Nutritional and Pharmaceutical Applications of Under-Explored Knottin Peptide-Rich Phytomedicines

Phytomedicines reportedly rich in cystine knot peptides (Knottins) are found in several global diets, food/herbal supplements and functional foods. However, their knottin peptide content has largely been unexplored, notably for their emerging dual potentials at both the food and medicine space. The nutritional roles, biological targets and mechanism(s) of activity of these knotted peptides are largely unknown. Meanwhile, knottins have recently been unveiled as emerging peptide therapeutics and nutraceuticals of primary choice due to their broad spectrum of bioactivity, hyper stability, selective toxicity, impressive selectivity for biomolecular targets, and their bioengineering applications. In addition to their potential dietary benefits, some knottins have displayed desirable limited toxicity to human erythrocytes. In an effort to appraise what has been accomplished, unveil knowledge gaps and explore the future prospects of knottins, an elaborate review of the nutritional and pharmaceutical application of phytomedicines rich in knottins was carried out. Herein, we provide comprehensive data on common dietary and therapeutic knottins, the majority of which are poorly investigated in many food-grade phytomedicines used in different cultures and localities. Findings from this review should stimulate scientific interest to unveil novel dietary knottins and knottin-rich nutraceutical peptide drug candidates/leads with potential for future clinical application.


Introduction Description, Distribution, Diversity, Abundance and Type of Knottin Peptides
An understanding of the role of plants as human-environmental co-tenants, which occupy the dual position of application as food and medicine, encouraged their domestication by man over 10,000 years ago [1]. Gradual domestication of plants over the years encouraged the careful selection of useful plants for nutritional purposes and to meet indigenous healthcare needs [2]. Such is the case of many plants including members of the grass family, legumes thought to have been domesticated over 2500 years ago in ancient India [3]. During this early human civilization in India and Africa, many of these plants currently occupying the interface of food and medicine [4] have been used by pastoralists to meet healthcare needs, nutritional requirements, feed farm animals or to improve the quality of polluted water [4]. The phytochemical content of plants informs their selection for nutritional or medicinal purposes. Toxic plants are often avoided except for well-informed applications such as for fishing, hunting etc. [5]. While the ubiquitously distributed primary plant metabolites including proteins, lipids and carbohydrates mainly serve nutritional purposes, the species-specific secondary compounds have been shown to Primary structure showing diagnostic intercysteine connectivity as well as free N-and C-termini for linear knottins and connected termini without free amino and carboxyl termini expressed naturally by cyclic knottins; (B) Secondary structural backbone showing antiparallel sheet stabilized by a cystine knot. Strands are shown in orange and the six cysteine residues that form the cystine knot are labeled 1-6. While the disulphide bonds are known to be responsible for the extremely stable nature of knottins, the continuous circular configuration in cyclic knottins additionally makes the N-and C-termini unavailable for attacks by external digestive agents.
Unlike cyclotides, hevein-like peptides are linear and simply fall under a class of peptides, known as antimicrobial peptides (AMPs), which are distributed wholly in plants and are known to confer resistance to phytopathogens, exclusively fungi. This was elucidated to be due to their ability to specifically bind to chitin, an essential part of the fungal cell wall. Some identified hevein-like peptides, like Pn-AMP1 and Pn-AMP2 isolated from Morning Glory (Pharbitis nil), have activity against fungi possessing chitin and even fungi without chitin in their cell wall. Others have also shown activity against Gram-positive and Gram-negative bacteria such as Fa-AMP2, isolated from Buckwheat (Fagopyrum esculentum), and AvesinA, isolated from Oats, at micromolar concentrations [21]. Hevein-like peptides are named as such because their structures resemble Hevein, the first member of the family isolated from Hevea brasiliensis, and to date, only 20 members have been found intercysteine connectivity as well as free N-and C-termini for linear knottins and connected termini without free amino and carboxyl termini expressed naturally by cyclic knottins; (B) Secondary structural backbone showing antiparallel sheet stabilized by a cystine knot. Strands are shown in orange and the six cysteine residues that form the cystine knot are labeled 1-6. While the disulphide bonds are known to be responsible for the extremely stable nature of knottins, the continuous circular configuration in cyclic knottins additionally makes the N-and C-termini unavailable for attacks by external digestive agents.
Unlike cyclotides, hevein-like peptides are linear and simply fall under a class of peptides, known as antimicrobial peptides (AMPs), which are distributed wholly in plants and are known to confer resistance to phytopathogens, exclusively fungi. This was elucidated to be due to their ability to specifically bind to chitin, an essential part of the fungal cell wall. Some identified hevein-like peptides, like Pn-AMP1 and Pn-AMP2 isolated from Morning Glory (Pharbitis nil), have activity against fungi possessing chitin and even fungi without chitin in their cell wall. Others have also shown activity against Gram-positive and Gram-negative bacteria such as Fa-AMP2, isolated from Buckwheat (Fagopyrum esculentum), and AvesinA, isolated from Oats, at micromolar concentrations [21]. Hevein-like peptides are named as such because their structures resemble Hevein, the first member of the family isolated from Hevea brasiliensis, and to date, only 20 members have been found in angiosperms. Although, recent findings have shown they may be present in gymnosperms with the discovery of ginkgotides, isolated from Ginkgo biloba, which also possess similar antifungal activity [22]. Subsequently, it is clear that the knottin peptides hold strong promise as drug molecules, drug scaffolds and intracellular drug delivery systems [19].
