Phytobioactive compounds as therapeutic agents for human diseases: A review

Abstract Phytobioactive compounds are plant secondary metabolites and bioactive compounds abundantly present in medicinal plants and have remarkable therapeutic potential. Oxidative stress and antibiotic resistance are major causes of present‐day ailments such as diabetes, atherosclerosis, cardiovascular disorders, cancer, and inflammation. The data for this review were collected from Google Scholar, PubMed, Directory of Open Access Journals (DOAJ), and Science Direct by using keywords: “Medicinal plants, Phytobioactive compounds, Polyphenols, Alkaloids, Carotenoids etc.” Several studies have reported the pharmacological and therapeutic potential of the phytobioactives. Polyphenols, alkaloids, terpenes, and polysaccharides isolated from medicinal plants showed remarkable antioxidant, anticancer, cytotoxic, anti‐inflammatory, cardioprotective, hepatoprotective, immunomodulatory, neuroprotective, and antidiabetic activities. This literature review was planned to provide comprehensive insight into the biopharmacological and therapeutic potential of phytobioactive compounds. The techniques used for the extraction and isolation of phytobioactive compounds, and bioassays required for their biological activities such as antioxidant, antimicrobial, anti‐inflammatory, and cytotoxic activities, have been discussed. Characterization techniques for the structural elucidation of phytobioactive compounds such as HPLC, TLC, FTIR, GC–MS/MS, and NMR have also been discussed. This review concludes that phytobioactive compounds may be used as potential alternative to synthetic compounds as therapeutic agents for the treatment of various diseases.


| INTRODUC TI ON
According to the Worlds Health Organization (WHO), any plants that have therapeutic potential or which are precursors of pharmaceutical drugs are referred to as medicinal plants .
Since the emergence of life, medicinal agents have been used for the treatment of ailments . Most of the traditional medicines are obtained from plants . In spite of huge progress in pharmaceutical industry, plants are major raw materials for synthetic drugs (Shang et al., 2021). Clinical advancements have increased the value of medicinal plants by determining active principles present in them (Noreen, Hussain, & Shahid, 2020).
Nowadays, the world is progressively turning toward effective herbal medicines (Aanouz et al., 2021). Synthetic drugs have not only side effects and are inadequate but are also expensive and unaffordable in developing nations .
In the last few decades, natural therapeutic compounds are explored by scientists from all across the world (Jilani et al., 2016).
Ethnomedicinal survey of plants sets the basis for discovering new medicinal compounds (Khalid et al., 2016). With the passage of time, a large amount of evidence has been gathered that demonstrates the use of plants in the field of pharmacology (Radha et al., 2021).
Since the appearance of life on Earth, plants are a major source of food, clothing, shelter, and medicine (Shahzadi et al., 2018;Yousaf et al., 2017). Due to their nutritive and medicinal potential, plants have been playing a vital role in human life (Yazarlu et al., 2021).
From the start of life, human civilization has been using plants, mainly as medicines, and most civilizations still use them (Bano et al., 2017). Currently, 25% of pharmaceutical drugs are derived from plants (Okpuzor et al., 2021). In developing countries, about 70%-95% population uses medicinal plants to treat health issues (Liaqat et al., 2019).
As stated by WHO, 80% of the world population uses phytochemical agents for the treatment of diseases. Bioactive compounds of plants have remarkable potential to treat diseases . Secondary metabolites, such as phenolics, flavonoids, alkaloids, saponins, and terpenoids produced by plants, are the constituents of plant's defense system but they have remarkable potential to treat various diseases (Aanouz et al., 2021;Anjum et al., 2020). Plant extracts have been gaining huge attention to control emerging antibacterial resistance (Abbas et al., 2020;Safdar et al., 2021). Crude plant extracts have remarkable antibacterial potential (Romano et al., 2021). Purified plant bioactive compounds are a major research topic among researchers (Lim et al., 2021;Shahid et al., 2021).
Phytobioactive compounds play a significant role in the adaptation of plants to their surrounding environment but are also a major source for the pharmaceutical industry (Merrouni & Elachouri, 2021).
Phytobioactives are also known as plant secondary metabolites and bioactive compounds . They are present ubiquitously in the plant kingdom and are considered nonnutritional but vital components for the maintenance of human health (Nafees et al., 2017). Phytobioactive compounds are not required for the basic metabolism of plants, are synthesized apart from the primary biosynthetic pathway, and are synthesized from the metabolites of biosynthetic routes . Several of them not only play important roles for the plants such as protecting, attracting, signaling, and adapting to their environment but also represent the main source of pharmaceuticals (Jahan & Shahid, 2019;Khairan et al., 2021). The foundation of modern therapeutics is based on the use of plants and their extract in preparing herbal drugs, enabling man in establishing an empirical medicinal system (Gumisiriza et al., 2021). Thus, screening of a number of plant species is being carried out for these herbal compounds (Waseem et al., 2021).

