against C. israelensis by CCE it was 25 mm; against H. salina by AARH it was 24 mm; against N. gonorrhoeae by AAA it was 21 mm; against S. sonnei by AAM it was greater against H. halophile (Table 6). SSFC represented higher activity than exhibited by the standard's activity against K. pneumonia strain; CCE exhibited activity of 32 mm against Staphylococcus aureus, which is very close to the activity value (35 mm) of the standard. Similarly, the order of the activity was AARH > AAM > AAA against N. gonorrhoeae, S. sonnei, and H. salina. Strains were similar or close to the antibacterial activity exhibited by the standard against these bacterial strains (Table 6). Based on FTIR and GCMS results, it was concluded that this fraction has hydroxyl-containing compounds which may be responsible for antimicrobial activity. Natalia et al. (2021) reported that phenolic composites are diverse groups of natural products present in medicinal plants, which may be widely employed against various disease-causing microbes [35]. In literature, it was reported that secondary metabolites such as saponins, alkaloids, and phenolics have proven potential medicinal impacts for the cure of many diseases such as heart-related illnesses, cancers, bacterial infections, tumors, and diabetes [24,36,37].

Antifungal Activity
Antifungal activity of all plant extracts was tested against Aspergillus flavus and Aspergillus niger estimated via disc diffusion method. Fluconazole was used as the standard (STD) against these fungal strains. The zone of inhibition was measured after 48 h and was incubated at 25 • C. The chloroform fraction of S. nigrum, the ethanolic fraction of A. americana, the ethanolic fraction of leaves and stem of S. surattense, and the chloroform fraction of leaves and stem of Solanum surattense showed significant activity against A. flavus. The butanolic fraction of A. arvensis, the hexane fraction of A. arvensis, the methanolic fraction of A. americana, the n-hexane fraction of A. americana, and the aqueous fraction of C. colocynthis exhibited good activity against A. niger fungi. The highest value of inhibition against A. flavus was exhibited by SNC and was 21 mm. Against A. niger the highest value was exhibited by AARB and was measured at 24 mm. Values of antifungal activity of SNC, AARB, and AARH were higher than that of the standard's value of inhibition, showing that these fractions have a higher potential to inhibit fungal growth. Results indicated chloroform fractions are mostly active against these fungal strains ( Figure 5). Infections due to fungal species are increasing day by day and are one of the main reasons for deaths [38]. The fungi which are toxic to human beings comprise Fusarium, Aspergillus, and Candida species; these species secrete hazardous materials such as mycotoxins. To minimize or protect from fungal infections, plants secret many types of secondary metabolites, which perform vital roles as protection against several infections. The use of plant-based therapeutic agents for different ailments is preferred by the local community due to its fewer side effects and excellent activity [39]. fraction of C. colocynthis exhibited good activity against A. niger fungi. The highest value of inhibition against A. flavus was exhibited by SNC and was 21 mm. Against A. niger the highest value was exhibited by AARB and was measured at 24 mm. Values of antifungal activity of SNC, AARB, and AARH were higher than that of the standard's value of inhibition, showing that these fractions have a higher potential to inhibit fungal growth. Results indicated chloroform fractions are mostly active against these fungal strains ( Figure  5). Infections due to fungal species are increasing day by day and are one of the main reasons for deaths [38]. The fungi which are toxic to human beings comprise Fusarium, Aspergillus, and Candida species; these species secrete hazardous materials such as mycotoxins. To minimize or protect from fungal infections, plants secret many types of secondary metabolites, which perform vital roles as protection against several infections. The use of plant-based therapeutic agents for different ailments is preferred by the local community due to its fewer side effects and excellent activity [39].

Antidiabetic activity
The antidiabetic activity determined via the inhibition of α-amylase enzyme studies showed that the chloroform fraction of C. colocynthis, S. nigrum and S. surattense (leaves), the S. surattense aqueous fraction of leaves and stem, the C. procera methanol fraction, the C. procera chloroform fraction, the A. arvensis ethanol fraction, the A. arvensis hexane fraction, the A. arvensis ethyl acetate fraction, the A. americana ethyl acetate fraction, the A. americana butanol fraction, and the A. americana aqueous fraction showed good inhibition against α-amylase. It is observed that chloroform fractions, ethanol fractions, and hexane fractions were mostly active against α-amylase ( Figure 6). These fractions have a rich source of hydroxyl and nitrogen containing compounds in the form of alcohol/phenol and amide or amines form which have the tendency to inhibit the targeted enzyme due to the strong binding on the active site of the enzyme.
The highest value of α-amylase inhibition was depicted by the chloroform fraction of S. surattense (SSFC) at 89.56%, while the chloroform fraction of C. procera has shown weak response. The highest inhibition among ethanolic extract was shown by AARE at 95.65% and the fraction of S. surattense exhibited low inhibition against diabetic enzymes. The aqueous fraction A. arvensis (AAA) represented the highest anti-diabetic activity (94.53%) while C. colocynthis had about no activity as presented in Figure 6. The antidiabetic results of the selected medicinal plants are tabulated in Table 7. Based on the results, it is depicted that understudied plant extracts/fractions have excellent potential for antidiabetic activity.

Antidiabetic Activity
The antidiabetic activity determined via the inhibition of α-amylase enzyme studies showed that the chloroform fraction of C. colocynthis, S. nigrum and S. surattense (leaves), the S. surattense aqueous fraction of leaves and stem, the C. procera methanol fraction, the C. procera chloroform fraction, the A. arvensis ethanol fraction, the A. arvensis hexane fraction, the A. arvensis ethyl acetate fraction, the A. americana ethyl acetate fraction, the A. americana butanol fraction, and the A. americana aqueous fraction showed good inhibition against α-amylase. It is observed that chloroform fractions, ethanol fractions, and hexane fractions were mostly active against α-amylase ( Figure 6). These fractions have a rich source of hydroxyl and nitrogen containing compounds in the form of alcohol/phenol and amide or amines form which have the tendency to inhibit the targeted enzyme due to the strong binding on the active site of the enzyme.
The variation in the activity may be due to differences in qualitative and quantitativ tochemicals.   The highest value of α-amylase inhibition was depicted by the chloroform fraction of S. surattense (SSFC) at 89.56%, while the chloroform fraction of C. procera has shown weak response. The highest inhibition among ethanolic extract was shown by AARE at 95.65% and the fraction of S. surattense exhibited low inhibition against diabetic enzymes. The aqueous fraction A. arvensis (AAA) represented the highest anti-diabetic activity (94.53%) while C. colocynthis had about no activity as presented in Figure 6. The antidiabetic results of the selected medicinal plants are tabulated in Table 7. Based on the results, it is depicted that understudied plant extracts/fractions have excellent potential for antidiabetic activity. The variation in the activity may be due to differences in qualitative and quantitative phytochemicals.
Natural products chiefly derived from a plant source are also major excavations for lead compounds and play an imperious part in new drug discovery [40,41]. The rural community is mainly dependent on the natural product due to cheaper prices, fewer side effects, and easy availability. The plant-based materials are the key player in all available therapies, especially in rural areas. They keep dominant pharmacological activities to ward off many diseases [42]. Many plants are a rich source of different therapeutic agents necessary for various diseases and, currently, several drugs on the market are either natural products or