Plants 2022, 11, x FOR PEER REVIEW 4 of 30 in angiosperms. Although, recent findings have shown they may be present in gymnosperms with the discovery of ginkgotides, isolated from Ginkgo biloba, which also possess similar antifungal activity [22]. Subsequently, it is clear that the knottin peptides hold strong promise as drug molecules, drug scaffolds and intracellular drug delivery systems [19]. Findings from these literature reports strongly emphasize the therapeutic roles of knottins in the overall bioactivity of these standardized indigenous phytomedicines. However, there is a lack of a comprehensive review of this documented work appraising what has been accomplished, unveiling the huge knowledge gaps and shining the light on the future prospects of knottins at the interface of food and medicine. Herein, we aim to provide a comprehensive review on the nutritional and pharmaceutical potentials of the poorly investigated knottins-rich phytomedicines used across the globe in order to trigger scientific interest for further in-depth investigation, particularly in regions where they are least investigated. Findings from this review could drive the renewal of scientific interest in the discovery of novel knottin peptide drug candidates, and thus increase the potential for their clinical application. Figure 2. 2D representation of a typical knottin peptide, a cyclic protease inhibitor (Trypsin inhibitor, MCoTI-II) isolated from the seeds of an important medicinal plant, Momordica cochinchinensis (Cucurbitaceae). This gene encoded cyclotide is abundantly expressed at the peptide level. The two nearly parallel disulfide bonds have been cross linked by a third one which passes through each of the two to form a knotted structural configuration that induces enormous stability in this bioactive knottin-like peptide. PDB:1HA9.
Findings from these literature reports strongly emphasize the therapeutic roles of knottins in the overall bioactivity of these standardized indigenous phytomedicines. However, there is a lack of a comprehensive review of this documented work appraising what has been accomplished, unveiling the huge knowledge gaps and shining the light on the future prospects of knottins at the interface of food and medicine. Herein, we aim to provide a comprehensive review on the nutritional and pharmaceutical potentials of the poorly investigated knottins-rich phytomedicines used across the globe in order to trigger scientific interest for further in-depth investigation, particularly in regions where they are least investigated. Findings from this review could drive the renewal of scientific interest in the discovery of novel knottin peptide drug candidates, and thus increase the potential for their clinical application.

Nutritional Applications of Knottin-Rich Phytomedicines
This family of chemically identical knottins, also known as cystine-knot miniproteins, does have a diversified amino acid composition and varied biochemical roles [23]. The role of "edible" cystine knot peptides (knottins) as innovative food peptides is largely yet to receive scientific assessment for their nutritional and functional roles. The dietary quality of food-derived knottins has not been documented. Meanwhile, knottins have been identified and characterized from several food/herbal recipes important in human diets [24]. Common examples of cystine knot peptides and Knottin peptide-rich plants commonly taken as food have been summarized in Table 1; they include cyclotides from Clitoria ternatea [25], PA1b peptides from Pisum sativum [26], knottin-like protease inhibitors from Beta vulgaris [13], knottin-type Roseltides from Hibiscus sabdariffa [27], Morintides from Moringa oleifera [28], knottin-type antimicrobial peptides from Amaranthus spp. [12,29] and antibacterial knottin-like peptides from Ghost pepper [30] and other Capsicum species recently reviewed by Oliveira and colleagues [31]. The contributory nutritional role of these knottins in the diets requires deliberate scientific attention. For instance, knottin peptides, although considerably stable to enzymatic attack, when hydrolyzed in the gut may act as a good source of dietary amino acids while the unhydrolysed stable peptide has the potential to trigger changes in the gut microbiota [32] owing to their broad bioactivity, antimicrobial activity in particular [33]. Medicinal plants and phytomedicines rich in knottin-like peptides commonly taken as food appear to be good sources of essential amino acids and low molecular weight peptides [34] needed for the proper physiological functioning and prevention of several metabolic diseases [35]. These essential and other types of important dietary amino acids have thus been classified as functional amino acids [35].
Clitoria ternatea (Darwin Pea): The plant, known as the butterfly pea, has been grown for many years as a fodder plant, but recently it is being explored for its dietary applications as well as potential uses in medicine and agriculture [11]. C. ternatea is a plant with flowers, having an abundance of anthocyanins, which predisposes its use as a food coloring agent and additive to improve aesthetic appeal [11]. Locally, its flowers have been used to color a popular Kelantan dish known as the nasi kebaru blue, which can be eaten with grilled chicken, fried fish coated with flour, fish crackers, salted egg and other local herbs [36] The plant could serve as a source of essential amino acids and protein whose content has been reported to be around 40% of mean weight [37]. The novel discovery of abundant ultrastable circular knottins (cyclotides) in C. ternatea opened the doors to potentially greater innovation that could unveil the dietary roles of these cystine knot peptides. One of such cyclotide-rich extract, Sero-X, was approved for use in Australia in 2017 as an eco-friendly biopesticide with an overall advantage of improving agricultural practices [11].