| HIS TORY
The history of medicinal plants is as old as humankind. In the beginning, people exploited herbs for nutrition but following the discovery of medicinal attributes, medicinal plants became a beneficial source for the improvement of health among human communities . Herbal products were introduced by the Romanian pharmacopeia during the 19th century by establishing the first institute of medicinal herbs in Cluj city in 1904 (Vinatoru, 2001).
The herbal plants' usage in ancient times truly illustrates the history of bioactive molecules (Swargiary et al., 2021). People were unaware of plants' bioactives but the usage of these molecules is sufficiently diverse in various prospects (Zeidali et al., 2021). Typically, secondary metabolites are the bioactive compounds produced by plants.
The use of different parts of plants (bark or flowers, leaves, berries, roots, and seeds) for healthcare in treating various diseases is called herbal medicine .
During the late 19th century, researchers began to isolate, purify, and identify phytobioactive compounds from plants, and their efforts led them to discover vital drugs from plants that are the base of modern medicine (Tanweer et al., 2018). In this view, for the preparation of semi-synthetic drugs, bioactive compounds isolated from medicinal plants have been modified to make them effective.
In 1953, aspirin was synthesized through the structural modification of an active constituent of a number of medicinal plants, which is salicylic acid, having pain-relieving effects .
in English were only considered for this review study. Publications that extensively investigated the biological activities and therapeutic effects of bioactive compounds present in plants were included. Moreover, the published research conducted on animal models for in vitro and in vivo experimentations were also included.

| PL ANT-DERIVED B IOAC TIVE COMP OUNDS AND THEIR THER APEUTI C P OTENTIAL
Phytobioactive compounds are the compounds present in plants that provide protection against various diseases but are not of dietary importance. These compounds obtained from plants having distinct structural and functional properties are referred to as phytochemicals or secondary metabolites of plants. These are found abundantly in vegetables, grains, fruits, seeds, and nuts. These metabolites are usually classified into six large-molecule families: phenolics, terpenes, alkaloids, saponins, glycosides, and polysaccharides based on their biosynthetic pathways. The schematic classification of phytobioactive compounds is shown in Figure 1, whereas the overview of the therapeutic potential of phytobioactive compounds is presented in Figure 2. Phytobioactive compounds have been extensively used in traditional medicines for the treatment of various diseases including type 2 diabetes. Higher antidiabetic potential of these phytobioactives has been shown in various animal models, and plant bioactive-based medicines currently are in great demand in the market due to their multiple efficacies and higher availability.
The possible mechanism of molecular action of phytobioactives for the treatment of type 2 diabetes is shown in Figure 3. There are many complications associated with type 2 diabetes, and few of the regularly used phytobioactive compounds through food have shown antidiabetic potential in a variety of ways including the reduction in drug loads during treatment (Ganesan et al., 2017). A summary of the biological/pharmacological properties of phytobioactive compounds and their action mechanism is given in Table 1.

