Chalcones are also known as benzyl acetophenones. Scientists describe chalcones as α, β-unsaturated carbonyl compounds, which are ketones. These ketones hold two rings namely A and B. These two rings are interconnected by a 3-carbon atom ketone. In Balkan states, plant species loaded with chalcones had been used as therapeutic agents. These plants include Ruscus, Angelica, Piper, and Glycyrrhiza genus. In one research, scientists found out that chalcone induces apoptosis by triggering endoplasmic reticulum (ER) stress through the sulfonation of IRE1α by an enzyme called NADPH oxidase 4 (NOX4). This sulfonation leads to the degradation of mRNA, known as “regulated IRE1α-dependent decay” (RIDD), instead of the typical splicing of XBP1. One consequence of IRE1α sulfonation-induced RIDD is the degradation of miR-23b, which in turn increases the expression of NOX4. The expression of NOX4 is also elevated in breast and prostate cancer tissue. In mouse models treated with chalcone, tumor growth was reduced through the consistent mechanisms involving NOX4-IRE1α sulfonation-RIDD. Additionally, NOX4 activation and IRE1α sulfonation were significantly increased under severe ER stress conditions (29–34).

Flavanols fall under the main subtypes of bioflavonoids. Scientists report that flavanols are found in a variety of food such as cereals, legumes, tea, cocoa, red wine, grapes, fruits, beer, and grapes among others. It is reported that 121 flavanols have been found in plants distributed in 59 species and 29 families. Examples of the species are Pronephrium penangianum, Camellia sinensis, Theobroma cacao, and Astragalus membranaceus. Green tea(Camellia sinesis) catechins are reported to have antioxidant, reproductive system therapeutic effects, anti-inflammatory, antitumor, cardioprotective effects, anti-obesity properties, neuroprotective properties, and so on. In a certain study (35), investigated the effects of catechin on heart function and the expression of a long non-coding RNA called MIAT in two different experimental models: MI/R rats and H/R-induced H9C2 cells. They found that catechin improved heart function in MI/R rats and decreased the expression of MIAT in myocardial tissue. In H/R-induced H9C2 cells, catechin protected against cell apoptosis (programmed cell death), while overexpression of MIAT attenuated this protective effect of catechin. The researchers also discovered that the transcription factor CREB could bind to the promoter region of MIAT, and catechin increased the expression of CREB, resulting in the suppression of MIAT expression. Catechin further enhanced mitochondrial function and reduced apoptosis by activating the Akt/Gsk-3β signaling pathway. Conversely, MIAT inhibited Akt/Gsk-3β activation and promoted cell apoptosis in H/R-induced H9C2 cells. Ultimately, the study concluded that catechin promoted Akt/Gsk-3β activation by inhibiting MIAT expression in H/R-induced H9C2 cells. These findings suggest that catechin has beneficial effects on heart function and mitochondrial function, and its mechanism of action involves the regulation of MIAT and the Akt/Gsk-3β pathway (36–44).

Flavones are found in many plant species. The popular genus from which flavones and their derivatives are found in Scutellaria. The genus belongs to the mint or Lamiaceae family. Plants like mints, lavender, basil, and rosemary belong to this family. It is reported that the Scutellaria genus has 360 species, and these species are distributed in East Asia, Europe, and the United States of America (United States). Among the Traditional Chinese Medicine herbs, Scutellaria baicalensis (Baikal skullcap) has been used to treat insomnia, diarrhea, dysentery, hypertension, inflammation, respiratory infections, and hemorrhage. Different in vitro studies showed that S. baicalensis root extracts have anticancer properties in various types of cancer such as myeloma, lymphoma, colon cancer, brain tumor, and lymphocytic leukemia. Recently, scientists (45) discovered that Apigenin exerts its effects by influencing the immune responses mediated by dendritic cells (DCs) and T-cells. The findings indicate that Apigenin shifts the balance of T-cell responses from pro-inflammatory Th1 and Th17 types toward regulatory T-cell responses. This shift is characterized by decreased expression of T-bet and IFN-γ, and IL-17, as well as increased expression of IL-10, TGF-β, and FoxP3. The protein RelB, a key component of the NF-κB pathway, plays a central role in DC maturation, antigen presentation, and DC-mediated T-cell activation. Apigenin was found to reduce the levels of RelB mRNA and protein, as well as inhibit its nuclear translocation. Furthermore, silencing RelB using siRNA enhanced the effects of Apigenin, confirming the involvement of RelB in Apigenin-mediated regulation of DC biology (21, 46–48).