Pisum sativum: The Garden Pea is a staple that makes up several diets worldwide due to its high protein and amino acid content, within a range of 21-30% of mean weight [38]. This nutritional content makes the plant a valuable food source that has even been proposed to meet the dietary needs of about 800-900 million undernourished people worldwide [39]. It is now considered as an add-on for cereals to boost their nutritional value and as a substitute for animal proteins [38]. The garden pea is also rich in phenolics, natural antioxidants which would contribute to normal general wellbeing [39]. The Garden Pea has been reported to be a rich source of cysteine-rich knotted peptides known as Pea Albumin 1 subunit b peptides (PA1b), a highly selective entomotoxin abundantly expressed in the edible seeds of the pea plant [26]. P. sativum has also been reported to produce peptide constituents that confer antimicrobial resistance; Psd1 and Psd2, isolated from P. sativum, are some plant defensins which have already been proven to possess some antifungal activity [40]. However, the nutritional role of these knottin peptides expressed in the plant and related species like Cajanus cajan, which potentially express cysteine knot peptides, await scientific investigation. Hibiscus Sabdariffa: The calyces of Hibiscus Sabdariffa are a source of proteins with 7.51% protein on a dry weight basis [41]. For a very long time, crude extract and calyx powder has been employed as a food source and nutraceuticals in most parts of Africa and Asia [42]. Most nutritional and pharmacological activity has been linked to bioactive constituents such as organic acids, anthocyanins, polyphenol and flavonoids. Only recently, roseltides, a knottin-rich neutrophil elastase inhibitor, were discovered [43]. Necessary scientific attention has not been placed on the contribution of these ultra-stable knottins to food and medicines. Moreover, it is yet to be investigated the percentage of the proteins that are knottin-rich and how they could contribute to nutritional and pharmacological benefits. Though knottins are stable in the gastrointestinal tract, protease can break down the knottin-rich peptide after an hour. These enzymes could help break down knottins into the individual amino acid residue, which could then be absorbed, and this contributes to its nutritional benefit. On the other hand, the undigested knottins after absorption tend to retain the conserved cysteine-knot motif and could become an excellent chemical entity contributing to the reported pharmacological activity of the crude extract and powdered calyx. However, while that study is yet to be investigated, it deserves research attention.
Beta Vulgaris (beetroot) has been used industrially as a source of sugar and traditional medicine in treating constipation, decreased libido, gut and joint pain and dandruff [44]. The nutritional content is due to a high protein constituent of approximately 13.23 mg/100 g [45]. Retzl and colleagues [13] recently identified a novel knottin-rich inhibitor peptide, bevuTI-I, from beetroot and showed that it belongs to the trypsin inhibitors family with a potential therapeutical advantage in the management of autoimmune diseases and cancer. Beetroot is majorly grown for food uses (pickles, salad, juice) rather than for sugar production. The extract has been employed in the management of cancer and coronary heart diseases due to its anti-inflammatory effect [46]. Further studies are needed to investigate the potential contributions of the knottin-rich peptide to the nutritional and pharmacological benefit derivable from the plant, as nature is not wasteful in natural product biosynthesis.
Amaranthus spp., also known as Amaranths, constitute a group of edible plants known as Pseudo-cereals which are consumed not only for their food use but also for their medicinal benefits [47]. This dual character as a food and drug serves as the basis for its economic importance, even in poor countries. Amaranthus constitutes a cosmopolitan genus with common species such as Amaranthus hypochondriacus, Amaranthus cruentus, Amaranthus hybridus, Amaranthus caudatus and Amaranthus tricolor [48]. While some of these species are known for insecticidal activity, some are known for an antimicrobial property. For example, Amaranthus caudatus contains known characterized Knottin peptides which are very effective in inhibiting the growth of fungi and Gram-positive bacteria [49]. The seeds of A. hypochondriacus contain α-amylase inhibitors which are effective for their insecticidal property against the larvae of various insects such as Prostephanus runcates, and Tenebrio molitor. The amaranth alpha-amylase inhibitor's (AAI) three-dimensional structure adopts an abcabc knottin fold [50]. Mammalian amylases are not affected by the α-amylase inhibitors due to selective activity [49]. Amaranths are useful nutraceuticals across the different species due to the variety of proteins they contain such as Ar-AMP from Amaranthus retroflexus, which is an effective antifungal against pathogens like A. consortiale, B. cinerea, etc. Ay-AMP from A. hypochondriacus which is also a useful antifungal against C. albicans, G. candidum, A. alternate, F. solani, etc. [51]. Studies have clearly summarized the wide importance of the various species under the Genus Amaranthus with properties across insecticidal activity, fungicidal activity and bactericidal activity. The presence of knottin-rich peptides in the seeds of this plant underlies its use as a Nutraceutical and its usefulness in Phytomedical applications.
Capsicum species, an important dietary ingredient used globally as a spice to add flavors to dishes, has been documented as a rich source of cystine knot antimicrobial peptides [52] which have not been elaborately studied. In Nigeria, particularly south-west Nigeria, Capsicum species are highly valued in diets as the plant species have strong historical claims of disease prevention and health restoration during infections and viral pandemic (personal communication). However, the complementarity and nutritional roles of these peptides in Capsicum-rich diets await scientific investigation.
Moringa oleifera Lam. (Moringaceae) is a tropical plant which has been domesticated in various parts of Africa and Asia where it is consumed as a nutrient-endowed food and medicine [4]. The reported antimicrobial knottin peptides [28] MO1 and MO2 and other water clarifying polypeptides have attracted scientific attention [53]. However, the nutritional relevance of these knottin-like peptides in maintaining gut integrity, improving food digestibility as well as other physiological functions have not been explored. However, these peptides are constantly released into water during water clarification treatment or when Moringa leaf is taken as tea. For instance, in northern Nigeria, the knottin peptide-rich leaf has a history of use as a traditional food/salad by the Hausa-Fulani ethnic nationality (personal communication).
Proteins and peptides from many sources have been studied extensively to develop important foods [54]. When testing protein-rich products, emulsification is a vital step in the process. Emulsifying properties of food bioactive peptides byproduct depend on their amphiphilic nature [55]. Emulsifying properties have been observed in plant-derived active peptides such as in potato, flaxseed, and soybean [56]. The potential emulsifying property of knottin peptides isolated from phytomedicines used as food additives have not been investigated.