| Polyphenols
Polyphenols are plants' secondary metabolites or the plants' nonnutritional natural products found in fruits, seeds, and vegetables.
Polyphenols are common in the plant kingdom and are a large family of bioactive metabolites derived from secondary metabolism. Most of the polyphenols are derived through phenylpropanoid pathway from l-phenylalanine. These are characterized by the presence of one or more phenolic groups that show highly diverse structures, of which, flavonoids, phenolic acids, lignans, stilbenes, tannins, and coumarins are the major structural type of polyphenols (Quideau et al., 2011). Some of the polyphenols are responsible for the color, aroma, and antioxidant properties of nuts, vegetables, fruits, and seeds that we consume in our daily life. The importance of polyphenols is increasing particularly because of their health benefits.
Their antioxidant role in preventing and treating various diseases is increasing particularly against oxidative stress, cancer, cardiovascular, inflammatory, neurodegenerative, and age-related degenerative diseases (Hano & Tungmunnithum, 2020 of uses as pharmaceuticals, food supplements, and cosmetic additives. Identification of more than 8000 polyphenols has been carried out from plants, and several hundred have been characterized from grains, vegetables, and fruits (Shen et al., 2017). Various classes of polyphenols are shown in Figure 1 and are described below.
Polyphenols play an important role to prevent carcinogenesis leading to the decreased incidence of some cancers. They have widespread medicinal applications with reduced toxicity and hence, can be used as chemopreventive agents. Any of the endogenous or exogenous carcinogens, tumor promoters, and inflammatory cytokines can take part in the activation of carcinogenesis through the regulation of transcription factors, AP-1 and NF-kB; proapoptotic proteins, PARP and caspases; the protein kinases, MAPK, c-Jun N-terminal kinase (JNK), and IκB kinase (IKK); growth factor signaling pathways; and the cell cycle proteins, CDK and cyclins (Shen et al., 2017). During the initiation, carcinogens cause damage by interacting with cellular DNA, therefore, blocking the damage caused by genotoxic chemicals can be an effective approach for the prevention of cancer that might be achieved through the elimination of ROS and induction of phase II conjugating enzymes. In the next stage, inhibition of cell proliferation is a useful strategy, such as through the modulation of apoptosis and cell cycle arrest. In progression step, prevention of malignant cells' progression to metastasis and invasiveness and/or the interruption of angiogenesis are particularly important (Ramos, 2008;Shen et al., 2017). Evidence indicates that polyphenols of dietary importance play a protective role in a multistep process of carcinogenesis through phase I and phase II enzymes, cell proliferation, cell cycle progression, DNA repair, metastasis, angiogenesis, and apoptosis (Shen et al., 2017). The possible action mechanisms through which the carcinogenic processes are inhibited by polyphenols are illustrated in Figure 4.
Polyphenols scavenge the excess free radicals (ROS) inhibiting the oxidative stress through interaction with protein kinase-like endoplasmic reticulum kinase (PERK) dependent on RNA, phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC), and mitogenactivated protein kinases (MAPKs) as these kinases upregulate F I G U R E 2 Overview of the therapeutic potential of phytobioactive compounds.
Nrf2 through phosphorylation. Polyphenols have the capability to suppress the activation of NF-κB and inhibit the expression of COX2, interleukin (IL)-1β, and inducible nitric oxide synthase (iNOS), hence decreasing the adoptive cellular responses (Shen et al., 2017).
Polyphenols like curcumin, caffeic acid, quercetin, EGCG, and resveratrol have been reported to inhibit oxidative stress. EGCG enhances the antioxidant capacity of cells through MAPK protein activation.
Quercetin increases the antioxidant enzymes expression such as γglutamylcysteine synthetase (γGCS), glutathione peroxidase (GPX), and superoxide dismutase (SOD), upregulates glutathione reductase (GSH) level, enhances the stabilization of Nrf2, and promotes the transcription activity mediated by Nrf2. Quercetin also inhibits the activation of NF-κB. Resveratrol inhibits oxidative stress through the upregulation of γGCS and hemoxygenase-1 (HO-1) expression and increases the Nrf2 transcriptional activity (Csiszar et al., 2011;Shen et al., 2017). EGCG reduces the cyclin D1 expression, induces the expression of p27Kip1 and p21waf1/Cip1 genes, and inhibits CDKs 2 and 4 thereby blocking the progression of cell cycle MCF-7 at G1 phase. Many polyphenols such as curcumin, ellagic acid, apigenin, resveratrol, EGCG, and quercetin induce apoptosis to inhibit carcinogenesis (Shen et al., 2017). Quercetin, theaflavin, catechin, curcumin, and resveratrol through AP-1 inhibition can inhibit tumor metastasis. Polyphenols including ellagic acid, genistein, quercetin, resveratrol, curcumin, and ECGC can act as inhibitors of NF-kB (Aggarwal & Shishodia, 2006). ther subdivided into family in order to hierarchize their differences (Naz et al., 2016). The increasing complexity of basic skeleton of the members characterized the first five classes while the sixth class includes a separate group of compounds called hybrid phenolics (Sharif et al., 2018).