Scientists say that flavanones’ antioxidant properties are attributed to phenolic moieties in their chemical structure (49, 50). The medicinal properties of flavanones are not limited to antioxidant and antimicrobial activities but also include neuroprotective, antidiabetic, anticancer, anti-psoriasis, and so on. The Erythrina genus is loaded with flavanones, like the Erythrina crista-galli. In 2014, scientists investigated the combined effects of a racemic flavanone called Naringenin (NG) and the protein sericin as blockers of TNF-α. Microparticles of sericin (SMPs) and (R/S) NG-loaded sericin (NGSMPs) were created through spray-drying, and their morphology, particle size distribution, and encapsulation efficiency were examined. The presence of NG did not affect the morphology or particle size distribution of the microparticles. Biological testing was conducted using lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (hPBMC), comparing the effects of (R/S) NG, SMPs, and NGSMPs. At the highest dose (200 μg/mL), NGSMPs exhibited cytotoxicity, surpassing the effects of (R/S) NG alone. Furthermore, while sericin alone did not effectively reduce LPS-induced serum TNF-α levels, NGSMPs loaded with (R/S) NG (93% encapsulation efficiency) significantly demonstrated greater potency than (R/S) NG alone. In conclusion, this study demonstrated the potential of sericin-based microspheres loaded with TNF-α blockers to regulate this cytokine and presents a foundation for developing new topical formulations for treating mid-stage psoriasis (51–57).

Several in vivo and in vitro studies showed cardioprotective properties of flavonols, especially quercetin. In addition, medicinal properties such as anti-diabetic, anticancer, antioxidant, antimicrobial, antiviral, and anti-inflammation were also reported. There are many sources of flavonols. Examples of plant species rich in flavonols are Allium tricoccum and Arabidopsis thaliana. Recently, scientists reported the anti-inflammatory property of quercetin. They investigated the impact of quercetin on macrophages. The results demonstrated that quercetin effectively suppressed the production of various inflammatory molecules, including nitric oxide (NO), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), interferon-inducible protein 10 (IP-10), Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted (RANTES), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), tumor necrosis factor-alpha (TNF-α), leukemia inhibitory factor (LIF), lipopolysaccharide-induced CXC chemokine (LIX), and vascular endothelial growth factor (VEGF). Moreover, quercetin also hindered the release of calcium ions in dsRNA-induced RAW 264.7 mouse macrophages. Additionally, quercetin showed significant inhibition of the mRNA expression of signal transducer and activator of transcription 1 (STAT1) and STAT3 in dsRNA-stimulated RAW 264.7 cells. Collectively, these findings suggest that quercetin possesses beneficial properties in combating viral inflammation by impeding the calcium-STAT pathway, leading to the suppression of NO, cytokines, chemokines, and growth factors in dsRNA-induced macrophages (58–61).

Isoflavones are commonly found in leguminous plant species like Medicago truncatula, Pisum sativum, Phaseolus vulgaris, Glycine max, Trifolium pratense, Trifolium repens, Medicago sativa, and so on. Isoflavone content varies in different plant species. In soybean, for example, isoflavones amount to 1.2 -4.2 mg/dry weight, mainly daidzein, genistein, and their derivatives. Isoflavones are chemoprotective. In addition, isoflavones are used to treat many hormonal disorders, cardiovascular diseases, osteoporosis, and/or menopausal symptoms. Genistein as a member of isoflavones has antioxidant, anticancer, antibacterial, antiviral, anti-inflammatory, and antidiabetic properties. Scientists investigated the impact of genistein, a compound with chemopreventive properties, on the progression of existing prostate cancer. The researchers used transgenic mice with prostatic intraepithelial neoplasia lesions and found that feeding them genistein resulted in a more aggressive advancement of prostate cancer, including increased numbers of poorly differentiated prostates and a higher incidence of pelvic lymph node metastases. The same effects were observed in human prostate cancer cells treated with genistein, which led to enhanced cell proliferation, invasion, and activity of matrix metalloproteinase-9. The researchers also identified osteopontin as a key mediator of genistein’s effects. However, a high pharmacologic dose of genistein counteracted these effects. The study suggests a biphasic regulation of prostate cancer growth and metastasis by genistein, highlighting the need for cautious examination of its effects on hormone-dependent cancers in a chemotherapy context (62–65).