Functional diets based on the usage of phytomedicines rich in knottin peptides are widely used across the continents but are under investigated. Knottin peptide-rich phytomedicines have proven to be particularly beneficial as food additives, in beverages and many other yet-to-be validated nutritional applications including as sources of essential amino acids. For example, these peptides may hold some promise as sugar substitutes or some color stabilizers, a thickener or an anti-caking factor. It is not known if knottin peptides have the potential to enhance the flavor or influence the acidity of food. By altering the water and oil retention, colloidal stability, viscosity and foam production in the end product, knottin peptides may improve food quality [57]. However, this informed guess begs for scientific investigation. Table 1. Reported knottin-rich phytomedicines used in various parts of the globe as both food and medicine (nutraceuticals) and their botanical sources.

Knottins Present Nutraceutical Applications Scientific Evidence References
Ewa/ Clitoria ternatea L. Indonesia Cyclotides, e.g., Cter M C. ternatea flowers are used as a natural coloring for food and beverage. It helps to improve food quality by affecting the water and oil retention capacity, colloidal stability, viscosity, and foam generation in the finished product. It possesses anti-osteoporotic, antihypertensive activity and reduces cardiovascular complications.
Andersson Brazil Caripe Cyclotides (Caripe 1-13) It helps to minimize the cooking/frying loss and shrinkage and enhances foaming stability in chicken sausages. Amoebic dysentery, cough, whooping cough and bronchitis can be treated using root extracts and powdered leaf of Carapichea. C. ipecacuanha contains emetine and cephaeline, to induce vomiting. It helps to reduce the risk of certain chronic diseases such as leukemia.
The natural action of the bioactive compounds protects the biological attributes such as anti-aging, anti-inflammatory, anti-viral, anti-microbial, and anti-cancer properties.  In silico, in vitro and in vivo (clinical trials) [68] Pea/ Pisum sativum L.
Middle East specifically to Turkey and Iraq The major components of peas are protein, starch and fiber which help in breaking down carbohydrates, hence are beneficial in the prevention and management of type-2 diabetes. Coumestrol in peas helps to lower the risk of stomach cancer. Addition of peas to food in combination with inulin fiber supplement greatly increases bowel movement frequency. It is a functional ingredient in vegan foods and dried seeds of pea are used as staple foods. Eating green peas regularly helps to lower blood pressure and blood cholesterol level due to the mineral content (Mg, K and Ca) present in them.
In silico, in vitro and in vivo [39,69]

Pharmaceutical Applications of Phytomedicines Rich in Knottins
Organic compounds and protein biologics have a lot in common, but knottins have evolved as intriguing prospects for therapeutic development because of their ability to adhere to clinical substrates with strong affinity and specificity [23]. Knottins also show promise in challenging pharmaceutical research goals, nutraceutical uses such as oral distribution, and the capacity to access cellular therapeutic targets due to their extraordinarily high tenacity and unique structural properties [54].
Degree of Evidence: the pharmaceutical applications of the underexplored knottinsrich peptides have been presented based on scientific evidence documented to support the pharmacological action. As a result, to unveil the extent of scientific evidence supporting the reported knottins, the following classifications have been used: Class I-At least one clinical investigation has validated the evidence (most significant degree of scientific evidence).
Class II-An in vivo experiment was used to validate a documented pharmacological activity.
Class III-In vitro experiments provide evidence for the reported pharmacological activity. Class IV-Evidence supported by in silico study or extrapolated from the crude extract. In this review, these established evidence levels are denoted by square brackets, such as [Class I] for claims obtained from at least one clinical investigation [77].

Antimicrobial
Among the cyclotides identified from coffee plants are Kalata B1, Circulin A, Circulin B, and Cyclopsychotride A, which were the first knottin-rich peptides with antibacterial properties to be described [78,79]. More cyclotides with antibacterial activity were subsequently discovered (Table 2).
Recent studies [82] showed that the cycloviolacin group of bracelet cyclotides (CyO2, CyO3 and CyO-19) exhibited broad spectrum activity, with significant activity against S. aureus. Similar to this, CyO2 and Kalata B2 were tested for their anti-staphylococcal efficacy against S. aureus. CyO2 showed the highest in vitro activity (MIC 25 µM), whereas Kalata B2 displayed somewhat lesser potency (MIC 50 µM). Considering the broad spectrum of activity and potency of CyO2, further studies are needed to demonstrate its antimicrobial activity against microorganisms resistant to commonly available antibiotics. This could open the floor for developing knottin-rich antimicrobial peptides with potential clinical application. The result was consistent with the first in vivo experiment conducted on a surgical S. aureus wound infection mouse model (Class II) [83].
Cyclotides from O. affinis displayed considerably different antimicrobial properties. Kalata B1, a popular uterotonic agent, was inactive against E. coli, whereas moderate activity was reported for P. aeruginosa. On the other hand, Kalata B7 displayed potent activity against P. aeruginosa (Class III) [78].
Generally, P. aeruginosa, E. coli, and S. aureus were the most vulnerable to cyclotidebased antibacterial activity [82]. Experiments have shown that the antimicrobial properties of cyclotides depend on various salt concentrations. In general, low salt concentration resulted in increased activity while a high salt concentration decreases or abolishes activities. Only the antimicrobial cyclotides circulin B, cyclopsychotride A, and cliotide T15 retained antimicrobial activities, both in low and medium salt concentration. Hence, they are promising lead compounds that can be explored for peptide-based antimicrobial agents [25]. In addition, ref. [84] isolated and characterized potent and selective bactericidal peptide Echinopsis pachanoi antimicrobial peptide (Ep-AMP1) with extremely low cytotoxicity. This peptide demonstrated significant activity against Gram-positive bacteria, weak activity against Gram-negative bacteria, and no activity against C. albicans even at concentrations exceeding 160 M. (Table 2) [84].