| Phenolic acids
Phenolic compounds have important properties, including antimicrobial activity, stabilization of ascorbic acid, inhibition of lipid peroxidation, and carcinogenesis (Sharif et al., 2018). Flavonoids are found in garlic and are known to be helpful in reducing cholesterol level, coronary thrombosis, atherosclerosis, and a variety of other life-threatening diseases (Ali et al., 2021). The radical-scavenging activity of flavonoids is ascribed to their capacity of donating hydrogen. Flavonoids contain the phenolic groups which act as a source of hydrogen atoms readily available (Tanweer et al., 2018). Flavonoids have the potential to inhibit fluids that caused diarrhea by targeting the intestinal cystic fibrosis transmembrane regulator (Ahmad et al., 2021). The therapeutic functions of flavonoids comprise protection against allergies, platelet aggregation, free radicals, inflammation, ulcers, microbes, viruses, and tumors (Abbas et al., 2012).
Due to physiological, pharmaceutical, and ecological roles, phenolic compounds have been explored and manipulated extensively .
Bioavailability of phenolic compounds is limited due to their less water solubility and instability at low pH values (Malode et al., 2021). The bioavailability of polyphenols can be increased by using nanoparticles (Irshad et al., 2020). One of the flavonols called quercetin belongs to flavonoids ubiquitously present in F I G U R E 3 Schematic mechanism of phytobioactive compounds in treating type 2 diabetes. TA B L E 1 Biological/pharmacological properties of phytobioactive compounds and their mechanism of action.   (Afzal, Shahid, & Jamil, 2014). Quercetin has antioxidant activity due to its potential to scavenge free radicals, metal cation chelating ability, and hydrogen atoms or electrons donating ability (Irshad et al., 2017). Quercetin and its glycosidic metabolites modulate the biological processes due to its antioxidant properties and these processes include the reduction of oxidative deoxyribonucleic acid (DNA) damage and cell signaling pathways (Sohaib et al., 2017).
Phenolic compounds have shown significant glucosidase inhibition potential which can be used to treat type 2 diabetes mellitus.
Cholinesterase inhibition potential has been used for the treatment of Alzheimer's disease (Rahman et al., 2021). Inflammation is a natural biochemical reaction of the body in response to infections and injuries caused by physical and chemical stimuli (Soulimani et al., 2021). The immune system responds to harmful stimuli by generating proinflammatory mediators, but moderate production of proinflammatory mediators leads to many lethal diseases. Phenolic compounds work in tandem with nonsteroidal anti-inflammatory drugs (NSAIDs) to inhibit the gene expression or activity of proinflammatory mediators, including cyclooxygenase (COX). Phenolic compounds inhibit the excessive production of proinflammatory cytokine which leads to asthma and cancer (Devi et al., 2015). Phenolic compounds have also been demonstrated to treat skin diseases due to their anti-inflammatory properties (Działo et al., 2016), rheumatoid arthritis (Nguyen et al., 2019), and inflammatory bowel disease (Singh & Easwari, 2021).

| Lignans and stilbenes
Lignans belong to the wide class of phenolic compounds found in higher plants and are derived from dibenzylbutane as well as produced in vivo from human gut microorganisms. These are phytoestrogens present in legumes, cereals, vegetables, and fruits as glycosides and bio-oligomers aglycones. The richest sources of lignans are sesame seeds, flaxseed, and linseeds. The most common lignans are syringaresinol, pinoresinol, sesamolin, sesamol, sesamin, matairesinol, secoisolariciresinol, 7-hydroxymatairesinol, and lariciresinol. Lignans have antifeedant, antiviral, antioxidants, and insecticidal properties (Banwo et al., 2021).
Resveratrol is the most widely studied stilbene known to have cardioprotective and anticarcinogenic activities. Resveratrol is found in berries, grapes, vine, and peanuts. The cardioprotective function of resveratrol stilbene is achieved by the prevention of vascular smooth muscle cell proliferation (Delgado et al., 2019).

| Alkaloids
Plant alkaloids are one of the largest groups of natural entities, representing a diverse group of chemical products. The term alkaloid was introduced for the first time by W. Meisner in the early 19th century to name the natural substances behaving like bases. There is no precise definition of alkaloids, and it is difficult to differentiate alkaloids from other natural metabolites containing nitrogen as an essential component . Alkaloids are defined as nitrogen-containing nonnucleosidic and nonpeptidic compounds.
Alkaloids are organic bases that have nitrogen-containing heterocyclic rings and majority of them possess definite pharmacological activities (Bari et al., 2012).
Alkaloids have a 3000 years golden history in human medicine used as laxative, astringent, and sedatives for snake bites, fever, and insanity. About 5500 alkaloids are known, comprising the largest class of phytobioactive compounds, and are widely distributed (Mehmood et al., 2012). They are known to have therapeutic potential and are used as medicational and recreational drugs. Most of the alkaloids are very toxic and bitter in taste which makes them useful for plants to be used as defensive agents against invertebrate pest attacks, microbial pathogens, and herbivores (Khadka et al., 2021).

Several medicinal plants containing alkaloids have been manipulated
by human beings as pain relievers .
Alkaloids form salts with organic acids and mineral acids. Plants containing berberine alkaloids are known to be used as antiseptics, analgesics, antistomatitis, and sedatives in Chinese folk medicine. In Islamic and Indian folk medicine, such plants are used for eye diseases and bleeding disorders, and as uterine muscle depressants, sedatives, and antiseptics. Both quaternary and their tetra-alkaloids derivatives possess several biological and therapeutic properties, for example, tetrahydropalmatine, jatrorrhizine, and palmatine have been reported for their in vitro antimalarial activity (Adhikari et al., 2021). In China, tetrahydropalmatine has been tabulated to exhibit sedative, hypotensive, and bradycardial activities and is used as an analgesic. About 40% of modern drugs are derived from natural sources and a wider pharmacological potential is exhibited by alkaloids, especially isoquinoline .