Anthocyanidins are found in many fruits, especially dark-colored fruits. The list of the plant species rich in anthocyanidins is long: Brassica campestris L, Ipomoea batatas L, Rheum spp., Raphanus sativus L, Allium cepa L, Solanum tuberosum L, Capsicum annuum L, Arachis hypogaea L, Pisum sativum L, Lens culinaris Medic, Zingiber officinale, Solanum melongena L, Daucus carota L, Brassica spp., Phaseolus spp., Asparagus officinalis L, Vaccinium myrtillus L, Rubus spp., Fragaria x ananassa Duch, and so on. Anthocyanidins have medicinal properties like anticancer, antidiabetic, neuroprotective, anti-inflammatory, antipsychotic, and so on. In one study (66), investigated the cardiovascular effects of malvidin, a polyphenol found in red grape skins. They quantitatively analyzed the polyphenolic content of red grape skin extract and found a high concentration of malvidin. Using isolated rat hearts, they observed that the extract and malvidin induced positive effects on heart contractions, relaxation, and coronary dilation. The mechanism of action involved the activation of the PI3K/NO/cGMP/PKG pathway, leading to increased levels of intracellular cGMP and the phosphorylation of various proteins involved in cardiovascular regulation. They also discovered that malvidin exhibited cardioprotective properties against ischemia/reperfusion damage. These findings contribute to understanding the cardiovascular benefits of food-derived polyphenols and highlight malvidin as a potential cardioprotective compound, which is significant for nutraceutical research (67–72).

There are several ways employed to extract bioflavonoids from different sources (73). Yao and colleagues reported the extraction of 21 bioflavonoids using ultra-high-performance liquid chromatography and rapid microwave extraction in tandem with a triple quadrupole-linear ion trap mass spectrometry (1). Mangang et al. (74) extracted bioflavonoids from the bark of Albizia myriophylla using a microwave-assisted method. Chahyadi & Elfahmi examined the impact of different extraction techniques on rutin yield from cassava leaves. The duo extracted rutin from the leaves using microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), reflux, boiling, and maceration. Manzoor and colleagues reviewed different methods of luteolin extraction like UAE, MAE, soxhlet, maceration, reflux, ultrasound microwave-assisted extraction, supercritical fluid extraction (SFE), high-speed counter-current chromatography extraction techniques, hydrodistillation, and enzyme-assisted extraction. Needless to say, each and every extraction technique has pros and cons (74–77).

Vitrification (Figure 2) is a technique that allows glass state formation without crystallization. This is one of the major cryopreservation principles. Scientists employ this technique to preserve plants. However, the major challenge is that most plant species have high water content and/or lack glassy state-inducing substances. Such plants do not have the ability to vitrify naturally. Only a few plants can vitrify naturally. Cryoprotectants used during the vitrification must have at least three principal properties: (a) minimal toxicity to the plant cells, (b) high glass-sate forming capacity, and (c) sufficient dehydration strength to vitrify the plant cells among others. The commonly used cryoprotectants used are glycine, dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, sucrose, and sorbitol in this order. The concentrations vary though. There are different versions of vitrification techniques like encapsulation-vitrification, and droplet-vitrification (14, 95).

Scientists came up with plant vitrification solutions (PVS), which are chemicals mixed in different proportions to optimize the vitrification technology. These solutions are used in modern plant cryopreservation techniques because of their protective effects and their osmotic characteristics. The PVS solutions increase viscosity at lower temperatures, leading to an amorphous glassy state, without forming ice. The common four PVS solutions are PVS1, 2, 3, and 4. It is reported that Uragami pioneered the use of PVS1(0.06 DMSO, 0.22 Glycine, 0.13 Ethylene glycol, and 0.13 Propylene glycol) to culture cells derived from Asparagus officinalis L mesophyll tissue. We suggest one can look at an article written by Zamecnik et al. (14) for a better understanding of PVS. The solutions come into contact with the cell and then form PVS solute compounds changing the glass transition temperature, and/or with the bound water, which hinders the intercellular ice crystals-formation (14, 96–99).