Recently, Ref. [85] synthesized cyclotide VarcA previously identified from Viola arvensis and demonstrated their antimicrobial activity against bacterial infection in aquaculture. Similar, cyclotides isolated from Sambucus nigra L. flowers demonstrated effectiveness against several pathogenic Gram-negative bacteria that threaten fisheries. Though the peptide is yet to be characterized, their proposed mode of action results from peptide interaction with the lipid membranes of the bacteria resulting in membrane disruption, leakage of cellular content and ultimate death of the pathogenic organism [86].

Anticancer
Knottins-rich peptides present a good protein scaffold that could be explored for potential application in cancer drug treatment and delivery [87]. The exceptional thermal, chemical and enzymatic stability, coupled with a distinct structural-activity-relationship (SAR) which is amenable to chemical modification, present a unique field that could be exploited to develop promising drugs for the management of cancer. This class of peptide not only possesses anticancer properties but also the ability to serve as a carrier in cancer drug delivery as peptides-drug conjugates, thereby facilitating its entry into the bloodbrain barrier, improving target affinity and specificity, and also the ability to bypass drug resistance mechanisms associated with conventional anticancer drugs [88,89].
Cytotoxic properties of knottin-rich peptides have been thoroughly documented, particularly for cyclotides; Grover et al. [89]. compared this activity and found that around 74% of the cyclic knottins demonstrated action at a relatively low micromolar concentration (7 M), confirming their efficacy against the tested cancer cell line. CyO2 isolated from various Viola spp., including Viola odorata, appear to be the most potent, promising and well-studied. In addition to being one of the most promising antimicrobial peptides, their high cytotoxicity presents an exciting opportunity for potential development as antitumor therapeutics. However, an in-depth understanding of possible interaction with the DNA of cancer cells needs to be established. Three novel cyclotides (Psyle A, C, and E) from Psychotria leptothyrsa (Rubiaceae) were compared to CyO2 in terms of their cytotoxicity, and it was found that they are both effective against breast cancer cells [90]. Additionally, they reported that they could create pores in drug-resistant breast cancer, which facilitates the uptake of the chemotherapeutic drug Doxorubicin. The synergistic effect of this combination requires further investigation to determine toxicity and efficacy, with the potential development as either monotherapy or fixed combinational therapy with an existing chemotherapeutic agent.
Among the cyclotides isolated from Hedyotis biflora Hornem. (Accepted name: Leptopetalum biflorum (L.) Neupane and N.Wikstr.), Hedyotide B7 is the most potent in both in vitro and in vivo experiments against pancreatic cell lines ( Table 2) [91]. However, when tested using human prostate cancer xenografts (Class II and III), the H. diffusa cyclotides derived from the root and leaves of H. diffusa showed potent cytotoxic activity.
Though the crude extract H. diffusa has been approved clinically for the management of colorectal cancer, its cyclotides are yet to be investigated for this activity (Class I) [92]. Hemolytic activity is often a significant setback for the potential development of knottinrich chemotherapeutic agents; however, lysine-rich cyclotides from M. latifolius displayed limited hemolytic activity even at a very high peptide concentration (Table 2) [93]. Hence, they are promising candidates for anticancer drug delivery/design since toxicity to erythrocytes is a major setback in some toxic knottin-rich peptides.
Cliotide T1 showed the most potent cytotoxicity in HeLa cell line among the twelve cyclotides isolated from C. ternata with IC 50 -0.6 µM [80,82]; Vigno 5 from Viola ignobilis recently demonstrated dose-dependent cytotoxicity against Hela cells by apoptosis linked to the release of cytochrome C and increased activity of caspase-9 and -3 in Hela cells [94].
Numerous pharmacological properties of Hibiscus sabdariffa's crude extract have been scientifically demonstrated, including the ability to lower blood pressure, improve memory, prevent cancer, reduce inflammation, and act as an antioxidant [42]. However, knottinrich extracts have not yet been studied for these properties. Recently, Loo et al. [43] identified Roseltides (rT1-rT8), a new knottin-rich human neutrophil elastase inhibitor with promising therapeutic relevance, in neutrophil elastase-related disorders. In addition, rT1 and rT7 is a cell-penetrating peptide that selectively targets the mitochondria and enhances ATP production, which could present a therapeutic option in managing disorders associated with ageing, such as cancer [27]. Further studies are needed to investigate these peptides' contribution to the pharmacology of the crude extract. Exploring how these peptides contribute to the neutraceutical properties of the extract after being subjected to high-temperature conditions during production will be intriguing, given their outstanding thermal and enzymatic stability.
The cysteine-knot Metallo carboxypeptidase inhibitors have been reported for Solanum lycopersicum, Solanum tuberosum, Nicotiana tabacum and Hyoscyamus niger, with various interesting yet unexploited pharmaceutical applications ( Table 2). The tomato cysteineknot mini-proteins (TCMP-2) have potent antiangiogenic activity via modulating vascular endothelial growth factor receptors [95]. Therefore, tomato consumption may help prevent various types of cancer and vascular disease due to the existence of TCMP-2 and other secondary metabolites with strong antioxidant properties [96].