| Terpenes
Terpenes, also called terpenoids or isoprenoids, constitute the largest class of natural products with over 55,000 known compounds. Terpenes are widely applied in the industrial sector as spices, fragrances, and flavors and are used in scents, cosmetic products, and as food additives. They are active components of drugs in pharmaceutical industry . Increasing interest in the therapeutic application of alkaloids among these pharmaceuticals, the antimalarial (artemisinin) and anticancer (paclitaxel) drugs are two of the well-known terpene-based drugs (Ashraf et al., 2020).
Monoterpenes in essential oils have the ability to damage microbial cells and inhibit microbial growth (Aslam et al., 2011).
Terpenes have remarkable anti-inflammatory potential, it perform anti-inflammatory activity by inhibiting inflammatory pathways associated with numerous diseases . Terpenes isolated from turmeric species (Curcuma sp.) showed anti-inflammatory properties, both in vitro and in vivo analyses (Tanweer et al., 2018).
Terpenes have enzyme inhibition activities as they have potential to inhibit lipoxygenase and proteinase, principal enzymes of inflammatory pathways . Terpenes inhibit oxidative stress by inhibiting the catalytic activity of enzymes involved in reactive oxygen species generation and by forming metal-ion chelates.
Camphor is a monoterpene widely used in cosmetics, pharmaceutics, and the food industry .

Terpenoids inhibit apoptosis of hepatic cells through the inhibi-
tion of cytosolic release of cytochrome c, reducing the Bax/Bcl-2 ratio, and by the inhibition of I-κB, ERK, and JNK phosphorylation.
They are also involved in the management of disorders caused by obesity, such as insulin resistance, type-2 diabetes, and hyperlipidemia (Chatterjee et al., 2019). Antioxidant properties of terpenoids are also associated with their hepatoprotective activity (Ielciu et al., 2021). Terpenoids are supposed to be a safe and promising agent for the treatment of diabetes (Panigrahy et al., 2021).

| Saponins
Saponins are known as surface-active compounds and are widely distributed in the plant kingdom. Saponins include five-ring triterpene saponins and four-ring triterpene saponins which are reported to have cardioprotective potential through diverse mechanisms involving calcium homeostasis, regulation of energy metabolism, and the inhibition of inflammation and oxidative stress (Chen et al., 2017).
A steroidal saponin called polyphyllin I (PPI) is extracted from Paris polyphylla roots. PPI is anticancer and cardioprotective. Its anticancer activity is through the inhibition of tumor cell growth and proliferation. It activates NF-κB and reduces oxidative stress and inflammation resulting in decreased myocardial death (Huang et al., 2020).

Platycodin D is another saponin found in Platycodon grandiflorus,
which has antioxidant potential and anti-inflammatory and antiatherosclerotic activities (Wang, Che, et al., 2018). Gypenoside A from Gynostemma pentaphyllum possesses anti-inflammatory, antioxidative, and antitumor activities (Chang et al., 2020). Ginsenoside Rb3 is a saponin mainly found in Panax ginseng C. A. Meyer plant which is antiapoptotic inhibiting apoptosis through the activation of NK/ NF-κB signaling pathway (Ma et al., 2014).

| Polysaccharides
Polysaccharides are generally found in vegetables and fruits. Several studies reported the cardioprotective potential of polysaccharides through various mechanisms like anticancer, antioxidative stress, anti-inflammatory, immunomodulatory, and regulating metabolism. The biologically important polysaccharides are gums and fucoidan (Chen et al., 2021). The possible mechanism of polysaccharides action is shown in Figure 6.
Gums are polysaccharides obtained through extraction or natural exudation from different plant parts (Naqvi et al., 2011). Different monosaccharide units are linked together through glycosidic linkages forming the gums Hussain, Bakalis, et al., 2015). On the basis of structure, composition, behavior, and production differences, gums have been classified into subcategories (Munir et al., 2015), broadly, exudates gums (synthesized by plants for protection against microbial attacks or in response to mechanical injury and process is named as gummosis) and nonexudate gums (artificially procured from plant by suitable extraction method). Gums have remarkable applications at commercial scale (Munir et al., 2016).
Plant gums possess several applications in food industry. Their use in food products to improve quality has been reported in the literature (Munir et al., 2017). However, in addition to numerous functionalities, gums have also been known for their health benefits (Ayub et al., 2018). Gums act as dietary fiber. Consumption of dietary fibers contributed to decreased risk of cardiac issues, promotes immunity and satiety, and contributes to the management of body weight (Irshad et al., 2011). Several studies have documented the role of gums as bioactive such as antioxidant and scavenging activity against free radicals which are necessary for decreasing oxidative stress (Ullah et al., 2019).
Guar gum like other natural gums is hydrophilic nontoxic, economical, and easily available. Being biodegradable has several applications in pharmaceutical, textile, and food industries. Due to biodegradation, it is less stable and cannot be used in its natural form. Surface modification via grafting enhances its uses as grafted gum is an important medium for controlled drug release . Crude, purified, and hydrolyzed guar gum characterized through TGA, XRD, FTIR, and SEM demonstrated that hydrolyzed and grafted gums are more crystalline and increase thermal stability Bukhari et al., 2014).

