Phytochemicals: Extraction, Isolation, and Identification of Bioactive Compounds from Plant Extracts

www.yra.ijaar.co.in ISSN – 2277-7911 Impact Factor – 5.077 Oct-Nov -Dec 2022 Vol. 11 No. 4 113 Phytochemicals: Extraction, Isolation, and Identification of Bioactive Compounds from Plant Extracts Dr. Kumar Gourav Faculty of Science. Magadh University Bodh Gaya. Bihar. India. Corresponding Author: Dr. Kumar Gourav DOI - 10.5281/zenodo.17579429 Abstract: There are concerns about using synthetic phenolic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) as food additives because of the reported negative effects on human health. Thus, a replacement of these synthetics by antioxidant extractions from various foods has been proposed. More than 8000 different phenolic compounds have been characterized; fruits and vegetables are the prime sources of natural antioxidants. In order to extract, measure, and identify bioactive compounds from a wide variety of fruits and vegetables, researchers use multiple techniques and methods. This review includes a brief description of a wide range of different assays. The antioxidant, antimicrobial, and anticancer properties of phenolic natural products from fruits and vegetables are also discussed. Keywords: Antimicrobial, Antioxidants, Medicinal Plants, BHT Introduction: Many antioxidant compounds can be found in fruits and vegetables including phenolics, carotenoids, anthocyanins, and tocopherols [1]. Approximately 20% of known plants have been used in pharmaceutical studies, impacting the healthcare system in positive ways such as treating cancer and harmful diseases [2]. Plants are able to produce a large number of diverse bioactive compounds. High concentrations of phytochemicals, which may protect against free radical damage, accumulate in fruits and vegetables [3]. Plants containing beneficial phytochemicals may supplement the needs of the human body by acting as natural antioxidants [4]. Various studies have shown that many plants are rich source of antioxidants. For instance, vitamins A, C, E, and phenolic compounds such as flavonoids, tannins, and lignins, found in plants, all act as antioxidants [3]. The consumption of fruits and vegetables has been linked with several health benefits, a result of medicinal properties and high nutritional value [5]. Antioxidants control and reduce the oxidative damage in foods by delaying or inhibiting oxidation caused by reactive oxygen species (ROS), ultimately increasing the shelf-life and Young Researcher Vol. 11 No. 4 OctNov - Dec 2022 Dr. Kumar Gourav 114 quality of these foods [2]. Beta carotene, ascorbic acid, and many phenolics play dynamic roles in delaying aging, reducing inflammation, and preventing certain cancers [3]. Increasing the consumption of fruits and vegetables has been recommended by many agencies and health care systems throughout the world [2]. The objective of this paper is to provide a review of phytochemical studies that have addressed extracting, measuring and identifying bioactive compounds of plants. This review includes an overview of the lipid oxidation process, details of plants known to be antioxidant and antimicrobial sources, phenolic compounds, antioxidants from vegetables and fruits, cancer prevention, extraction techniques for phenolic compounds, isolation and purification of bioactive molecules, and techniques for structural classification of bioactive molecules. Methods Used for Bioactive Compound Extraction, Isolation, and Purification Extraction of Phenolic Compounds Using Solvents: Scientists have studied and analysed the impact of different types of solvents, such as methanol, hexane, and ethyl alcohol, for the purpose of antioxidant extraction from various plants parts, such as leaves and seeds. In order to extract different phenolic compounds from plants with a high degree of accuracy, various solvents of differing polarities must be used [9]. Moreover, scientists have discovered that highly polar solvents, such as methanol, have a high effectiveness as antioxidants. Anokwuru et al. reported that acetone and N,N dimethylformamide (DMF) are highly effective at extracting antioxidants, while Koffi et al. found that methanol was more effective in at a large amount of phenolic contents from walnut fruits when compared to ethanol [1,2]. It has been reported that ethanolic extracts of Ivorian plants extracted higher concentrations/amount of phenolics compared to acetone, water, and methanol [1]. Multiple solvents have been commonly used to extract phytochemicals, and scientists usually employed a dried powder of plants to extract bioactive compounds and eliminate the interference of water at the same time. Solvents used for the extraction of biomolecules from plants are chosen based on the polarity of the solute of interest. A solvent of similar polarity to the solute will properly dissolve the solute. Multiple solvents can be used sequentially in order to limit the amount of analogous compounds in the desired yield. The polarity, from least polar to most polar, of a few common solvents is as follows: Hexane < Chloroform < Young Researcher Vol. 11 No. 4 OctNov - Dec 2022 Dr. Kumar Gourav 115 Ethylacetate < Acetone < Methanol < Water. Microwave-Assisted Extraction (MAE): MAE has attracted the attention of researchers as a technique to extract bioactive compounds from a wide variety of plants and natural residues Microwaves have electromagnetic radiation that occurs at frequencies between 300 MHz to 300 GHz, and wavelengths between 1 cm and 1 m. These electromagnetic waves consist of both an electrical field and a magnetic field. These are described as two perpendicular fields. The first application of microwaves was to heat up objects that can absorb a part of the electromagnetic energy and convert it into heat. Commercial microwave instruments commonly use the frequency 2450 MHz, which corresponds to an energy output of 600–700 Watts. Recently, advanced techniques have become available to reduce the loss of bioactive compound without increasing the extraction time. Therefore, microwave-assisted extraction is demonstrated to be a good technique in multiple fields, especially in the medicinal plant area. Moreover, this technique reduced the losses of the biochemical compounds being extracted Microwave-assisted extraction (MAE) has been used as an alternative to conventional techniques for the extraction of antioxidants because of its ability to reduce both time and extraction solvent volume In fact, the main objective of using MAE is to heat the solvent and extract antioxidants from plants with a lesser amount of these solvents [13]. Li et al. reported that conventional methods using various solvents presented less antioxidant activity and phenolic content than MAE [16]. Therefore, the finding confirmed that MAE was more effective at increasing antioxidant activity by measuring ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), and total phenolic content (TPC). The efficiency of the microwave extraction can be changed through some factors such as extraction temperature, solvent composition, and extraction time. The extraction temperature was usually studied more than other factors due to its ability to increase the efficiency of the microwave extraction. Tsubaki et al. reported that 170 °C was the most effective temperature for extracting phenolic compounds from Chinese tea. In addition, increasing the extraction temperature beyond this point resulted in a reduced extraction yield [17]. Recently, Christophoridou et al. used a new microwave-assisted extraction (MAE) process, which converts energy to heat, thereby cooperating with solvents in order to extract a specific compound. Williams et al. showed many Young Researcher Vol. 11 No. 4 OctNov - Dec 2022 Dr. Kumar Gourav 116 advantages of MAE, including lower solvent consumption. Ultrasonic-Assisted Extraction: Ultrasound-assisted extraction (UAE) has been used in diverse applications of food-processing technology to extract bioactive compounds from plant materials Ultrasound, with levels greater than 20 kHz, is used to disrupt plant cell walls, which helps improve the solvent’s ability to penetrate the cells and obtain a higher extraction yield. UAE can use a low operating temperature through processing, maintaining a high extract quality for compounds. UAE is known to be one of the easiest extraction techniques because it uses common laboratory equipment such as an ultrasonic bath. In this technique, a smashed sample is mixed with the suitable solvent and placed into the ultrasonic bath, while temperature and extraction time are controlled UAE of various organic and inorganic samples can use a wide range of solvents. Common equipment used in ultrasoundassisted extraction includes an ultrasonic bath and an ultrasonic probe system. Unfortunately, ultrasonic probe has two main negative properties mainly related to experimental repeatability and reproducibility. Tabarak et al. noted that green technology is necessary to protect the environment from toxic substances, Therefore, extraction of phenolic compounds by ultrasound has grown during recent years due to its role in reducing the amount of solvent and energy used. Corrales et al. have shown that UAE can break down plant tissue and work properly during the production process and release of active compounds in solvents with a high efficiency Results showed an increase in antioxidant activity from 187.13 μmol TE g−1DM to 308 μmol TE g−1DM by using UAE as an effective method to extract antioxidants from different sources. Recently, Albu et al. studied and applied the use of ultrasound to extract phenolic compounds from rosemary [23]. Multiple criteria have been compared including ultrasonic bath extractions, ultrasonic probe system, a shaking water bath at various temperatures, and different solvents to select the most efficient method. In all situations, the operation time was dramatically decreased by applying and using the ultrasonic bath and probe systems. Similar behaviour was reported by Cho et al. when extracting resveratrol from grapes [24]. In another