In general, knottin-rich peptides possess potent cytotoxic properties, good cell penetration and a diverse SAR that could be modified for the development of the ideal chemotherapeutic drug candidate in the search for anticancer agents. For instance, the analysis of hyen D cyclotide ( Figure 4S6) characterized from Hybanthus enneaspermus (Linn) has shown the functionally and structurally critical residues important for membrane binding and cytotoxicity [97]. antiviral activity against HIV and were identified from the crude extract of Chassalia Parvifolia [99]. The proposed antiviral mechanism of action of the prototypical Kalata B1 is by interaction with the viral particles before entry, thereby preventing its fusion into the host cell membrane. By the early 2000s, numerous knottin-rich peptides had been identified with significant activity against HIV [100][101][102]. The prototypical Kalata B1 also showed strong activity against HIV by destroying the viral particles prior to entry, and also inhibiting fusion of the virus to host cell membrane [103][104][105]. Similarly, a kalata B1-inspired peptide produced from the amino acid modification of Kalata B1 presents significant activity against dengue virus (Class III) [106]. Alstotides and Allotides, botanical cystine knot α-amylase inhibitors, respectively, from Alstonia scholaris (Apocynaceae) and Allamanda carthatica L. (Apocynaceae), Produced α-amylase inhibition, while the Alstotides additionally showed significant membrane permeability and early phase antiviral activity against infection of bronchitis virus and Dengue infection (DENV2). For clarity of name, the solution structure of Allatide C4 (Figure 4, S7) is an example of a characterized Allotide from Allamanda carthatica. These groups of knottin-rich peptides were shown to be highly stable in the metabolic environment; they selectively target the viral cell membranes and are non-cytotoxic even at peptides concentration up to 100 μM [107].

Antiviral
Knottin-rich peptides, particularly cyclotides have been extensively investigated for their activity against viruses that cause diseases in humans, including the human immunodeficiency virus (HIV), the H1N1 influenza virus, and dengue (DENV) [98].
Circulin A and B (Figure 4) are the first group of cyclotides discovered to possess antiviral activity against HIV and were identified from the crude extract of Chassalia Parvifolia [99]. The proposed antiviral mechanism of action of the prototypical Kalata B1 is by interaction with the viral particles before entry, thereby preventing its fusion into the host cell membrane. By the early 2000s, numerous knottin-rich peptides had been identified with significant activity against HIV [100][101][102]. The prototypical Kalata B1 also showed strong activity against HIV by destroying the viral particles prior to entry, and also inhibiting fusion of the virus to host cell membrane [103][104][105]. Similarly, a kalata B1-inspired peptide produced from the amino acid modification of Kalata B1 presents significant activity against dengue virus (Class III) [106].
Alstotides and Allotides, botanical cystine knot α-amylase inhibitors, respectively, from Alstonia scholaris (Apocynaceae) and Allamanda carthatica L. (Apocynaceae), Produced α-amylase inhibition, while the Alstotides additionally showed significant membrane permeability and early phase antiviral activity against infection of bronchitis virus and Dengue infection (DENV2). For clarity of name, the solution structure of Allatide C4 ( Figure 4S7) is an example of a characterized Allotide from Allamanda carthatica. These groups of knottinrich peptides were shown to be highly stable in the metabolic environment; they selectively target the viral cell membranes and are non-cytotoxic even at peptides concentration up to 100 µM [107].
Anti-HIV activity has also been described for Cycloviolacin (O2, O13, O14, O24, Y1, Y4 and Y5) isolated from various Viola spp. Cycloviolacin Y5 is the most potent of the cyclotides isolated from Viola philippica Cav. while cycloviolacin Y1 is the least potent [108]. Gerlach et al. [90] showed that combining protease inhibitors (Saquinavir and Nelfinavir) with CyO2 increased their antiviral activity. This peptide accelerates pore formation in HIV-infected T-cells and monocytes, thereby increasing the absorption of the antiviral drugs. The antiviral mechanism of action of cyclotides is similar to that of antibacterial, mainly through interaction with the cell membrane resulting in cell shrinkage, leakage of cellular content and subsequent death of the pathogenic organism [18]. In the case of enveloped viruses such as HIV, these peptides destroy viral particles by disrupting the lipid membrane and thereby preventing its fusion into the host cell [105].
Despite the promise knottin-rich peptides have shown, before they are employed as antiviral agents, there is a need to assess their hemolytic effect on erythrocyte and cytotoxicity to human cells. Interestingly, these groups of peptides are amenable to structural modification to optimize their activity, improve pharmacokinetics profile and reduce toxicity.

Antifungal
The knottin-rich peptides displayed potent antifungal activity against pathogenic fungal species from plants and animals Table 2). The first reported antifungal activity from four knottin-like cyclotides (kalata B1, circulin A, circulin B, cyclopsychotride A- Figures 3 and 4) were reported against Candida kefyr, C. tropicalis and C. albicans under different salt conditions. Under low salt conditions the four cyclotides showed moderate activity against C. kefyr and C. tropicalis, whereas no activity was reported for C. albicans [78]. Cycloviolacin O2, cycloviolacin O3 and cycloviolacin O19 showed potent activity against C. albicans with 99% activity seen at Minimum Fungicidal Concentration (MFC) of 10 µM (Class III) [109]. The same studies also reported the MFC of 40 µM for tricyclon A against C. albicans and low fungicidal activity was reported for cliotides (cter B, cter E, cter G) and kalata cyclotides (KB1, KB2, KB7 and KB13) [109].
Apart from their activity against fungal infection in humans, knottin-rich peptides also possess considerable activity against fungal infection in plants. It is unsurprising since this class of peptides primarily serve as defensive 'molecular machines' in plants. Slazak et al. [109] demonstrated that the antifungal activity of knottin-like cyclotides is similar to that of bacteria by selectively targeting and disrupting membranes containing phosphatidylethanolamine. Recently, the cycloviolacin (O2, O3, O8 and O19) are under scientific investigation as potential bio-fungicide against Fusarium head blight in crops [109,110].