| E X TR AC TI ON OF B I OAC TIVE COMP OUNDS
Considering the variations among phytobioactive compounds and variety of plant species, it is essential to formulate a standard technique for the screening of phytobioactives from plants extract. Different extraction techniques have been used for the selective extraction of bioactive compounds from natural sources (Abbas et al., 2014). Most of the techniques used for bioactive extraction remain almost same through hundreds of years . All these assays have com-

| B I OA SSAYS FOR PHY TOB I OAC TIVE COMP OUNDS
After a plant has been identified as a medicinal plant, the bioactive compounds are detected by using so-called "Bioassays" (Zahid et al., 2016).
There are several procedures available for in vitro and in vivo screening of plant extract. In this process, successive extraction of medicinal plants is carried out using solvents of increasing polarity and tested by a range of bioassays according to the activity of bioactive molecules (Ali et al., 2016).
When the bioactivity has been identified, combination of chromatographic methods has been performed for further separation (Rehman et al., 2013). After obtaining a pure compound, a variety of spectroscopic methods like MS, IR, and NMR are used for the structural elucidation, and X-ray crystallography is used in certain cases (Riaz et al., 2017). After

F I G U R E 6
Possible action mechanism of plant polysaccharides as antioxidant, anti-inflammatory, antidiabetic, and immunomodulatory agents. Plant polysaccharides increase the endogenously produced antioxidant enzymes like catalase and superoxide dismutase levels, whereas significantly reduce ROS-induced free radicals, which are responsible for the destruction of important biomolecules found in body tissues that lead to apoptosis. These polysaccharides are primarily involved in the downregulation of toll-like receptor 4 (TLR-4) and NF-κB, a nuclear transcription factor involved in inflammatory diseases by triggering the inducible NO synthase, TNFα, IL-6, and IL-1β. Antidiabetic activity of plant polysaccharides has been shown through various mechanisms including antioxidative and anti-inflammatory properties by alleviating β-cell dysfunctioning, increasing insulin secretion, and inhibiting the α-amylase and α-glucosidase activity to improve glucose metabolism. Plant polysaccharides exhibit immune-modulatory activity by inducing ROS and cell proliferation and increasing cytokines and macrophages phagocytosis.
toxicity study, clinical trial is the last, but the most important part to determine and compare the efficacy and doses of the new medicinal product to the already available products in the market (Rubab et al., 2016).

| Antioxidant assays
Antioxidants are molecules that have the ability to scavenge free radicals produced during oxidative stress . All organisms have natural defense systems that scavenge free radicals but present-day lifestyle causes overproduction of free radicals which are responsible for oxidative stress. Oxidative stress imposed several deleterious effects such as damaging the cell structures, including proteins, lipids, and DNA (Hina et al., 2017). Many pharmaceutical drugs have been formulated for the treatment of oxidative stress (Iqbal et al., 2017). Synthetic antioxidants have a few limitations as they cause carcinogenicity and are expensive. Natural antioxidants (vitamin C, vitamin E, xanthophylls, tannins, carotenes, and phenolic) have several biological activities (Cheema et al., 2011), which is why scientists have been exploring natural antioxidants for use in medicine and food to replace the synthetic compounds . Bioactive compounds of medicinal plants have antioxidant potential (Irshad et al., 2012) and are being explored by researchers extensively . Different bioassays have been performed to explore the antioxidant properties of phytobioactives . Some bioassays are as follows: 6.1.1 | DPPH assay The radical scavenging DPPH bioassay (2, 2-diphenyl-1-picrylhydrazine) was described by Blois in 1958 and was slightly modified by numerous researchers (Afzal, Shahid, Iqbal, & Hussain, 2014;Afzal, Shahid, & Jamil, 2014). DPPH assay is one of the most commonly used assays to determine the antioxidant activity of plant extract. The stable free radical DPPH reacts with hydrogen-donating compounds. In its reduced form, DPPH is violet in color and turned into pale yellow color upon oxidation by an antioxidant. The activity of antioxidants is calculated by a reduction in optical density. In this method, DPPH solution (0.004%) in methanol is prepared and 10 μL of the sample was mixed with 1 mL of 0.004% DPPH solution (0.004%). Following incubation for 30 minutes, absorbance was measured at 517 nm (Irshad et al., 2017).
where A 1 is the unknown sample absorbance and A 0 is the blank absorbance.