study, Barbero et al. suggested the use of ultrasound in different industries because of its positive effects in the extraction of capacious of hot peppers Moreover, the ultrasonic method had the ability to decrease the degradation of phenolics. Multinacci et al. compared the extraction time of phenolic compounds from strawberries with Young Researcher Vol. 11 No. 4 OctNov - Dec 2022 Dr. Kumar Gourav 117 other extraction methods such as solid– liquid, subcritical water, and microwave-assisted method The results confirmed that UAE was the most effective method. Techniques of Isolation and Purification of Bioactive Molecules from Plants: Purification and isolation of bioactive compounds from plants is a technique that has undergone new development in recent years ,This modern technique offers the ability to parallel the development and availability of many advanced bioassays on the one hand, and provided precise techniques of isolation, separation, and purification on the other. The goal when searching for bioactive compounds is to find an appropriate method that can screen the source material for bioactivity such as antioxidant, antibacterial, or cytotoxicity, combined with simplicity, specificity, and speed . In vitro methods are usually more desirable than in vivo assays because animal experiments are expensive, take more time, and are prone to ethical controversies. There are some factors that make it impossible to find final procedures or protocols to isolate and characterize certain bioactive molecules. This could be due to different parts (tissues) in a plant, many of which will produce quite different compounds, in addition to the diverse chemical structures and physicochemical properties of the bioactive phytochemicals ,Both the selection and the collection of plant materials are considered primary steps to isolate and characterize a bioactive phytochemical. The next step involves a retrieval of ethno-botanical information to discern possible bioactive molecules. Extracts can then be made with various solvents to isolate and purify the active compounds that are responsible for the bioactivity. Column chromatographic techniques can be used for the isolation and purification of the bioactive compounds. Developed instruments such as High-Pressure Liquid Chromatography (HPLC) accelerate the process of purification of the bioactive molecule. Different varieties of spectroscopic techniques like UVvisible, Infrared (IR), Nuclear Magnetic Resonance (NMR), and mass spectroscopy can identify the purified compounds. Purification of the Bioactive Molecule: Many bioactive molecules have been isolated and purified by using paper thin-layer and column chromatographic methods. Column chromatography and thin-layer chromatography (TLC) are still mostly used due to their convenience, economy, and availability in various stationary phases. Silica, alumina, cellulose, and polyamide exhibit the most value for separating the phytochemicals. Plant materials include high amounts of Young Researcher Vol. 11 No. 4 OctNov - Dec 2022 Dr. Kumar Gourav 118 complex phytochemicals, which make a good separation difficult. Therefore, increasing polarity using multiple mobile phases is useful for highly valued separations. Thin-layer chromatography has always been used to analyse the fractions of compounds by column chromatography. Silica gel column chromatography and thin-layer chromatography (TLC) have been used for separation of bioactive molecules with some analytical tools. Structural Clarification of the Bioactive Molecules: Determination of the structure of certain molecules uses data from a wide range of spectroscopic techniques such as UV-visible, Infrared (IR), Nuclear Magnetic Resonance (NMR), and mass spectroscopy. The basic principle of spectroscopy is passing electromagnetic radiation through an organic molecule that absorbs some of the radiation, but not all. By measuring the amount of absorption of electromagnetic radiation, a spectrum can be produced. The spectra are specific to certain bonds in a molecule. Depending on these spectra, the structure of the organic molecule can be identified. Scientists mainly use spectra produced from either three or four regions—Ultraviolet (UV), Visible, Infrared (IR), radio frequency, and electron beam for structural clarification. UV-Visible Spectroscopy: UV-visible spectroscopy can be performed for qualitative analysis and for identification of certain classes of compounds in both pure and biological mixtures. Preferentially, UV-visible spectroscopy can be used for quantitative analysis because aromatic molecules are powerful chromophores in the UV range. Natural compounds can be determined by using UV-visible spectroscopy. Phenolic compounds including anthocyanins, tannins, polymer dyes, and phenols form complexes with iron that have been detected by the ultraviolet/visible (UVVis) spectroscopy. Moreover, spectroscopic UV-Vi’s techniques were found to be less selective and give information on the composition of the total polyphenol content. The UV-Vis spectroscopy was used to determine the total phenolic extract (280 nm), flavones (320 nm), phenolic acids (360 nm), and the total anthocyanins (520 nm). This technique is not timeconsuming, and presents reduced cost compared to other techniques. Infrared Spectroscopy: Some of the frequencies will be absorbed when infrared light passes through a sample of an organic compound; however, some frequencies will be transmitted through the sample without any absorption occurring. Infrared absorption is related to the vibrational changes that happen inside a molecule when it is exposed to infrared radiation. Therefore, infrared spectroscopy can essentially be described as a vibrational spectroscopy. Young Researcher Vol. 11 No. 4 OctNov - Dec 2022 Dr. Kumar Gourav 119 Different bonds (C–C, C=C, C≡C, C–O, C=O, O–H, and N–H) have diverse vibrational frequencies. If these kinds of bonds are present in an organic molecule, they can be identified by detecting the characteristic frequency absorption band in the infrared spectrum [35]. Fourier Transform Infrared Spectroscopy (FTIR) is a highresolution analytical tool to identify the chemical constituents and elucidate the structural compounds. FTIR offers a rapid and non-destructive investigation to fingerprint herbal extracts or powders. Nuclear Magnetic Resonance Spectroscopy (NMR): NMR is primarily related to the magnetic properties of certain atomic nuclei, notably the nucleus of the hydrogen atom, the proton, the carbon, and an isotope of carbon. NMR spectroscopy has enabled many researchers to study molecules by recording the differences between the various magnetic nuclei and thereby giving a clear picture of what the positions of these nuclei are in the molecule. Moreover, it will demonstrate which atoms are present in neighbouring groups. Ultimately, it can conclude how many atoms are present in each of these environments [33]. Several attempts have been made in the past by using preparative or semi preparative thin-layer chromatography, liquid chromatography, and column chromatography to isolate individual phenols, the structures of which are determined subsequently by NMR offline [4]. Mass Spectrometry for Chemical Compounds Identification: Organic molecules are bombarded with either electrons or lasers in mass spectrometry and thereby converted to charged ions, which are highly energetic. A mass spectrum is a plot of the relative abundance of a fragmented ion against the ratio of mass/charge of these ions. Using mass spectrometry, relative molecular mass (molecular weight) can be determined with high accuracy and an exact molecular formula can be determined with a knowledge of places where the molecule has been fragmented [4]. In previous work, bioactive molecules from pith were isolated and purified by bioactivityguided solvent extraction, column chromatography, and HPLC [4]. The techniques of UV-visible, IR, NMR, and mass spectroscopy were employed to characterize the structure of the bioactive molecule. Furthermore, molecules may be hydrolysed and their derivatives characterized. Mass spectrometry provides abundant information for the structural elucidation of the compounds when tandem mass spectrometry (MS) is applied. Therefore, the combination of HPLC and MS facilitates rapid and accurate identification of chemical compounds in medicinal herbs, Young Researcher Vol. 11 No. 4 OctNov - Dec 2022 Dr. Kumar Gourav 120 especially when a pure standard is unavailable [37,38,39,40]. Recently, LC/MS has been extensively used for the analysis of phenolic compounds. Electrospray ionization (ESI) is a preferred source due to its high ionization efficiency for phenolic compounds. Lipid Oxidation: Lipid oxidation can occur during the processing, shipping, and storing of many foods. Lipids (such as triglycerides, sterols, and phospholipids) readily become oxidized with exposure to an oxidative environment [41]. Lipid molecules, especially those carrying polyunsaturated double bonds (i.e., linolenic acids), readily undergo oxidation within foods. Oxidatively stable oil with high melting temperature is necessary for solid fat application, and thus, highly saturated seed oil (palmitic acid and stearic acid) would be suitable for this end use [2]. Soybeans provide 56% of the world’s oilseed production. However, the percentage of saturated oil is very low in seed plants (about 10%), if compared to unsaturated oil (about 90%) [3]. Palmitic acid improves the oxidative stability of soybean oil, and can also be used to produce transfat-free shortening, margarine, and cosmetic products. However, this saturated short-chain fatty acid is undesirable for nutrition because its consumption results in an unfavorable lipoprotein profile in blood serum [4]. Stearic acid does not exhibit these cholesterolemic effects on human health [5]. Stearic acid is less likely to be incorporated into cholesterol esters and has a neutral effect on the concentration of blood serum LDL cholesterol .Extensive research has been performed in order to increase stearic acid content oil production in the most widely consumed legume crop in the world, soybeans. By employing induced mutagenesis, seed stearic acid content was increased by up to 7 times . Lipid oxidation in food systems can be caused by oxygen free radicals or reactive oxygen species. Free radicals are molecules with one or more unpaired electrons that work independently to cause oxidation [49]. Reactive oxygen species are a perfect example of oxygen free radicals. Reactive oxygen species do not solely contain free radical molecules, but also some non-free radicals that can influence lipid oxidation. Examples of non-free radical reactive oxygen species are hydrogen peroxide (H2O2), hydrochloric acid (HCl), ozone (O3), and molecular oxygen (O2) [42]. Molecular oxygen can react with linoleic acid about 1450 times faster than triplet oxygen. One of the major causes of oil rancidity is molecular oxygen. Lipid oxidation caused by the chain reaction of free radicals can be illustrated in three stages: initiation, propagation, and termination: Young Researcher Vol. 11 No. 4 OctNov - Dec 2022 Dr. Kumar Gourav 121 (1) Initiation: RH + initiator → R ROOH + initiator → ROO• (2) Propagation: R + O2 → ROO ROO + RH → ROOH + R• (3) Termination: R + R → R-R ROO• + R → ROOH The processes above occur in response to several physical or chemical factors including heating, radiation, temperature, metal ion catalysts, reactive oxygen species, and photosensitizers such as chlorophyll. The initiation step, shown in Equation (1), often happens at either an allytic methylene group of an unsaturated fatty acid (RH) or a lipid-hydroperoxide (ROOH). Next, the generated free radical (R•) reacts with oxygen to form a peroxy radical (ROO•). This product can directly react with another lipid molecule to produce a lipid hydroperoxide (ROOH), and thus a lipid free radical (R•). This causes continuously cascading chain reactions to occur until the free radicals are neutralized by other free radicals. This whole stage is shown in Equation (2). In the termination step, there are two radicals that have converted into nonfree radical products, and thus will stop the cascade mode of the chain reaction according to Equation (3). Moreover, the reaction chain can also be terminated by some antioxidants or free radical scavengers. Metal ions, especially those of iron and copper, effectively catalyze these reactions [50]. Lipoxygenases (EC 1.13.11) can also act, causing oxidation to produce the peroxides in food materials that contain lipids. Hydrogen peroxide is one of the primary products of the oxidation, and it is very unstable and easily converts into secondary products. The final product of oxidation may include different chemical groups such as aldehydes, ketones, alcohols, acids, or hydrocarbons. These kinds of compounds can have a negative effect on the appearance, quality, and edibility of a food product by changing the texture, colour, flavour, and safety of foods, or also by producing unacceptable off Odors or off tastes, even negatively affecting the nutritional value [50]. Plants as a Source of Antioxidants: Antioxidants can be defined as bioactive compounds that inhibit or delay the oxidation of molecules [2]. Antioxidants are categorized as natural or synthetic antioxidants. Some synthetic antioxidants commonly used are: BHT, BHA, propyl gallate, and tertbutyllhydroquinine. Many scientists have concerns about safety because synthetic antioxidants have recently been shown to cause health problems such as liver damage, due to their Young Researcher Vol. 11 No. 4 OctNov - Dec 2022 Dr. Kumar Gourav 128 Eleven Algerian medicinal plants have been measured for phenolic compound content and antioxidant activity using the ABTS method. The tested plants showed antioxidant activity. Artemisia campestris L. had better antioxidant activity than caffeic acid and tocopherol. Moreover, HPLC analyses exhibited a good correlation between the antioxidant activity and hydroxycinnamic derivative content. Evaluation of Vitex negundo Linn seed antioxidant activity using different methods such as superoxide, hydroxyl, and DPPH scavenging activity has been carried out [87]. The highest antioxidant activity was in both raw and dry heated seed extracts, while lower antioxidant activity was observed in the hydrothermally processed samples. Characterization of Antioxidants from Other Eudicots: The nitric oxide and superoxide scavenging activity of green tea have been studied by Nakagawa and Yokozawa , who concluded that certain tannins had the ability to exhibit excellent antioxidant activity. Zin et al. estimated the antioxidant activity of the extracts from various parts of Mengkudu (Morinda citrifolia L.), including the leaves, fruits, and roots, using different solvents such as methanol and ethyl acetate [89]. Ferric thiocyanate and thiobarbituric acid were used as models to observe and evaluate the antioxidant activity. The results exhibited a higher antioxidant activity in the methanol extract of Mengkudu root, although it was not significantly different from tocopherol and BHT extracts. The methanol extracts of the fruits and leaves showed unassuming activity. According to these scientists, the antioxidant activity in the roots resulted from polar and non-polar compounds, but the antioxidant activity in leaves and fruits was only due to nonpolar compounds. Increase of the antioxidant activity of fennel (Foeniculum vulgare) seed extracts in vitro has been shown to be proportional to the increase in the concentration of extract [89]. Nine other extracts of Bolivian plants have been measured for radical scavenging and antioxidant activity using the DPPH and β carotene bleaching methods . It was found that the ethyl acetate fractions had higher radical scavenging and antioxidant activity compared to the other extracts. It has been reported that the bioactive compounds of Rhodiola rosea extracted in methanol showed a significant yield of phenolics, about (153 ± 2 mg/g) . Wangensteen et al. investigated the antioxidant activity of Ss globosus using DPPH scavenging and inhibition of lipoxygenase . Coriander had a high capacity to inhibit oxidation. There was also a positive correlation between total phenolics and antioxidant activity. Moreover, it was observed that the leaves of the coriander had higher antioxidant activity than the seeds [91]. Young Researcher Vol. 11 No. 4 OctNov - Dec 2022 Dr. Kumar Gourav 129 Antioxidant activity of Phyllanthus niruri was estimated using methanol and water as a solvent. The extracts of leaves and fruits exhibited high antioxidant activity by using the inhibition of lipid peroxidation and DPPH scavenging . The results also noticed a higher superoxide scavenging activity in the aqueous extract compared to the methanol extract. Moreover, the antioxidant and free radical scavenging activity of Phyllantus species from India in an aqueous extract has been also evaluated . The antioxidant activity was estimated using DPPH, β carotene, superoxide, nitric oxide scavenging, and reducing power methods. The extract of Coleus aromaticus exhibited a moderate inhibition on DPPH and nitric oxide scavenging activity. Panax exhibited strong iron chelating and weak superoxide scavenging. Ajila et al. carried out bioactive compounds and antioxidant activity of mango peel extract . The results showed a higher concentration of anthocyanins and carotenoids in the ripe peel compared to the raw peel, while the raw peel exhibited higher polyphenol content. The range of IC50 values of lipid peroxidation and DPPH were 1.39–5.24 μg of gallic acid equivalent. Chen and Yen investigated the antioxidant activity and free radical scavenging capacity of dried guava leaves and fruit [94]. The results confirmed that guava leaf and guava tea extracts had the ability to inhibit oxidation by 94–96% at a concentration of 100 μg/mL. Fruit extracts exhibited less activity compared to leaf extracts, while the scavenging effect increased with an increase in the concentration. Also, there was a correlation between antioxidant activity and phenolic compounds. Dastmalchi et al. investigated the chemical composition and antioxidant activity of watersoluble Moldavian balm (Dracocephalum moldavica) in vitro by using DPPH, ABTS, and superoxide activity [95]. The finding confirmed that polar compounds such as caffeic acid and rosmaric acid were responsible for the antioxidant activity observed. Mulberry leaves were investigated to determine the antioxidant activity using different solvents [95]. The procedure used DPPH and inhibition of lipid peroxidation methods to evaluate its activity. The results showed that the methanolic extract exhibited the highest yield of total phenolics, and it was the most essential antioxidant in all the methods used. The antioxidant activity of kale (Brassica obraceae L.) has been screened after removing a fat fraction from the samples [96]. The extraction process used methanol to investigate its antioxidant activity while using DPPH scavenging activity as tested method. The works successfully isolated nine phenolic acids using HPLC and MS, and confirmed that the total phenolic Young Researcher Vol. 11 No. 4 OctNov - Dec 2022 Dr. Kumar Gourav 130 content was correlated with DPPH scavenging activity. In another study, ethanol has been used to estimate the antioxidant activity of sun-dried cashew nuts (Anacardium occidentale L.) skin [97]. First, bioactive compounds were extracted with a protocol including lipid peroxidation, ABTS, and DPPH methods to measure the capability to inhibit oxidation. The results found that epicatechin was the major polyphenol in the extract, which was responsible for antioxidant activity. Kaviarasan et al. measured the antioxidant and antiradical activity of fenugreek (Trigonella foenum ssp. graecum) seeds in vitro; the results showed that there was a positive relationship between the antiradical activity and phenolic compound content in the extract. Hexane and methanol were used to extract the bioactive compounds and measured the antioxidant activity of Pueraria tuberosa by using ABTS, lipid peroxidation, and superoxide and hydroxyl scavenging activity. An independent study has shown an inhibition of the lipid peroxidation. The rhizome of the lotus (Nelumbo nucifera Gaertn.) has been measured for its antioxidant activity in various solvent extracts using β Carotene bleaching and DPPH methods. Methanol extraction had a higher DPPH scavenging activity than acetone. Helichrysum pedunculatum has been tested to determine the antioxidant activity, and total phenolic and flavonoid content. The results demonstrated that whenever the amount of phenolic content and flavonoid content was increased, higher antioxidant activity was obtained. MeotDuros and Magn screened the leaves of Crithmum maritmum to show if there was any correlation between the antioxidant activity and phenolic content and found a significant correlation between antioxidant activity and phenolic content when methanol was used as the solvent. Another dicot, Tricholepis glaberrima L. (Asteraceae), has been investigated for antioxidant activity using different kinds of extracts [101]. Higher antioxidant activity was found by methanol, and a lower antioxidant activity in both chloroform and aqueous extracts. Sakat et al. investigated Oxalis corniculata L. to measure the antioxidant and anti-inflammatory activity employing methanol as a solvent. The IC 50 values of DPPH and nitric acid were about 93 and 73.07 μg/mL, respectively. Jain et al. studied Taberna Montana divaricate L. to determine the phytochemical and free radical scavenging activities in vitro. The results indicated that the antioxidant activity was the same in both ethanol and water extracts, but less in petroleum ether. Young Researcher Vol. 11 No. 4 OctNov - Dec 2022 Dr. Kumar Gourav 131 It has been reported that Asclepiadaceae and Pteroptochidae presented high antioxidant activity, with the presence of a strong correlation between antioxidant activity and phenolic content . Laitonjam and Kongbrailatpam studied the chemical composition and antioxidant activities of Smilax lanceafolia by isolating the flavonol glycoside and steroidal saponin, which showed high antioxidant activity . Spinach (Spinacea olerace L.) is among the most popular vegetables in the world. It was domesticated and first cultivated in West Asia. According to analytical chemistry, spinach is a source of violaxanthin and neoxanthin antioxidants that cannot be commercially produced. Although they may be present, pigments such as carotenoids can be masked by chlorophyll in greenish vegetables such as spinach [106]. B-carotene, lutein, violaxanthin, and neoxanthin are the major carotenoids in raw spinach. Pumpkins belong to the family Cucurbitaceae. This family is classified depending on the texture and shape of stems, such as in Cucurbita pepo, Cucurbita moschata, Cucurbita maxima, and Cucurbita mixta. Nowadays, the market offers a wide variety of vegetables, with pumpkin being one of them because of its many applications for nutrition or decoration Plants Vitamins and Phenolic Compounds as Antioxidants: Phenolic Compounds: Phenols and Phenolic Acid: Phenolic acids contain carboxylic acid in the chemical composition. Hydroxycinnamic and hydroxybenzoic acids are both main pillars of phenolic acids, according to Figure 1A. Moreover, scientists have noted that p-coumaric, caffeic, ferulic, and sinapic acids are main components of the hydroxycinnamic acids (Figure 1A). Results and Discussion: Young Researcher Vol. 11 No. 4 OctNov - Dec 2022 Dr. Kumar Gourav 132 Hygrophila spinosa Andres leaves showed significant antibacterial activity when collected between September to October, with less activity seen during other months. Artemisia pallens L. has been studied for its antimicrobial activity against seven species of bacteria . The results found that Bacillus cereus was more sensitive to A. pallens extracts. Also, a methanolic extract exhibited higher antibacterial activity than the other solvents used. Akroum indicated the antimicrobial activity of some Algerian plants . The results expressed higher antibacterial activity in methanolic extracts of Linum capitatum, Camellia sinensis, Allium schoenoprasum, Vicia faba, Citrus paradise, Lippia citriodora, Vaccinium macrocarpon, and Punica granatum. Bajpai et al. screened the antibacterial activity of Pongamia pinnata leaves by using methanol and ethyl acetate extracts to confirm its ability against certain pathogenic bacteria . The results exhibited significant inhibition compared to streptomycin. It has been demonstrated that Memecylon edule has higher antibacterial activity in chloroform extracts compared to other extracts Gram-negative bacteria were more susceptible to the crude extracts compared to Gram-positive bacteria. Bansal et al. studied plants found in arid zones in order to determine the antibacterial efficiency. An ethanolic extract of Tinospora cordifolia L. inhibited Bacillus cereus and Staphylococcus aureus. Kumar et al. reported Andrographis serpyllifolia L. to have significant antimicrobial activity against tested organisms in methanol extracts of both aerial parts and