The hevein-like, knottin-rich peptide Ginkgotides (gB1-gB11) and Vaccatide from Ginkgo biloba and Vaccaria hispanica, respectively, also showed potent activity against phytopathogenic fungi with an IC 50 value within micromolar concentration ( Table 2) [22]. This class of extremely stable knottin-rich peptide presents potential, but are currently underexploited as potential bio-fungicides and orally active and metabolically stable peptidebased therapeutics.
Plant defensin are a superfamily of knottin-rich antimicrobial peptide with cis configuration which majorly function as component of plant innate immune system This class of peptides confers protection to plants against pathogenic fungal and bacterial infection, mainly through membrane permeabilisation, generation of free radicals, specific lipid interaction, and initiation of cell wall stress and cellular leakage [111]. In addition, this class of peptide is involved in the development of metal tolerance and possesses inhibitory activity against α-amylase and trypsin. For instance, Arabidopsis thaliana Plant Defensin Type 1.1 (AtPDF1.1) is a secreted protein that can chelate apoplastic iron, and it presents a strong antibacterial activity against necrotrophic bacterium (Pectobacterium carotovorum) via an iron-deficiency defense response [112]. The plant defensin RBAFP isolated from the seed of pinto bean and red bean showed numerous potential pharmaceutical applications ( Table 2) in addition to their use as functional foods [113]. These peptides may contribute significantly to the nutritional value of the bean. In addition to antifungal activity, antiproliferative activity towards tumor cells including human liver hepatoma cells Bel-7402 and neuroblastoma cells SHSY5Y was reported for Phaseolus limensis (Table 2) [114].
PA1b (Pea Albumin 1, subunit b), a 37aa peptide first isolated from garden pea seeds, has been reported to possess strong bioinsecticidal activity against cereal weevils such as (Sitophilus oryzae, S. zeamais, S. granarius) or the pea aphid (Acyrthosiphon pisum) via induction of apoptosis through PA1b/V-ATPase interaction resulting in insect death [115]; however, this highly selective pea peptide and its analogues have not been investigated for potential therapeutic application following from the ethnopharmacological uses of their host legume plants, especially the genus Cajanus (pigeon peas) widely used in African and Asian ethnomedicines. 100 µg/mL gave the strongest antimicrobial activity. [25,86] Clitoria ternatea L. Cationic cT15, cT19 and cT20 Potent activity against Gram-negative bacteria (E. coli), antibacterial activity diminished by high salt concentration, except for cT15. Immunomodulating activity presents an opportunity to be used as an adjuvant in vaccines.

MCoTI-II
The linear 9mer peptide grafted into the loop 6 of MCoTI-II showed reduced antibacterial activity but extreme stability in 100% human serum. Antimicrobial activity of the cyclotides was also slightly reduced in 10% human serum.

Kalata B1
In bioengineering, they become excellent scaffolds for radioactive imaging probes to be used in positron emission tomography (PET), single photon emission computed tomography (SPECT). These scaffolds have also been combined with non-radioactive probes for fluorescence and ultrasound imaging. Useful as drug delivery vehicles for bioactive therapeutic peptides. [23,124] Momordica cochinchinensis (Lour.) Spreng.

Cystine-knot peptide
This peptide has been engineered to bind to cytotoxic T Lymphocyte-associated antigen-4 (CTLA-4) which happens to be a target in the treatment of metastatic melanoma.

Limitations to the Pharmaceutical Applications of Knottin-Rich Peptides in Clinical Settings
Knottin-rich peptides and phytomedicines rich in knottins have demonstrated numerous applications and clinical uses that range from insecticidal, cytotoxic, antimicrobial, antiviral, antineoplastic and much more [17], yet limitations to their adoption abound ( Figure 5). These limitations include unwanted properties, characteristics or effects that are clinically undesired. The broad range of activity of some of these peptides, however important, may sometimes lead to unwanted effects. One of such effects is hemolysis. Though some knottins, such as plant defensins, have been reported to be non-toxic to human cells [126], a few cyclotides have shown hemolytic potential to mammalian erythrocytes [81]. This ability to lyse red blood cells occurs via its interaction with membranes-the same mechanism by which it kills microbes. So far, it has been observed that the level of hemolytic activity varies from peptide to peptide and often correlates with parasiticidal activity [127]. Cycloviolacin H4, Cycloviolacin Y5, VarvE, characterized from Viola hederacea, Viola yedoensis, and Viola tricolor are examples of peptides which have shown hemolytic activity [128]. As expected, this hemolytic effect limits the applications of some knottin peptides (particularly cyclotides) in clinical therapy, but fortunately knottins are easily modified and eliminating this unwanted property using a synthetic biology technique known as site-directed mutagenesis is achievable. This will be crucial in enhancing its therapeutic suitability and wider adoption.
Generally, knottins such as cyclotides are known to be target specific. However, the usefulness of some knottins as cytotoxic agents in the treatment of cancer may be limited in its clinical application due to target non-selectivity. Some of the cytotoxic knottins affect both healthy and unhealthy cells. This is a major problem faced in cancer therapy. The majority of existing cancer treatment options are faced with this drawback which reduces beneficial outcomes for patients. The cytotoxic effect is also related to their ability to bind to cell membranes. On the bright side, recent studies have reported a higher selectivity among some cyclotides [94]. Another potential limitation in clinical application is low immunogenicity. Some cyclotides were discovered to be immunologically unresponsive without antigen conjugation [129]. Although these prospective clinical uses have piqued interest in cyclotides, a better understanding of their immunological characteristics is required and demands closer scientific attention. modified and eliminating this unwanted property using a synthetic biology technique known as site-directed mutagenesis is achievable. This will be crucial in enhancing its therapeutic suitability and wider adoption.
Generally, knottins such as cyclotides are known to be target specific. However, the usefulness of some knottins as cytotoxic agents in the treatment of cancer may be limited in its clinical application due to target non-selectivity. Some of the cytotoxic knottins affect both healthy and unhealthy cells. This is a major problem faced in cancer therapy. The majority of existing cancer treatment options are faced with this drawback which reduces beneficial outcomes for patients. The cytotoxic effect is also related to their ability to bind to cell membranes. On the bright side, recent studies have reported a higher selectivity among some cyclotides [94]. Another potential limitation in clinical application is low immunogenicity. Some cyclotides were discovered to be immunologically unresponsive without antigen conjugation [129]. Although these prospective clinical uses have piqued interest in cyclotides, a better understanding of their immunological characteristics is required and demands closer scientific attention. Many knottin peptides are still in pre-clinical trials, but Table 3 highlights knottin peptides that have made it to clinical trials. Prominent among them is the nature-inspired T20K kalata B1, originally characterized from the African traditional plant Oldenlandia affinis. This knottin-like cyclotide has demonstrated therapeutic promise in inflammatory- Many knottin peptides are still in pre-clinical trials, but Table 3 highlights knottin peptides that have made it to clinical trials. Prominent among them is the nature-inspired T20K kalata B1, originally characterized from the African traditional plant Oldenlandia affinis. This knottin-like cyclotide has demonstrated therapeutic promise in inflammatoryrelated and autoimmune disorders [13,120]. A linear knottin, bevuTI-I, isolated from the aqueous extracts of Beta vulgaris, showed interesting mammalian enzyme inhibitory activity against trypsin (IC 50 = 471 nM) and human prolyl oligopeptidase (IC 50 = 11 µM), a drug target for neurodegenerative and inflammatory disorders [13]. It is of note that the same aqueous extracts of this plant are currently under clinical investigation for blood pressure, inflammatory markers and oxidative stress in type 2 diabetes (Table 3). Knottins such as cyclotides and their linear variants are well reported as highly specific and stable peptide-based protease inhibitors. The documented inhibition of prolyl oligopeptidase by knottin-like cyclotides [13,130] should trigger scientific investigation of the potential of knottin-like peptides, to inhibit the enzymes involved in a cascade of reactions leading to the extracellular plaques of beta-amyloid peptide (Aβ) deposits in the brain observed in Alzheimer's disease [131]. As a first step, Kalmankar and colleagues [132] showed that cyclotides from Clitoria ternatea can inhibit the aggregation of amyloid β peptide (Aβ). This computational study showed that cyclotides can prevent Alzheimer's progression by binding to peptide structures and inhibiting their aggregation. Following a thorough molecular dynamics simulation analysis, it was discovered that cyclotides reduce the inter-strand hydrogen bonds between the Aβ peptide by forming hydrogen bonds with the hazardous amyloid assemblies. While there is a dearth of clinical trials on the use of knottin-like peptides in Alzheimer's disease, this computational study suggests its possible potential in Alzheimer's therapy and highlights the need for further research. Other knottin peptide-rich phytomedicines that are in clinical trials for at least one human disease include Zufa (containing Viola odorata), Alstonia scholaris, Moringa oleifera and Momordica cochinchinensis (Table 3).
Findings from keywords were downloaded and archived. Papers excluded from the list are those which do not meet the inclusion criteria for knottins: molecular weight of 2-6 kDa, scientifically sound and phytomedicines rich in knottins occupying the interphase of food and medicine. Knottin peptides from animal sources were excluded.

Conclusions and Prospects
Knottin peptides, biosynthesized in several edible botanical sources, have been poorly investigated for their benefits, bioactivity and nutritive properties. In the future, knottins with validated, acceptable levels of safety should be investigated for use in creating functional food products with an extended shelf life; this is already evident by their reported property as antioxidants in fatty foods and as anti-infective/antimicrobial agents in food packaging. In-depth and elaborate pre-clinical and clinical research is needed to discover which knottin peptides are favorable for clinical use, their dose/response relationship, absorption, pharmacokinetic and compatibility with various foods.
Knottins are peptide-based phytoconstituents whose contributory nutritional/pharmacological roles are largely unknown in many standardized phytomedicines. The therapeutic and nutritional roles of knottin peptide components of these phytomedicines have not received the attention of the scientific community despite their amazing potential in the food-medicine space. Thus, one of the future prospects in the use of knottins may be to investigate their potential as alternative dietary protein source and functional amino acids that could produce a therapeutic benefit at the highest level of clinical evidence.
Future prospects for the effect of knottin in antimicrobial studies would be to test many more and establish their activities against multi-resistant strains of various microorganisms as well as their clinical isolates. It would also be important to establish their mechanism of actions in executing these antimicrobial activities and justify their safety for inclusion as functional food additives.
Knottins are also enriched with excellent protein scaffolds that can be explored in the search for new drug entities in cancer drug management and drug delivery. They can equally serve as a carrier in drug delivery, especially in overcoming the blood-brain barrier which could be difficult for several bioactive conventional drug candidates.
Members of the knottin peptides are amenable to molecular imaging applications owing to their rapid renal clearance. This property is relevant to pharmacokinetic studies and could fast track novel peptide drug discovery for the management of the most challenging metabolic diseases. Knottin peptides therefore seem to fulfill the 21st-century requirement as non-invasive diagnostic agents awaiting beneficial exploitation.
In conclusion, reported knottin peptides are mainly gene encoded making them suitable candidates for engineering applications. This increases their potential for numerous therapeutic and nutraceutical applications via synthetic biology approaches and the precision tool of site-directed mutagenesis. Although the database for knottin peptidebased molecules is continuously growing, more elaborate research on knottins is highly encouraged to expand the database and explore novel aspects of their nutraceutical and therapeutic applications.