| ABTS assay
The ABTS radical scavenging assay was developed by Rice Evans and Miller in 1994 and then modified by Re in 1999. The modification is based on the production of a radical cation through metmyoglobin activation with H 2 O 2 in the presence of ABTS•+ . This assay is extensively used, generating a blue/green ABTS•+ chromophore via ABTS and potassium persulfate reaction.
ABTS radical scavenging method together with DPPH assay is

| Antimicrobial activity
Antibiotics are used for the treatment of microbial infections and also as chemotherapeutic agents against infectious diseases (Hussain et al., 2010). However, few pathogens become resistant to antibiotics with the passage of time . These drug-resistant pathogens contribute to morbidity, mortality, and increased health issues (Mansoor et al., 2019). Resistance to antibiotics has become a global concern (Anwar et al., 2009). The therapeutic potential of several available antibiotics is being threatened by the appearance of multidrug-resistant pathogens (Shahzadi et al., 2019). The use of crude plant extract and phytobioactive compounds are of huge significance to cope with multidrug-resistant pathogens (Irshad et al., 2020).
Biofilm formation is a significant virulence strategy for microorganisms to survive in harsh environments (Iram et al., 2013). Bacteria form biofilms both on biotic and abiotic surfaces. Biofilms help bacteria in spreading infections by preventing them from antibiotics and the host immune system (Maqbool et al., 2019). Quorum sensing (QS) is an intercellular communication process adopted by both Gram-negative and Gram-positive bacteria which is based on the secretion and detection of external signal molecules (Misbah Bhatti et al., 2021). In the form of biofilm, bacteria communicate with each other via quorum sensing. Quorum sensing is based on bacterial cell density. Inhibition of quorum sensing has the ability to inhibit virulence of bacteria and biofilms also (Afzal et al., 2021).
Compounds having anti-quorum-sensing properties are able to control microbial infections, which is why researchers in the medical field are interested to explore more antimicrobial compounds (Abbasi et al., 2020). Phytobioactive compounds have remarkable antimicrobial properties (Shah et al., 2019). Lipophilic compounds isolated from Moringa oleifera bind with cytoplasmic membrane and inhibit the growth of filamentous fungi, especially by causing membrane permeabilization (Jabeen et al., 2008;Latif et al., 2011).
Proteins purified from Croton tiglium L. showed remarkable antimicrobial activity . Phytobioactive compounds isolated from sunflower seeds demonstrated high antibacterial properties (Den et al., 2019).
Broadly speaking, two types of bioassays are available for evaluating the antibacterial activity of natural compounds (Riaz, Rasool, Bukhari, Shahid, Zahoor, et al., 2012). Bioassays used for the detec-

| Cytotoxicity analysis
When phytobioactive compounds are isolated as antioxidant and antimicrobial compounds, their toxicity analysis is quite necessary Sadaf et al., 2009). Most lethal poisons in the world, for example, botulinum, maitotoxin, batrachotoxin, and ricin, are naturally occurring compounds (Nighat et al., 2020;Shahid et al., 2020). In vitro bioassays for toxicity testing have increased considerably (Naseem et al., 2020). In vitro assays available for cytotoxicity testing are AMES assay (Jabbar et al., 2012;Rubab et al., 2015), DNA damage protection assay , inflammatory assay , hemolytic assay (Gul et al., 2017;JaEeen et al., 2015;Shahid et al., 2016), thrombolytic assay (Rizwan et al., 2014;Sharif et al., 2018), etc. Several of the commonly used cytotoxicity assays are discussed below: 6.3.1 | AMES assay AMES test has been used to evaluate the mutagenic potential of medicinal plant extract. It was standardized for the identification of mutagenic potential of chemicals in 1970s (Rasgele & Dulger, 2021). For the last few years, this test has been extensively used to assess herbal products. This mutagenic assay uses two mutant strains of Salmonella typhimurium (TA98 and TA100 The test was executed in triplicate (Kauser et al., 2018).

| CHAR A C TE RIZ ATION OF PHY TOB IOAC TIVE COMP OUNDS
Phytobioactive compounds are separated and purified from plant extracts employing chromatographic techniques (Qazi et al., 2021).
The most common chromatographic methods used are thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC; Alim et al., 2010;Naseem et al., 2020). Gas chromatography (GC) is another technique used for separating, identifying, and quantifying phytobioactive compounds such as flavonoids, phenolic acids, and condensed tannins (Sajid et al., 2016). GC technique is used for the analysis of volatile compounds only (Qasim et al., 2020).
FTIR and NMR are used to determine the functional groups of compounds present in the crude plant sample and to detect the number of protons present in the compound.