root [134]. Memecylon malabaricum, Cochlospermum religiosum, and Andrographis serpyllifolia have been rested for their possible antimicrobial activity . Moderate activity against both Grampositive and Gram-negative bacteria was observed. The antimicrobial activity of an ethanolic extract of Anethum graveolens was better than the aqueous extract. Khanahmadi et al. found a higher antibacterial activity against Gram-positive bacteria compared to Gram-negative bacteria when an ethanolic extract of Smyrnium cordifolium Boiss was used . Koperuncholan et al. studied some medicinal plants of the south eastern slopes of the Western Ghats [138]. Gram-positive bacteria were more sensitive than Gram-negative bacteria to the plant extracts. Niranjan et al. screened Schrebera swietenioides Roxb to measure the effectiveness against human pathogenic bacteria [139]. Water and methanol extracts were most effective to prohibit growth of all the harmful bacteria tested. Different studies have isolated tannins and saponins from some Indian Young Researcher Vol. 11 No. 4 OctNov - Dec 2022 Dr. Kumar Gourav 133 medicinal plants, testing the antibacterial activity against Klebsiella pneumoniae . Ethanol extracts of Tinospora cordifolia strongly inhibited Bacillus cereus and Staphylococcus aureus. Also, significant antibacterial activity from ethanolic extracts of Coleus aromaticus L. has been found. The most effective range of inhibition was at concentrations of 25–39 μg/mL. Vinothkumar et al. evaluated a Andrographis paniculata L. leaf extract’s ability to inhibit the growth of Gram-positive and Gram-negative bacteria. The results found that aqueous extracts inhibited harmful microbes [134]. A positive effect of pumpkin has been observed by investigating its antimicrobial activity against Staphylococcus aureus, Bacillus subtillus, Escherichia coli, and Pseudomonas. aeruginosa. Three different solvents were used to prepare the extracts: water, chloroform, and alcohol. The results showed that the alcohol extract was more powerful than both water and chloroform extracts. Staphylococcus aureus was sensitive to all extracts. Recently, the novel antimicrobial activity of ultrasonicated spinach leaf extracts using random amplification of polymorphic DNA (RAPD) markers and electron microscopy against both Grampositive and Gram-negative bacteria has been revealed [134]. RAPD is an emerging technique used for diagnostic mutation detection within a genome. The range of the minimum inhibitory concentrations (MICs) of the extracted leaf spinach antimicrobial substances against Escherichia coli and Staphylococcus aureus was observed between 60 and 100 mg/mL. The optimal extraction conditions were at 45 °C, ultrasound power of 44%, and an extraction time of 23 minutes. The study showed that the treated bacterial cells appeared to be damaged by a reduction in cell number. In fact, it was inferred that spinach leaf extracts exert bactericidal activity by inducing mutations in DNA and causing cell wall disruptions. Conclusions: In summary, plant extracts showed strong antioxidant capacity both in vitro and in vivo, and the extracts can be considered a good source of natural antioxidants and antimicrobials. Polyphenol extraction from plants using fast and appropriate techniques is a low-cost method due to the reduction in the amount of solvent used, in addition to avoiding the need for longer extraction times compared to the conventional extraction method. Moreover, natural bioactive compounds have been found to interfere with and prevent all kinds of cancer. Flavonoids have been shown to work as anti-tumor (benign, melanoma) agents involving a free radicals quenching mechanism (i.e., Young Researcher Vol. 11 No. 4 OctNov - Dec 2022 Dr. Kumar Gourav 134 OH, ROO). In fact, many studies have shown that flavonoids play significant multiple roles including mutagenic, cell damage, and carcinogenic, due to their acceleration of different aging factors. In addition to antioxidant activity, the inhibition of cancer development by phenolic compounds relies on a number of basic cellular mechanisms. More comprehensive studies related to these compounds will enhance pharmaceutical exploration in the field of carcinogenic disease prevention. References: 1. Jugde H., Nguy D., Moller I., Cooney J.M., Atkinson R.G. Isolation and characterization of a novel glycosyltransferase that converts phloretin to phlorizin, a potent antioxidant in apple. FEBS J. 2008;275:3804–3814. doi: 10.1111/j.17424658.2008.06526.x. 2. Huang Y.F., Doligez A., FournierLevel A., Le Cunff L., Bertrand Y., Canaguier A., Morel C., Miralles V., Veran F., Souquet 3. J.M., et al. Dissecting genetic architecture of grape proanthocyanidin composition through quantitative trait locus mapping. BMC Plant Biol. 2012;12:30. doi: 10.1186/14712229-12-30. 4. Takos A.M., Ubi B.E., Robinson S.P., Walker A.R. Condensed tannin biosynthesis genes are regulated separately from other flavonoid biosynthesis genes in apple fruit skin. Plant Sci. 2006;170:487–499. doi: 10.1016/j.plantsci.2005.10.001.