Osmoprotective solutions are concocted by mixing non-penetrating and low or moderately-concentrated penetrating cryopreservants. In plant cryobiology, the most used osmoprotective solution is known as loading solution 1 (LS1). This solution is a mixture of 0.4 M sucrose and 2.0 M glycerol. Other loading solutions are composed of ethylene glycol, DMSO, sucrose, and glycerol in different combinations and concentrations. As such, there is LS2, 3, and 4. The purpose of the LS is to prepare the plant specimens biophysically to osmotic stress by exposure to a moderately concentrated osmoticum, prior to severe dehydration with highly concentrated cryoprotectants (CPAs) (91, 100).

The slow freezing technique involves subjecting plant cells to a mixture of membrane-permeating cryopreservants (pCPAs) and non-cell membrane permeating CPAs (npCPAs) before cooling at a controlled rate to an average low temperature and lastly plunging the vials into liquid nitrogen. Scientists report that the pCPAs prohibit intracellular ice formation whereas npCPAs cause net efflux of water from the cells. The use of one type of CPA mitigates post-thaw regrowth rates to less than 30%, whereas both types in concert scored more than 60% regrowth as demonstrated for wheat (Triticum aestivum) and brome (Bromus inermis) cells. In spite of the protective properties of the CPAs, the direct plant cell freezing in liquid nitrogen has not yet so far resulted in their regrowth following thawing. This implies that an intermediate cooling step between –25°C and –40°C is mandatory. The commonly used cooling rate is – 0.2°C min^–1 (91, 101–107).

Scientists argue that the success of plant cryopreservation depends on factors like the origin of plant cell cultures (PCCs) and the balance between the mixtures of pCPAs and npCPAs among others. PCCs are typically sourced from plants through an intermediate callus. The callus is an unorganized mass of dedifferentiated plant cells that form if excised tissues (explants) are subjected to a mixture of auxins and cytokinins in vitro. Auxins prohibit organogenesis whereas cytokinins promote cell division. As a result, the presence of these two phytohormones promotes continuous cell division without cell differentiation. A callus is normally generated from flowers, leaves, petioles, and roots. Seedlings, buds, and young leaves are not conducive to generating a callus because they contain actively dividing cells called meristems. In one research, the Gentiana tibetica PCCs were prepared in a 0.4 M sucrose CPA for a week. The follow-up protocol employed was vitrification. The scientists observed that the post-thaw viability of the cells was 90%. In a Gentiana. tibetica generated PCCS that were subjected to 0.4 M sorbitol for 2 days, a follow-up technique was slow freezing. The post-thaw viability of these cells was found to be 3%. It was also observed that adjusting cell density properly resulted in plant growth following cryopreservation. There is what is referred to as the minimum cell density threshold which is the cell density that results in plant growth following cryopreservation. The most common packed cell volumes (PCVs) for slow freezing and encapsulation-dehydration techniques used are 20-30%. These PCVs yield around 80% plant growth following cryopreservation. The usual PCVs for the vitrification technique are within the range of 40–50% (107).

Different preservation techniques have different impacts on plants’ content and bioactivity of bioflavonoids. In one study, after cryopreservation, the antioxidative activity of two Hypericum species that have been grown in vitro namely H. rumeliacum Boiss. and H. tetrapterum Fr. was estimated. Following a vitrification-based cryopreservation procedure, both species were successfully regenerated. In response to cold temperatures, flavonoids assemble in leaves and stems. Cold stress is said to boost the biosynthesis of flavonoids, according to recent reports. Additionally, flavonoid compounds were primarily blamed for some of the beneficial qualities of Hypericum species. The increased flavonoid production in the two experimental species is an unanticipated positive outcome of cryopreservation. In H. tetrapterum tissues, there was a 237% increase in flavonoid content compared to the control. Total antioxidant activity was found to vary between control values in cryopreserved H. tetrapterum while increasing (33% above control) in regenerated H. rumeliacum plants, which showed the presence of oxidative stress and a lower tolerance for cryopreservation (108).

In another investigation into the effects of various drying methods, including freeze-drying and hot air oven drying, on flavonoids, specifically, flavanone glycosides, antioxidant potential, and total phenol content in immature dropped fruits of Citrus reticulata Blanco, bioactive compounds were identified and quantified. Using high-performance liquid chromatography (HPLC), flavonoids were measured. Azino-bis [3-ethylbenzthiazoline-6-sulfonic acid] (ABTS), 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), the ferric reducing ability of plasma (FRAP), and total phenol concentration were used to test the antioxidant activity. In comparison to hot air-dried samples (17.99%), freeze-dried samples of 12- and 14-mm size retained the highest amounts of hesperidin flavonoid content (27.03 and 27.20%) and higher phenolic content, which ranged from 50.54 to 54.19 mg GAEL^–1 (109).

Freeze-drying (Figure 3) is a widely used method of encapsulation that involves dehydrating a frozen sample through sublimation. It is particularly suitable for preserving sensitive bioactive compounds since it does not subject the substances to high temperatures like spray-drying. Freeze-dried products can be quickly and easily reconstituted, making them valuable for emergency situations such as administering antibodies and vaccines promptly. Additionally, the freeze-drying technique is simpler compared to other microencapsulation methods, as it involves a limited number of steps (111).

The encapsulation process is crucial for maintaining the stability of freeze-dried powders, and the choice of coating or carrier materials plays a significant role in its efficiency and physicochemical properties. These materials encompass the active core materials during microencapsulation, and they can be sourced from natural or synthetic options, such as maltodextrins, modified starches, proteins, dextrins, among others. The encapsulating agents’ selection is vital in ensuring the overall effectiveness of the process and the preservation of the encapsulated substances. Milea and colleagues investigated the encapsulation efficiency of anthocyanins extracted from sweet cherry skins using two different wall materials, namely whey protein isolate (WPI) and chitosan. The study found that the encapsulation efficiency was approximately 77.68% with a narrow variation of ±2.57%. Comparing this to a similar study by Oancea et al., who worked with sour cherry skins, the encapsulation efficiency of anthocyanins microencapsulated in WPI and acacia gum was reported to be 70.28% with a standard deviation of ±2.17%. Furthermore, when a single protein was used as the wall material, the encapsulation efficiency ranged from 44.79 to 64.69% as reported by Mamani-Matsuda et al. In a separate investigation conducted by Stănciuc et al. with grape skin extract, the encapsulation efficiency was found to be remarkably higher, reaching 94% when using acacia gum and 99% when employing pectin as the wall material. These findings highlight the influence of both the fruit source and the choice of wall material on the encapsulation efficiency of anthocyanins, which are valuable natural compounds with potential health benefits (112–115).

In one study, scientists aimed to determine if phenolic compounds from Vaccinium species leaves, both as microencapsulated powder and aqueous extracts, are bioaccessible after being exposed to in vitro simulated digestion. The effect of maltodextrin and glucose microencapsulation carriers on the phenolic content of the extracts was also evaluated. Prior to encapsulation, the emulsions’ viscosity was demonstrated at a shear stress of 50 s^-1 dilatant, and above this point, they exhibited Newtonian behavior with a final viscosity ranging between 1.024 and 1.049 mPas. The samples’ final microencapsulation yield varied from 79 to 81%. The phenolic content dropped following gastrointestinal digestion, despite the microencapsulated forms offering a targeted release at the intestinal level. The microencapsulated extracts’ bioaccessibility displayed greater values than those of their non-encapsulated counterparts (116). Many other studies are also conducted on the microencapsulation of bioflavonoids (10, 111, 117–127).

Environmental conditions and microbial attacks are the two major challenges that affect bioflavonoids’ shelf-life in the food industry. Scientists report that cryopreserving and encapsulating the bioflavonoids extend their shelf-life. For example, Saqueti and colleagues conducted an experiment to investigate the shelf-life of cryopreserved and microencapsulated bioactive compounds from Acerola pulp over a period of 6 months. The findings demonstrated that microencapsulation was more effective in retaining the antioxidants present in Acerola pulp compared to other preservation methods utilized. This was attributed to the higher concentration of bioactive compounds (BCPs) within the microencapsulated form and the subsequent decrease in pH. Moreover, the results indicated that microencapsulation outperformed lyophilization in preserving the desired compounds in Acerola pulp over a span of 6 months, as evidenced by the superior BCPs content in the microencapsulated samples. These results highlight the potential of microencapsulation as an attractive option for the food industry (129, 130).