| Thin-layer chromatography (TLC)
TLC is the most commonly used adsorption chromatographic technique, employed for the separation and analysis of low-molecularweight phytochemicals where plant extract is separated based on the nature of stationary phase (Ullah, 2020).

| High-performance liquid chromatography (HPLC)
HPLC is an analytical technique for the separation and identification of organic and inorganic solutes in a variety of samples including pharmaceutical, biological, industrial, and environmental samples.
HPLC separation of compounds is based on the interaction of compounds with solid particles of tightly packed columns (stationary phase) and the mobile-phase solvent (Grybinik & Bosakova, 2021).
HPLC is useful for samples that cannot be vaporized or decomposed at high temperatures. Nowadays, this technique is gaining popularity among other analytical techniques for the analysis of medicinal plant extracts (Sahu et al., 2018).

| Gas chromatography (GC) and GC-MS
Gas chromatography is used for the analysis of volatile compounds which provides quantitative and qualitative information on individual components present in the compound such as essential oils, hydrocarbons, and solvents. Gas chromatography is based on the principle of adsorption and partition. In GC, sample is injected onto the top of the chromatographic column and then transported through the column (liquid stationary phase) by the flow of inert, Inhibition % = 100 × OD sample ∕ OD control − 1 % Hemolysis = OD sample ∕ OD control × 100 gaseous mobile phase (Al-Rubaye et al., 2017). GC-MS has become a highly recommended analytical technique in the pharmaceutical industry due to its robustness, selectivity, sensitivity, and reproducibility (Beale et al., 2018).

| UV-Visible spectroscopy
UV-visible spectroscopy is used for the identification and qualitative analysis of biological mixtures and plant extracts. Phytobioactives can be analyzed by using UV-visible spectroscopy because aromatic compounds act as chromophores in the UV range. This technique provides information about the total phenolic contents of plant mixtures (Rocha et al., 2018).

| Fourier transform infrared (FTIR) spectroscopy
FTIR spectroscopy is used for the identification of functional groups of the organic and inorganic compounds from plants extract by using infrared light. It helps in the identification, characterization, and structure determination of unknown molecules.
It is a high-resolution analytical technique for the identification and structural elucidation of phytobioactive compounds .

| Nuclear magnetic resonance (NMR) spectroscopy
NMR spectroscopy provides physical, chemical, and biological properties of matter. Due to its accuracy, rapidness, and intactness, NMR spectroscopy has gained a key role in determining the structures and dynamic properties of pharmaceutical drugs. Although NMR has remarkable advantages, sometimes it is combined with other techniques, such as HPLC, FT-IR, and GC-MS, for more accurate and effective results (Cao et al., 2021).

| CON CLUS I ON S AND FUTURE PER S PEC TIVE S
Since ancient times, phytobioactive compounds are being used as traditional medicine for the treatment of several diseases around the globe. Several studies on their therapeutic potential are documented, as they are considered to be the natural sources for the production of new drugs with greater efficacy and biocompatibility.
Recent publications are gradually adding up new characteristics to their pre-existing vast spectrum pharmaceutical value. In presentday lifestyle, oxidative stress has been continuously increasing and causing numerous disorders such as diabetes mellitus, cancer, and cardiovascular problems. Misuse of antibiotics results in antibiotic resistance which poses serious health issues, especially in hospitals. Synthetic drugs are insufficient to cope with these problems.
Exploration of natural phytobioactive compounds to cure presentday diseases is necessary. Polyphenols, alkaloids, and terpenoids have the potential to treat cancer, oxidative stress, inflammation, ulcers, diabetes, platelet aggregation, microbial resistance, and tumors.
This review study concluded that the use of bioactive compounds in foods, beverages, and pharmaceutical products plays a significant role in preventing various disorders including cancer, neurodegenerative diseases, oxidative stress, cardiovascular diseases, diabetes, obesity, etc. The antioxidant and other beneficial effects of phytobioactive compounds can be explained through the modulation of multiple pathways involved in cell signaling. These mechanisms of phytobioactive compound action include the modulation of caspases, NF-kB, MAPK, and Nrf2 as well as LOX and COX. An important approach to prevent pathological processes is the possible crosstalk between these signaling pathways. One of the critical factors that can affect the potential of these phytobioactives to counteract the oxidative damage in human organs induced by ROS is bioavailability. The application of these phytobioactive compounds as therapeutic agents against human diseases will be facilitated through the understanding of their absorption, distribution, metabolism, and excretion. The complications encountered while using phytobioactives, like their stability, solubility, and bioavailability, need to be addressed to use these metabolites as medicine.

ACK N O WLE D G E M ENTS
The authors acknolwledge their resposective institutions for giving them time to work on this review.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declared no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable-no new data are generated and this review article describes entirely the published literature. Phytochemical screening of plants used in folkloric medicine: