*Article* **Enhanced Anticancer Activity of** *Hymenocardia acida* **Stem Bark Extract Loaded into PLGA Nanoparticles**

**Oluwasegun Adedokun <sup>1</sup> , Epole N. Ntungwe 2,3, Cláudia Viegas 4,5,6 , Bunyamin Adesina Ayinde 7, Luciano Barboni <sup>8</sup> , Filippo Maggi <sup>9</sup> , Lucilia Saraiva <sup>10</sup> , Patrícia Rijo 2,11,\* and Pedro Fonte 4,5,12,13,\***


**Abstract:** *Hymenocardia acida (H. acida)* is an African well-known shrub recognized for numerous medicinal properties, including its cancer management potential. The advent of nanotechnology in delivering bioactive medicinal plant extract with poor solubility has improved the drug delivery system, for a better therapeutic value of several drugs from natural origins. This study aimed to evaluate the anticancer properties of *H. acida* using human lung (H460), breast (MCF-7), and colon (HCT 116) cancer cell lines as well as the production, characterization, and cytotoxicity study of *H. acida* loaded into PLGA nanoparticles. Benchtop models of *Saccharomyces cerevisiae* and *Raniceps ranninus* were used for preliminary toxicity evaluation. Notable cytotoxic activity in benchtop models and human cancer cell lines was observed for *H. acida* crude extract. The PLGA nanoparticles loading *H. acida* had a size of about 200 nm and an association efficiency of above 60%, making them suitable to be delivered by different routes. The outcomes from this research showed that *H. acida* has anticancer activity as claimed from an ethnomedical point of view; however, a loss in activity was noted upon encapsulation, due to the sustained release of the drug.

**Keywords:** anticancer activity; cytotoxicity; *Hymenocardia acida*; nanoencapsulation; nanoparticle; plant extract; PLGA

**Citation:** Adedokun, O.; Ntungwe, E.N.; Viegas, C.; Adesina Ayinde, B.; Barboni, L.; Maggi, F.; Saraiva, L.; Rijo, P.; Fonte, P. Enhanced Anticancer Activity of *Hymenocardia acida* Stem Bark Extract Loaded into PLGA Nanoparticles. *Pharmaceuticals* **2022**, *15*, 535. https://doi.org/ 10.3390/ph15050535

Academic Editor: Ilkay Erdogan Orhan

Received: 21 March 2022 Accepted: 22 April 2022 Published: 26 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Cancer is generally referred to as a lethal disease characterized by the uncontrolled growth and replication of cancer cells. It can occur in most organs of the multicellular organism and is reported as one of the major public health challenges. Additionally, it is the principal cause of morbidity worldwide among all age groups [1–4]. Cancer is the second leading cause of death in developing countries and the leading cause of death in developed countries [5]. Population growth, aging as well as the adoption of lifestyles associated with smoking, drinking, lack of physical exercise, and consumption of chemically contaminated foods have caused an increase in cancer incidence in developing nations [2,6,7]. Statistical reports show that by the year 2000, 10 million new cases of cancer had emerged with an increase of 25% every decade. Mortality rates associated with cancer might increase from 6 million to 16 million between the years 2000 and 2050, with 17 million and 7 million novel cases from developing and developed countries, respectively [8–10].

The management of cancer has been challenging despite the numerous methods of modern treatments available. These include radiotherapy, surgery, immunotherapy, and chemotherapy which can be used alone or in combination. Localized cancers are usually treated by surgery and radiation while cancer cells that have metastasized to other parts of the body are treated using chemotherapy, such as alkylating agents, antibiotics, hormones, and antimetabolites [8,10,11].

Despite the cytotoxic attributes of chemotherapeutic agents, they have significant limitations. For example, they display numerous side effects by affecting proliferating normal cells localized in the hair, bone marrow, and gastrointestinal tract. Other limitations include low absorption rate, development of secondary malignancy, high cost of drug/treatment, insolubility, instability, and tumor drug resistance.

All these limitations impose the search for natural drugs with improved efficacy, selectivity, reduced toxicity, and low secondary effects inherent in cancer management [12].

Plants are natural sources of drugs and have been used as medicines for at least 60,000 years. They can produce secondary metabolites with a wide range of pharmacological properties, including anticancer activity. They can be used as crude and/or their derived natural products or compounds and have been useful in cancer treatment, research, and development [13–15]. *Hymemocardia acida* Tul (Hymenocardiaceae) is a dioecious and deciduous shrub, mostly found in the Savannah region of the Southwestern part of Nigeria, normally 6–10 m in height. It is characterized by contorted and stunted growth, and it is widely known and used in African trado-medicine. It is called "heart-fruit" in English, "enanche" in Idoma, "ikalaga" in Igbo, "ii-kwarto" in Tiv, "emela" in Etulo, "Uchuo" in Igede, "jan yaro" in Hausa, "yawa satoje" in Fulani, and "Orunpa" in Yoruba [16–18]. Ethnomedicinal information suggests that the plant is used traditionally to treat hemorrhoids, chest pain, eye infection, migraine, skin diseases, and several infections, and as a poultice to treat abscesses and tumors [16,17,19]. Phytochemical studies indicate that these therapeutic applications result from their varied composition of secondary metabolites such as alkaloids, terpenoids, glycosides, flavonoids, saponins, and tannins [17].

Although experimental findings have shown that many natural products have a strong therapeutic value, their poor solubility and bioavailability (at the target organ) have been a challenge over time. Another problem associated with the use of conventional plant extractbased formulations is the presence of toxicity to other organs and tissues. To overcome this, some scientists have used the "green chemistry" approach to nanoparticle production that includes clean, non-toxic, and environmentally friendly methods. NP synthesized via green synthetic routes are highly water soluble, biocompatible, and less toxic [20]. Other strategies using hybrid systems combining nanoparticles and ionic liquids may be also used to improve the delivery of poorly soluble drugs [21,22].

Nanotechnology by the nanoencapsulation of natural products in a polymer to improve drug delivery to cancer targets has gained considerable interest over the past decades [17,23,24]. Thus, polymer-based drug delivery systems allow the control of drug release, enhance effective drug solubility, minimize drug degradation, contribute to reduced drug toxicity, and facilitate control of drug uptake, which significantly contributes to the therapeutic efficiency of a drug. Poly (lactic-co-glycolic acid) (PLGA) based nanoencapsulation has been shown to possess numerous advantages over other conventional delivery devices based on high biocompatibility, biodegradability, drug protection from degradation, sustained and controlled drug release, linkage of other molecules with PLGA for better interaction with biological materials, and the possibility to target specific organs or cells. Furthermore, PLGA will be degraded into nontoxic substances and the breakdown products are lactic acid and glycolic acid, which are hydrophilic, diffusible, and rapidly metabolized in the human body [25–27]. PLGA has also shown good results in improving the bioavailability of drugs delivered by the oral route, a non-invasive route that may be promising in cancer treatment [28], hence, the choice of PLGA base encapsulation in this research.

This research, therefore, aims to evaluate the cytotoxic activity of crude *H. acida* methanol stem extract using both benchtop assays as well as human cancer cell lines (breast, colon, and lung cancer cell lines). Additionally, nanoencapsulation of the extract was carried out using the sulforhodamine B (SRB) assay and reviewing comparative studies on the nanoencapsulated extract and crude extract on breast, colon, and lung cancer cell lines. This method allows the determination of the cell density, based on the measurement of cellular protein content and the cytotoxicity screening of compounds with adherent effect to 96-well format [29].

#### **2. Results and Discussion**

Surgery and or radiotherapy have been great tools for the management of diverse forms of cancer if diagnosed early. Studies have shown that half of all cancer patients use some form of integrative therapy when cancer cells are not responding to medical procedures to reduce pain as well as improve the overall wellbeing of the patients.

The use of medicinal plants for the treatment of diverse diseases, including cancer, cannot be overemphasized as they have served as the source for compounds of therapeutic importance [30]. Medicinal plants are a source for lead compounds and highly bioactive drugs useful in the management of diseases associated with man and animals [30,31]. Recent studies have shown that 55% of chemotherapeutics are directly or indirectly from natural products [32]. Side effects associated with present cancer therapeutics and increasing cancer cases have prompted the search for novel anticancer agents of plant extract or isolated compounds of natural origin, which needs to be studied using both in vitro and in vivo cytotoxicity models [30,33].

#### *2.1. In Vitro Cytotoxicity Assay of H. acida Using R. ranninus*

In this study, tadpoles (*R. ranninus*) were used in the in vitro cytotoxicity assay of *H. acida* crude extract due to their accessibility, mostly in the rainy season, allowing the simulation of a complete multicellular organism. The *H. acida* extract was exposed to *R*. *ranninus* for 24 h at a concentration range of 20–400 μg/mL. The cytotoxic potential against this model was verified by a reduction in the movement of the tadpoles and confirmed with consequent cessation of movement. The results reveal a significant difference in cytotoxicity activity (*p* ≤ 0.05) in all concentrations of *H. acida* examined relative to 5% DMSO (negative control), which has no cytotoxic effect on *R*. *ranninus*. Moreover, at a concentration of 20 μg/mL, the *H. acida* extract showed 89.52 ± 1.52% bioactivity while concentrations at 40–400 μg/mL indicated 100.00 ± 0.00% cytotoxic potential against *R*. *ranninus* (Figure 1).

**Figure 1.** Effect of crude *Hymenocardia acida* (*H. acida*) extract on % *Raniceps ranninus* (*R. ranninus*) mortality at concentrations ranging from 20–400 μg/mL–an index of cytotoxic effect. A 5% DMSO solution was used as negative control. Each bar represents the mean ± SEM of 10 (ten) independent experiments (*n* = 10). Samples with superscript \* indicate a significant difference at *p* < 0.05 relative to the negative control.

#### *2.2. In Vitro Cytotoxicity Activity of Crude H. Acida Using S. cerevisiae*

The yeast *S. cerevisiae* is one of the widely used eukaryotic models. Its rapid growth and ease of manipulation to evaluate multiple biological effects induced by the drugs under consideration make it a suitable model for cytotoxicity study [34]. In vitro preliminary cytotoxicity study on the crude stem bark extract of *H. acida* was performed against *S. cerevisiae* using nystatin as positive control and 2% DMSO in YPD as blank. The growth rate of *S. cerevisiae* in the blank was considered to be 100%, that is, a zero percentage of inhibition, so its absorbance was maximum at 300 min (0.328). The cells were exposed to *H. acida* extract and nystatin at different concentrations (7.81 to 500 μg/mL) and the absorbance of the specific cell growth rates was measured from 0 to 300 min.

According to the results (Table 1), *H. acida* extract showed a concentration-dependent effect. The extract significantly inhibited *S. cerevisiae* growth in all the concentrations tested over time when compared with Nystatin and negative control (DMSO). Overall, the percentage growth inhibition ranges from 71.70 to 100%. At a concentration of 500 μg/mL, 100% of inhibition was observed similar to Nystatin.


**Table 1.** General toxicity effect of crude extract of *H. acida* on the percentage of growth inhibition of *S. cerevisiae*.

The values above are presented by mean ± SEM of three replicates (*n* = 3). Values with superscript \* indicate a significant difference at *p* < 0.05 when compared to the corresponding percentage inhibition of solvent (DMSO a) for each concentration using one-way analysis of variance (ANOVA) and complemented with the Krustal–Wallis test (non-parametric), <sup>b</sup> = positive control, and <sup>a</sup> = negative control.

Considering that the higher the percentage growth of inhibition, the higher the general toxicity of the extract, we can conclude that *H. acida* extract is toxic against S. *cerevisiae* and *R. ranninus*. The correlation between the results of these two organisms validates their use for preliminary toxicity studies. These results can be supported by the composition of *H. acida* in cyclopeptide alkaloids, namely in hymenocardine, found by Tuenter et al. (2016). In their studies, this compound present in the root bark of *H. acida* showed cytotoxicity activity against MRC-5 cells (human lung fibroblasts) with an IC50 value of 51.1 ± 17.2 μM [35]. Moreover, an in vivo study carried out by Sowemimo et al. (2007) showed that *H. acida* steam bark extract is toxic to brine shrimps and caused chromosomal damage in rat lymphocytes, and consequently that it is mutagenic and has cytotoxic activity [31].

#### *2.3. Phytochemical Study of H. acida*

To understand the chemical composition of the bioactive *H. acida* crude extract, and to identify the main compound responsible for the tested bioactivity, this extract was subjected to several chromatographic techniques. 3β- lup-20(29)-en-3ol (Lupeol) was isolated from this extract (as a colorless crystal, mp 212–214 ◦C) and its structure was confirmed through a comparison of its spectroscopic data (Supplementary Materials) to those described in the literature (Figure 2) [36–38].

**Figure 2.** Lupeol isolated from *H. acida*.

#### *2.4. H. acida-Loaded PLGA Nanoparticles Production and Characterization*

PLGA nanoparticles are used to improve the pharmacokinetics, stability, and delivery of the extract [33]. Therefore, to enhance the drug delivery and therapeutic potentials of *H. acida* crude extract, *H. acida* nanoparticles (HA-Np) and blank nanoparticles (unloaded Np, negative control) were produced and characterized [39].

The mean hydrodynamic particle size of both HA-Np and unloaded Np were 210 ± 3 nm and 193 ± 2 nm, respectively, which showed good method robustness and ability to obtain a nanoparticle size suitable for different delivery routes [40–42]. The nanoparticles were further observed by scanning electron microscopy (SEM) to confirm the nanoparticles size and evaluate its morphology (Figure 3). The nanoparticles presented a spherical shape and smooth surface characteristic of PLGA nanoparticles [43]. No relevant differences were observed between unloaded and HA-Np, demonstrating the robustness of the production method.

The Pdl of the nanoparticles was also determined. Small values of PdI (near to zero) were desirable because this indicates a uniform size distribution and a monodisperse nanoparticle formulation [44]. In the case of HA-Np, a PdI of 0.231 ± 0.050 was obtained to 0.100 ± 0.010 observed in unloaded Np, which implies more heterogeneity between the HA-Np particles and a polydisperse formulation as shown in Table 2. Similar results for nanoparticle size distribution and PdI were obtained by our group in the encapsulation of other drugs [42,45]. Although, this is an expected known result for loaded Np because the particles have to contain the volume of the extract of *H. acida* [40].

**Figure 3.** SEM microphotographs of unloaded PLGA Np (**left**) and *H. acida*-loaded PLGA nanoparticles (**right**).

**Table 2.** Physical–chemical properties and characterization of blank nanoparticles (unloaded Np) and *H. acida* nanoparticles (HA-Np) (*n* = 3, mean ± SEM). Results are significantly different (*p* < 0.05).


The diffusion constant describes the quantity of a substance that is diffusing from one region to another through a unit cross-section per unit time when the volume–concentration gradient is constant. A higher diffusion constant of 2.34 × 10<sup>8</sup> ± 0.07 was observed in *H. acida* nanoparticles (HA-Np) relative to unloaded Np 2.55 × 10<sup>8</sup> ± 0.09, which implies a faster rate of diffusion due to the small particle size of HA-Np. Although the diffusion constant is a physical constant that depends on molecular size, temperature (high surface area to volume ratio), pressure, and other properties of the diffusing substance, a reduced diffusion constant of HA-Np will enhance rapid contact of nanoparticle to the targeted receptor for improved drug delivery. Additionally, both unloaded Np as well as HA-Np nanoparticles obtained were homogenous in aspect and form a homogenous colloidal formulation. Moreover, 61.71 ± 2.17% association efficiency was observed, which is a very good achievement. A similar refractive index of 1.33 ± 0.01 was observed in both blank-Np as well as HA-Np, which implies that light waves will pass through both particles in a vacuum by 1.3328 times slower, which also showed a good nanoparticle formulation as shown in Table 2.

To confirm that the extract is incorporated in the polymeric matrix of the PLGA nanoparticles and to assess drug–polymer interactions upon encapsulation, FTIR analysis of *H. acida* extract, unloaded Np, and HA-Np was carried out (Figure 4). The FTIR analysis is a powerful non-invasive technique to assess the structure of NP and its content [46]. Their data also confirms that the extract is incorporated in the polymeric matrix of the PLGA nanoparticles because the transmittance band in the range 3100–3600 cm−<sup>1</sup> present in the *H. acida* extract is reflected slightly in the HA-Np spectrum. Another characteristic band of the extract is found at 1600 cm−<sup>1</sup> in the HA-Np spectrum. On the other hand, in the HA-Np spectrum, the bands related to the nanoparticles at the 1000–1600 cm−<sup>1</sup> zone are attenuated. All these bands confirm that *H. acida* extract is incorporated in the polymeric matrix of the PLGA nanoparticles. It is also possible to check the spectra of both unloaded Np as well as HA-Np, the intense band relative to the carbonyl groups present in the two monomers of PLGA (C = O stretching vibrations) around 1750 cm<sup>−</sup>1, the band relative to ester bond (C-O-C stretching vibrations) around 1186 cm−<sup>1</sup> and the band relative to C–H stretches around 2285–3010 cm−<sup>1</sup> which does not appear in the *H. acida* extract spectrum [25,40].

**Figure 4.** ATR-FTIR spectra of crude extract of *H. acida* (HA extract), blank nanoparticles (unloaded Np), and *H. acida* nanoparticles (HA-Np).

#### *2.5. Cytotoxic Effect of H. acida and PLGA Nanoparticles on Human Cancer Cell Lines*

PLGA is an FDA-approved polymer known for its biomedical applications in drug delivery due to its versatility, biodegradability, and biocompatibility. It is used extensively to prepare nanoparticles to deliver a wide range of therapeutic agents, including active pharmacological molecules, peptides, and nucleic acids [47].

The PLGA nanoparticles were produced to protect the *H. acida* extracts and to allow controlled release of the extracts into the target cells. Therefore, it is important that the stability and also cytotoxic effect of the *H. acida* extracts are maintained and that the release of the contents of the PLGA nanoparticles occurs promptly. The in vitro cytotoxicity of the *H. acida* extract and HA-Np nanoparticles was assessed using Sulfordiamine (SRB) assay. The results for the cytotoxic effect of *H. acida* crude extract and HA-Np on colon colorectal carcinoma (H460), human breast adenocarcinoma (MCF-7), and lung cancer carcinoma (HCT116) using this assay are shown in Table 3.

**Table 3.** Cytotoxic effect (IC50 (μg/mL)) of *H. acida* nanoparticles in H460, MCF-7, and HCT116 cell lines of *H. acida* and *H. acida* nanoparticles using sulforhodamine B assay after 48 h of treatment. Data are presented by mean ± SEM (*n* = 4).


*H. acida* crude extract had an IC50 (μg/mL) of 20.80 ± 6.10 in the human lung (H460) cancer cell line, 38.70 ± 0.80 in MCF-7, and 42.90 ± 0.20, in colon (HCT116) cancer cell lines. The results obtained for cancer cell lines subjected to *H. acida* extract reveal a good cytotoxic effect of this extract, specifically in the H460 human lung cancer cell line. These

results are in comparison with those obtained by Calhelha et al. [48]. The cytotoxic effect (in GI50 values, μg/mL) of Portuguese propolis samples (collected in Aljezur) against the lung (NCI-H460), breast (MCF7), and colon (HCT15) cancer cell lines (37 ± 1, 47 ± 2, and 50 ± 11) once again affirm the potential that *H. acida* extract is cytotoxic. Those results from *H. acida* were also comparable with the study proceeded by Sharma et al. [49], which reveals similar results from the anticancer activity of essential oil from *Cymbopogon flexuosus* in lung cancer cell lines (IC50 values varied from 49.7 to 79.0 μg/mL for each line) and in colon cancer cell lines (IC50 values varied from 4.2–60.2 μg/mL for each line). Thus, although the mechanisms by which *H. acida* has cytotoxic effects are unknown, it appears that its extracts have an impact on cell viability, and thus in cancer treatment. This impact of the extract on cell viability could be partly explained due to the presence of the isolated lupeol. Lupeol is shown to have cytotoxicity in different cancer cell lines. The anti-leukemic activity of this compound was tested against the K562 cells and it was shown to decrease cell viability [50]. Similar results were observed in other studies against different cancer cell lines where lupeol was cytotoxic against MCF-7, Caco-2, SW620, KATO-III, HCT-116 cell lines [51–54]. Lupeol can thus contribute to the cytotoxicity of *H. acida* extracts.

However, an IC50 > 50 for HA-Nps was observed in all human cancer cell lines, which means a poor activity of *H. acida* loaded in PLGA nanoparticles. One hypothesis for this loss of inactivity might be a result of the delayed release of the drugs (*H. acida*) from the PLGA nanoparticles. To overcome this loss of activity, a lower PLGA concentration could be used since this will result in a thinner cover of the Nps, and the production of highly porous nanoparticles making it easier to release the content [26]. Another hypothesis for this loss of cytotoxic effect of *H. acida* loaded in PLGA nanoparticles could be due to some loss of stability of the extracts during the encapsulation process, and consequently loss of efficacy.

Contrary to our results for the activity of HA-Np, recently, Adlravan et al., in their study on the potential cytotoxic activity of *Nasturtium officinale* extract non-nanoencapsulated (free NOE) and PLGA/PEG nanoencapsulated (NOE-loaded) in human lung carcinoma A549 cells, found that NOE-loaded showed better cytotoxic effects than free NOE. This work reinforces the idea that the nanoencapsulation of the extracts improves the anticancer effects of the therapies, as well as allows a sustained and controlled release of NOE constituents from nanoparticles and increases intracellular concentrations. On the other hand, free NOE easily diffuses through the lipid bilayers, being more rapidly eliminated, leading to lower cytotoxicity on target cells [55].

Based on the above results, additional studies should be performed on the in vitro release study for HA-Np to understand if the extract is difficult to release from the HA-Np. Other concentrations of PLGA or combinations of polymers should also be studied since encapsulation of extracts into nanoparticles is known to be a promising strategy to enhance therapeutic efficiency, and consequently overcome these challenges.

#### **3. Materials and Methods**

#### *3.1. Materials and Cell Lines*

(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) (MTT), rhodamine 123 (Rho123), 5-fluorouracil, Pluronic F-68, and dimethylsulfoxide (DMSO) were from Sigma–Aldrich Chemie GmbH (Paris, France). PLGA Resomer® RG 503 H (was obtained from Evonik Industries, Essen, Germany. Phosphate buffer saline (PBS) was from Merck (Darmstadt, Germany). Fetal bovine serum (FBS) was from Gibco, Alfagene, Carcavelos, Portugal. RPMI-1640 medium (Roswell Park Memorial Institut), DMEM (Dulbecco's Modified Eagle Medium), penicillin–streptomycin solution, antibiotic–antimycotic solution, L-glutamine, and trypsin/EDTA were from PAA (Vienna, Austria). *Saccharomyces cerevisiae* (ATCC 2601) cell culture, yeast extract peptone (YPD), HCT-116 (lung), MCF-7 (breast), and H460 (colon) human cancer cell lines were from the National Cancer Institute (Frederick, MD, USA). All other chemicals used in this study were of analytical grade and were purchased locally.

#### Cell Culture Maintenance

The cells were grown and maintained in an appropriate medium, pH 7.4, supplemented with 10% fetal calf serum, glutamine (2 mM), penicillin (100 units/mL), and streptomycin (100 μg/mL). The cell cultures were grown in a carbon dioxide incubator (Heraeus, GmbH, Germany) at 37 ◦C with 90% humidity and 5% CO2 [56,57].

All cancer cells were cultured in RPMI-1640 medium with ultraglutamine (Lonza, VWR, Carnaxide, Portugal), and supplemented with 10% FBS. Cells were maintained at 37 ◦C in a humidified atmosphere of 5% CO2.

#### *3.2. Botanical Authentication and Extraction*

*H. acida* stem barks were collected from the Iwo community in Osun State, Nigeria. Botanical identification and authentication were carried out at the herbarium section of the University of Benin by Prof. MacDonald Idu (Professor of Phytomedicine and Taxonomy). The voucher specimen (UBH-R633) was deposited at the herbarium unit. The plant was grounded to a coarse powder using a laboratory milling machine. The extraction was carried out in methanol using 1.2 kg of the plant powder and the plant extract was obtained using a Soxhlet apparatus. The crude extract obtained was concentrated using Heidolph Rotavapor (LABORATA 4000) with a speed set at 120 rpm and a reduced temperature of 40 ◦C. The concentrated extract was removed from the round bottom flask with methanol and poured into weighed beakers [58].

#### *3.3. In Vitro Cytotoxicity Assay Using R. ranninus (Tadpoles)*

A preliminary cytotoxicity study was carried out on crude stem extract of *H. acida* using *R. ranninus*. The organisms were collected from pounds at Olomo beach, Uhonmora village, Edo State. Ten *R. ranninus* of similar sizes were placed into different beakers containing 30 mL of the freshwater from the habitat of tadpoles. The volume was completed up to 49 mL with distilled water and the extract was added to a total volume of 50 mL. The extract was tested at 20, 40, 100, 200, and 400 μg/mL dissolved in 5% DMSO. The experimental procedure was performed in triplicate and a control assay was performed using 50 mL containing 1 mL of 5% DMSO in distilled water [23,56,59]. The mortality rates of the tadpoles were observed for a maximum of 24 h.

#### *3.4. Isolation and Structural Characterization of Lupeol*

About 51.90 g of aqueous fraction of *H. acida* was subjected to vacuum liquid chromatography (VLC) using dichloromethane, ethylactetae, and methanol in increasing order of polarity. This yielded four (A to D) VLC fractions, based on similarities in their analytical TLC profile, A (1; 1.13 g), B (2–3; 1.89 g), C (4–6; 4.88 g), and D (7–8; 41.15 g). B was further fractionated by normal phase open column chromatography using Silica gel G (kieselgel 70–230 mesh size) and dichloromethane, ethylactetae, and methanol as eluent with increasing polarity. Detection was carried out using non-destructive (visible light and UV light (254 and 365 nm)) followed by spraying with concentrated sulphuric acid and heating at 110 ◦C). This resulted in seven fractions (BF12–8). Fraction BF3 obtained from column chromatography was subjected to a series of purification using preparative-TLC and this resulted in a colorless crystal, lupeol (12.4 mg).

The 1D and 2D NMR analysis of the compound were carried out using a Bruker Fourier spectrometer (600 MHz). The compound was dissolved in deuterated chloroform. 1H and 13C chemical shifts are expressed in part per million (ppm) while coupling constant (*J*) as Hertz (Hz) (Supplementary Materials).

#### *3.5. In Vitro Cytotoxicity Assay Using Saccharomyces Cerevisiae*

Further preliminary cytotoxicity study was carried out on the crude stem extract of *H. acida* using *Saccharomyces cerevisiae* (*S. cerevisiae*). Approximately 1.0 × 10<sup>7</sup> cells per mL of *S. cerevisiae* cell cultures were obtained by inoculating *S. cerevisiae* grown on YPD medium (yeast extract 1%, peptone 0.5%, and glucose 2%) containing 1.5% agar into 20 mL of YPD

and placed into an incubator 30 ◦C without agitation for 16–20 h. About 0.5 × 10<sup>6</sup> cells were transferred into 4 mL disposable cuvettes containing YPD medium and aliquots of stock solution of plant extract to obtain concentrations of 7.81 μg/mL, 15.6 μg/mL, 31.2 μg/mL, 62.5 μg/mL, 125 μg/mL, 250 μg/mL, and 500 μg/mL to a total volume of 2.2 mL. Nystatin, a known antifungal was used as the positive control while YPD medium and 5% DMSO were used as the negative controls. The cuvettes were incubated in a Heidolph Incubator 1000 with a shaker at 30 ◦C and 230 rpm agitation to ensure homogeneous suspensions for 5 h. Initial absorbance was measured at the start time (0 min) and every 60 min. The assay was performed in triplicates for each concentration. The reproducibility of the results was analyzed by repeating the assay on three different days. The absorbance at 525 nm of each sample (cell cultures) over the time (0–300 min) was measured. Growth curves were obtained from the number of cells per mL of YPD medium over time; the percentage growth inhibition rate of *S. cerevisiae* in the presence of *H. acida* stem extract and nystatin was determined. Statistical analysis was performed using one-way analysis of variance (ANOVA) and the Krustal–Wallis test (non-parametric) for comparison between groups. The values are presented as mean ± SEM; significant difference at *p* < 0.05 was considered. [57].

#### *3.6. Production of H. acida Loaded PLGA Nanoparticles*

*H. acida* loaded PLGA nanoparticles (HA-Np) were produced by solvent-evaporation o/w single emulsion technique [60]. Crude extract containing 20 mg of *H. acida* was added to 5 mL acetone: methanol (8:2), along with 50 mg PLGA resulting in *H. acida* organic solution. Then, this organic phase solution was added in a dropwise manner into a 10 mL aqueous solution containing the stabilizer Pluronic F-68 1% (*w*/*v*). This mixture was sonicated for 30 s at 70% of amplitude in a Q125 Sonicator (QSonica Sonicators, Newtown, CT, USA). The formed emulsion was then subjected to evaporation under reduced pressure for organic solvent removal. The formulations were washed three times and resuspended in ultrapure water. Then, the samples were freeze-dried for further use. Blank nanoparticles (unloaded Np) were also produced following the same procedure.

#### *3.7. H. acida Loaded PLGA Nanoparticles Characterization*

The freeze-dried samples were reconstituted with ultrapure water at the desired concentration and were lightly shaken in a vortex for 2 min for complete homogenization. The mean hydrodynamic particle size, polydispersity index (PdI), diffusion constant (D) and refractive index, and viscosity (cP) were evaluated by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS ZS (Malvern Instruments, UK). Each sample of unloaded Np and HA-Np formulation was analyzed in triplicate at 25 ◦C.

The drug loading into PLGA nanoparticles was quantified by evaluating the association efficiency percentage (%AE) by an indirect method, where the amount of *H. acida* encapsulated into PLGA nanoparticles was calculated by the difference between the total amount of *H. acida* extract used in the nanoparticle formulation and considering the free *H. acida* amount in the supernatant after centrifugation of HA-Np formulation in HERMLE Z323K ultracentrifuge at 15,000× *g* during 20 min at 4 ◦C. The quantification of free *H. acida* in the supernatant was performed by Folin–Ciocalteu's method by using a UV-Visible spectrophotometer.

The %AE of HA-Np was determined by the following Equation (1):

$$\% \text{AE} = \frac{\text{Total amount of H.acida} - \text{Free H.acida in supernantant}}{\text{Total amount of H.acida}} \times 100\tag{1}$$

The morphology of the PLGA nanoparticles was evaluated by SEM using a FEI Quanta 400 FEG SEM (FEI, Hillsboro, OR, USA). In a prior observation, the nanoparticles were placed on metal stubs, and vacuum-coated with a layer of Gold/Palladium for 60 s with a current of 15 mA.

#### *3.8. Fourier Transform Infrared Spectroscopy Spectroscopy*

The *H. acida* extract, HA-Np, and unloaded Np were evaluated by ATR-FTIR. All spectra were collected from 64 scans, in the 4000–500 cm−<sup>1</sup> range at 4 cm−<sup>1</sup> resolutions, on an ABB MB3000 FTIR (Zurich, Switzerland). All spectra were area-normalized for comparison using the Origin 8 software (OriginLab Corporation, Northampton, MA, USA).

#### *3.9. In Vitro Cytotoxicity Assay against Human Cancer Cell Lines*

The crude extract and nanoencapsulated *H. acida* extract were subjected to in vitro cytotoxicity assay using human cancer cell lines involving semiautomatic procedure using sulforhodamine-B (SRB) assay, as described earlier [29,30,61,62]. They were tested in different cancer cell lines: colon colorectal carcinoma (HCT116), human breast adenocarcinoma (MCF-7), and lung cancer carcinoma (H460). The procedure involves growing human cancer cell lines in tissue culture flasks at a temperature of 37 ◦C, 5% CO2 as well as 90% relative humidity in a complete growth medium. Flasks with a subconfluent stage of growth were selected and cells were harvested by treatment with trypsin-EDTA.

Cells were plated in 96-well plates at a density of 10,000 cells/100 μL cells/well and incubated for 24 h. *H. acida* and encapsulated samples were added to the 96-well plates. The extracts were tested at 10, 30, and 100 μg/mL, and prepared in DMSO (the final DMSO concentrations were between 0.001% (lowest) and 0.5% (highest)). The effect of the samples was analyzed following 48 h incubation, using the sulforhodamine B (SRB) assay. Briefly, following fixation with 10% trichloroacetic acid from Scharlau (Sigma–Aldrich, Sintra, Portugal), plates were stained with 0.4% SRB from Sigma–Aldrich (Sintra, Portugal) and washed with 1% acetic acid. The bound dye was then solubilized with 10 mM Tris Base and the absorbance was measured at 540 nm in a microplate reader (Biotek Instruments Inc., Synergy, MX, USA). The concentration of *H acida* and nanoencapsulated *H. acida* extract that causes a 50% reduction in the net protein increase in cells (IC50) was determined. Data are mean ± SEM of 4–5 independent experiments [62].

#### *3.10. Statistical Analysis*

All data collected from the entire study were analyzed using *Microsoft Excel* and GraphPad Prism 7 (developed by Dr. Harvey Motulsky, San Diego, USA). Relevant tables, charts, and descriptive statistics were used to present the pertinent points of the study. Data were expressed as the mean ± SEM. The data were subjected to statistical analysis using one-way analysis of variance (ANOVA) and complemented with the Krustal–Wallis test (non-parametric).

#### **4. Conclusions**

*H. acida* possesses significant toxicity in *S. cerevisiae* and *R. ranninus models.* It had *the* highest cytotoxicity (IC50 of 20.80 ± 6.10 μg/mL) against the lung cancer cell lines. The solubility of this extract was successfully improved through nanoencapsulation. However, a loss in cytotoxicity was observed with IC50 = >50 for all the human cancer cell lines tested. This may be due to the sustained delay in the release of the extract from the nanoencapsulation. The present results show that *H. acida* can be a promising source for possible anticancer compounds. Further research is ongoing to identify more bioactive principles using bio-guided isolation procedures, identify the mechanism of action and structure–activity relationship in the bioactive principle(s), and improve the methods for encapsulation and controlled delivery.

**Supplementary Materials:** Lupeol NMR Data. The following are available online at https://www. mdpi.com/xxx/s1, Figure S1: 1H-NMR spectrum of lupeol, Figure S2: 13C spectra of Lupeol, Figure S3: HSQC spectrum of Lupeol, Figure S4: COSY spectrum of Lupeol, Figure S5: HMBC spectrum of Lupeol, Figure S6: Compound isolated from *H. acida* suggested to be 3*β*- lup-20(29)-en-3ol (Lupeol), Figure S7: Numbering and melting point of Lupeol isolated from *H. acida*.

**Author Contributions:** Conceptualization, P.R. and P.F.; methodology, O.A., E.N.N., C.V., B.A.A. and L.S.; validation, P.R. and P.F.; investigation, O.A., E.N.N., C.V., B.A.A., L.B., F.M. and L.S.; writing original draft preparation, O.A. and E.N.N.; writing—review and editing, P.R. and P.F.; visualization, P.R. and P.F.; supervision, P.R. and P.F.; funding acquisition, P.R. and P.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by national funds from Fundação para a Ciência e a Tecnologia (FCT) in the scope of the projects UIDB/04326/2020, UIDP/04326/2020 from CBIOS – Research Center for Biosciences and Health Technologies, and LA/P/0101/2020 of the Research Unit Center for Marine Sciences–CCMAR, and UIDB/04565/2020 and UIDP/04565/2020 of the Research Unit Institute for Bioengineering and Biosciences–iBB, UIDB/50006/2020 (LAQV/REQUIMTE) and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy-i4HB. Cláudia Viegas also would like to thank FCT, Portugal for the Ph.D. grant (2020.08839.BD). Oluwasegun Adedokun thanks the fellowship from PADDIC – ALIES supervised by Patricia Rijo.

**Institutional Review Board Statement:** Not Applicable.

**Informed Consent Statement:** Not Applicable.

**Data Availability Statement:** Data is contained within the article and Supplementary Materials.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Javad Mottaghipisheh 1,\* , Hadi Taghrir <sup>2</sup> , Anahita Boveiri Dehsheikh 3, Kamiar Zomorodian 4, Cambyz Irajie 5, Mohammad Mahmoodi Sourestani <sup>3</sup> and Aida Iraji 6,7,\***


**Abstract:** Many flavonoids, as eminent phenolic compounds, have been commercialized and consumed as dietary supplements due to their incredible human health benefits. In the present study, a bioactive flavone glycoside linarin (LN) was designated to comprehensively overview its phytochemical and biological properties. LN has been characterized abundantly in the *Cirsium*, *Micromeria*, and *Buddleja* species belonging to Asteraceae, Lamiaceae, and Scrophulariaceae families, respectively. Biological assessments exhibited promising activities of LN, particularly, the remedial effects on central nervous system (CNS) disorders, whereas the remarkable sleep enhancing and sedative effects as well as AChE (acetylcholinesterase) inhibitory activity were highlighted. Of note, LN has indicated promising anti osteoblast proliferation and differentiation, thus a bone formation effect. Further biological and pharmacological assessments of LN and its optimized semi-synthetic derivatives, specifically its therapeutic characteristics on osteoarthritis and osteoporosis, might lead to uncovering potential drug candidates.

**Keywords:** flavonoids; linarin; chemotaxonomy; phytochemistry; bioactivities

#### **1. Introduction**

The application of plants for medicinal purposes is as old as humanity itself. Since many of them are considered as functional foods and extensively consumed in folk medicine, their biological and phytochemical assessments are pivotal attitudes [1,2]. By developing human knowledge, the study of plant constituents has led to the discovery of secondary metabolites (phytochemicals) as the major compounds responsible for the bioactivities. These biosynthesized compounds (both volatile and non-volatile) mostly possess defensive roles in plants to assist surviving them against abiotic and biotic stressors [3,4].

Investigation of phytoconstituents has been the target of many researchers in order to determine their health benefits. So far, many phytochemicals have been developed and consumed as successful drugs for the treatment of a diverse range of ailments and disorders, specifically cancer types [5–7]. Among the varied phytochemical classifications, flavonoids have been introduced as one of the largest natural phenolic compounds with broad valuable biological properties [8,9]. Based on the chemical structures, these compounds are divided into six main subclasses: flavones, flavanones, flavonols, flavan-3-ols, isoflavones, and

**Citation:** Mottaghipisheh, J.; Taghrir, H.; Boveiri Dehsheikh, A.; Zomorodian, K.; Irajie, C.; Mahmoodi Sourestani, M.; Iraji, A. Linarin, a Glycosylated Flavonoid, with Potential Therapeutic Attributes: A Comprehensive Review. *Pharmaceuticals* **2021**, *14*, 1104. https://doi.org/10.3390/ph14111104

Academic Editor: Daniela De Vita

Received: 1 October 2021 Accepted: 25 October 2021 Published: 29 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

anthocyanins [10,11]. Even though the phytochemical and biological characteristics of these compounds are being studied [11,12], they are still interesting target molecules to be explored.

Linarin (syn. acacetin 7-O-rhamnosyl(1'→6)glucoside, or acacetin 7-O-rutinoside), as a glycosylated flavone (Figure 1), has been identified from various plant species mainly belonging to the Asteraceae and Lamiaceae families. Regarding the potent bioactivities of this flavonoid reported by several experiments, and the importance of flavonoid consumption as drugs and/or supplements, the present study aims at comprehensively collecting all the phytochemical (i.e., chemotaxonomy and phytochemistry) and biological reports of this flavonoid.

**Figure 1.** The chemical structure of linarin.

The scientific databases including Web of Science, SciFinder, and PubMed were used to find the correlated data by utilizing the keyword of "linarin" within the English-language papers (access date: 25 May 2021).

#### **2. Phytochemistry and Chemotaxonomy of Linarin**

So far, among the 13 plant families containing linarin (LN), Asteraceae and Lamiaceae have been identified as the richest ones. The most LN contents have been reported in various *Cirsium* spp.; however, this compound has also been isolated from the genus *Micromeria* and *Buddleja* belonging to Lamiaceae and Scrophulariaceae, respectively. This glycosylated flavone has mainly been isolated and characterized from alcoholic (methanolic and ethanolic) and hydro-alcoholic extracts. In the following sections, the available data on the phytochemistry of this compound are discussed in detail (Tables S1 and S2).

#### *2.1. Isolation of Linarin from Plant Species*

#### 2.1.1. Asteraceae

LN has been isolated from diverse parts of *Cirsium* spp. By utilizing column chromatography on silica gel (CC) as the final separation step, this compound has been isolated from the methanolic extract of *C. arvense* aerial parts [13]. From the roots of *C. arvense* subsp. *vestitum* via application of vacuum column chromatography [14], and flowers of *C. canum* (L.) using reverse-phase high-performance liquid chromatography (RP-HPLC), LN has been isolated [15].

*C. japonicum* can be considered as one of the richest plant species of LN. Sephadex® LH-20 (SLH) has been applied to the isolation or purification of many flavonoid derivatives [12]. This technique has been employed to isolate LN from the aerial parts of *C. japonicum* [16].

Zhang et al. (2018) isolated LN from *C. japonicum* [17]; in addition, liquid chromato graphy-mass spectrometry (LC-MS/MS) was implemented to characterize it from the hydro-ethanolic (70%) extract [18]. Preparative-HPLC has been exploited to isolate LN from the ethanolic fraction of *C. japonicum* var. *maackii* [19]. Moreover, the methanolic extract of *C. japonicum* var. *ussuriense* (Regel) Kitam. ex Ohwi obtained from the aerial parts

has been subjected to isolate LN by applying the solvent system of CHCl3−MeOH–H2O (25:8:5) in CC on silica gel [20].

LN has been isolated from three other *Cirsium* species: from the leaf and flower methanolic extract of *C. rivulare* using preparative-HPLC [21]; from the flower methanolic fraction of *C. setidens* applying CC on Silica gel [22]; and from the aerial arts using liquid chromatography (LC) [23] and hydro-ethanolic extracts of *C. setosum* (Willd.) MB. (utilizing HPLC) [24]; however, LN has also been identified from the ethanolic extract of this species by applying ultra-performance liquid chromatography-mass spectrometry (UPLC–MS) [25].

*Chrysanthemum* species are considered as one of the major sources of LN. It has been isolated from the methanolic extracts of *Chrysanthemum boreale* (Makino) Makino flowers by utilizing CC on silica [26,27], and the hydro-ethanolic (95%) fractions obtained from the *Chrysanthemum morifolium* Ramat flowers [28].

*C. indicum*, famed as "Ye Ju Hua" in China, has a long history in the treatment of inflammation, hypertension, and respiratory diseases in traditional Chinese and Korean medicine; furthermore, it is traditionally used in tea preparations, tinctures, creams, and lotions [29].

This plant species (*C. indicum*) has been implemented to isolate LN conducted by several studies. It has also been isolated from its flower, using mostly CC on silica gel [30–33], from the dichloromethane extracts of aerial part and methanolic soluble-fraction of the whole part via application of CC on silica gel [34,35].

The purification of LN was also carried out by the solid-liquid extraction method from the hydroethanolic (75%) extract of the same plant species through utilization of various solvents including petroleum ether, ethyl acetate, ethanol, and water [36]. The whole herb and its aerial parts of *C. zawadskii* var. *latilobum* (Maxim.) Kitam. has been reported to possess LN, whereas CC on silica gel was used [37,38].

In the study of Li et al. (2016), high-speed counter-current chromatography (HSCCC) was applied in order to isolate this flavonoid from the hydro-ethanolic extract (80%) of *Flos Chrysanthemi indici* [39], however, it has also been identified from this species as reported by three other groups [40–42].

The whole part methanolic extract of *Artemisia capillaris* Thunb. has been chromatographed by CC on silica gel using CH2Cl2–MeOH (20:1) as solvent systems leading to isolate LN [43]; moreover, this compound was identified in a rare species *Picnomon acarna* (L.) Cass., where its aerial parts were separated in CC [44].

#### 2.1.2. Lamiaceae

Lamiaceae (syn. Labiatae), a large plant family consisting of perennial or annual herbaceous plants and shrubs, is majorly known for their aromatic characteristics [45]. Various genus belonging to this family are considered as natural flavonoid sources including LN. Among them, different species of *Mentha*, *Micromeria*, and *Satureja* can be mentioned.

LN has previously been isolated from the hydro-methanolic (80%) extracts of the flower [46] and aerial parts [47] of *Mentha arvensis* L.; however, it has been reported in *M. haplocalyx* Briq. in the ethyl acetate extracts of the aerial parts of three other *Mentha* species comprising *M. spicata*, *M. piperita*, and *M. villosonervata*, where CC on silica gel was applied as the final chromatography step [48].

Dai et al. (2008) isolated LN from a hydro-ethanolic (75%) soluble-fraction of *Dracocephalum peregrinum* L. aerial parts by hiring extensive chromatographic techniques [49]. This flavone has previously been isolated and characterized from other following species: ethanolic extract of *Leonurus japonicus* Houtt. aerial parts (via CC on silica gel CH2Cl2– MeOH (100:1–0:100) [50] as well as the leaves of methanolic extracts of *Calamintha officinalis* Moench [51] and *Calamintha glandulosa* (Req.) Benth. [52], where in the later study, semiprep-HPLC was utilized as the final separation step by using H2O–ACN (50 to 100%). LN has further been reported in the *Ziziphora clinopodioides* Lam. herb methanolic extract [53].

#### 2.1.3. Scrophulariaceae

The plants belonging to Scrophulariaceae can be considered as the third natural source of LN. Among them *Buddleja* spp. are the richest ones. Previously, from the leaf methanolic extract of *Buddleja davidii* Franch., LN was isolated by the utilization of centrifugal partition chromatography (CPC) and the solvent system of CHCl3–MeOH–H2O (45:33:22) [54]. El-Domiaty et al. (2009) also purified LN from the whole part hydro-ethanolic (95%) extract of *Buddleja asiatica* Lour., while CC on silica gel was used to separate it [55].

*Buddleja cordata* Kunth was subjected to isolate its phytoconstituents and LN was isolated and characterized from the leaves [56] and whole parts [57]. In three other investigations, LN was isolated from mostly alcoholic extracts of the flowers of *Buddleja officinalis* Maxim [58–60]. CC on silica gel using CHCl3–MeOH with ratios of 19:1, 9:1, 8:2 were applied to isolate LN from the aerial parts of *Buddleja scordioides* Kunth [61]. This compound was isolated from two *Linaria* species *L. japonica*, *L. vulgaris*, and *L. kurdica* subsp. *eriocaly*, while the whole parts were chromatographed [62–64].

#### 2.1.4. Miscellaneous Plants

LN has been isolated from the whole part methanolic extract of *Exacum macranthum* Arn. ex Griseb. (Gentianaceae) via the recrystallization method [65]. This phytochemical has also been isolated and identified from *Lobelia chinensis* Lour. (Campanulaceae) [66], *Ginkgo biloba* L. (Ginkgoaceae) [67], *Bombax malabaricum* DC. (Malvaceae) [68], *Avena sativa* L.(Poaceae) [69], *Thalictrum aquilegiifolium* L. [70], and *Coptis chinensis* Franch [71] (Ranunculaceae), *Zanthoxylum affine* Kunth (Rutaceae) [72] and *Lippia rubella* (Moldenke) T.R.S.Silva & Salimena (Verbenaceae) [73].

#### *2.2. Quantification and Qualification Analysis of Linarin in Plants*

By utilization of extensive analytical methods, LN has been qualified and quantified in plant species. So far, the plants belonging to Asteraceae, Lamiaceae, Scrophulariaceae, and Valerianaceae have been reported to be rich in LN content. Table S2 comprehensively lists all the information regarding the fingerprinting analysis of this compound throughout the plant species, however, the following sections describe them in brief.

#### 2.2.1. Asteraceae

Plants in the Asteraceae family, particularly *Cirsium* spp. and *Chrysanthemum* spp., have been characterized as the richest herbal sources of LN. It has been identified throughout six species of the *Cirsium* genus; HPLC coupled to an ultraviolet (UV) detector was used to qualify this compound in the methanolic extract of *C. arvense* [13], along with the report by Demirta et al. (2017), which quantified LN from its root via HPLC-MS (MicroTOF-Q) [14].

The LN content of various soluble-fraction extracted from the flower part of *Cirsium canum* (L.) All. has formerly been analyzed by HPLC-DAD (HPLC-diode array detector). Consequently, the hydro-methanolic (50%) and dichloromethane extracts possessed the highest and lowest contents with 121.75 and 1.94 μg/g, respectively [15].

*Cirsiumjaponicum* (Thunb.) Fisch. ex DC., Japanese field thistle, is renowned in Chinese pharmacopeia for the treatment of inflammation and bleeding [16] as well as application in Korean folk medicine as a uretic as well as antihemorrhagic and antihepatitic medication [74]. Nonetheless, Ganzera et al. (2005) analyzed pectolinarin as the main phytoconstituent of the *C. japonicum* methanolic aerial part extract, and LN was also quantified with a significant content of 0.26–1.15 mg/100 g through different plant samples by employing HPLC-MS [16].

From the alcoholic extracts of two different *C. japonicum* varieties (*C. japonicum* var. *maackii* Maxim and *C. japonicum* var. *ussuriense* (Regel) Kitam. ex Ohwi), LN was detected by employing HPLC-UV [19,20]. Moreover, the mixture of LN and pectolinarin was compared with the methanolic extracts obtained from the leaf (170 mg/g) and flower (20 mg/g) parts of *Cirsium rivulare* (Jacq.) All., whereas HPLC-UV was utilized as the analytical tool [21]. The methanolic extract of *Cirsiumsetidens*(Dunn) Nakai was phytochemically analyzed through HPLC-UV and a significant LN concentration of 120.3 mg/g was measured [22].

*Cirsiumsetosum* (Willd.) Besser ex M.Bieb. has further been elaborated to possess phytochemical contents in four studies. The LN content range of 0.3–2 mg/100 g has been recorded through analysis with HPLC-MS [16]. The methanolic soluble partitions of *Hemistepta lyrate* (Bunge) Bunge flower extracted from different plant samples were analytically assessed, and LN was subsequently quantified (0.06–4.26 mg/g) [26].

In a comparative phytochemical analysis of the ethanolic extract obtained from the *Chrysanthemum morifolium* Ramat. flower, LN was qualified and quantified in three cultivars by using HPLC-DAD-ESI/MS with the contents ranging from 0.117 to 0.583 mg/g [28]. HPLC-DAD analysis of the *Chrysanthemum zawadskii* var. *latilobum* (Maxim.) Kitam. extract showed LN as the marker compound with a 22.8 mg/g extract [75].

*Chrysanthemum indicum* L., as an edible medicinal plant, is famed for its consumption as a food supplement and herbal tea. It has a diverse range of therapeutic applications, specifically in Chinese and Korean folk medicine, for the treatment of immune-related disorders, to heal several infectious diseases, and hypertension symptoms [31]. In several studies reporting its phytoconstituents, LN has also been characterized as the major compounds. He et al. (2016) qualified this compound in the flower methanolic extract via utilization of HPLC-DAD [31].

In a comparative investigation, the phytochemical content of different parts of *C. ndicum* dichloromethane extract was analyzed. As the result, the leaf extract contained the highest LN content (1.47 g/100 g) compared to its stem and flower parts (0.65 g/100 g) [35]. In a similar study, HPLC-MS application led to the fingerprinting analysis of various *C. indicum* parts collected from China; consequently, the root and flower parts indicated the highest and lowest LN amounts (0.344 and 0.052 μg/mg FW), respectively [34]. Furthermore, the hydro-ethanolic extract (75%) of several *C. indicum* samples was phytochemically assessed by HPLC-DAD, and a diverse range of LN concentrations (2.08–55.68%) was recorded [36]. Apart from a qualification study, in which the LN content was determined in the flower methanolic extract of *C. indicum* [32], hydro-ethanolic partition (95%) of the flower and bud parts contained 48.3 mg/g, whilst acetonitrile and water (in formic acid 0.1%) in HPLC-DAD was used as the solvent system [30].

The flower hydro-ethanolic extract (80%) was analyzed via HPLC-UV and LN was accordingly quantified (32.8 mg/g) [39]. The impacts of several extraction conditions on LN contents of the *C. indicum* flower ethanolic extract [40] have been explored; the highest LN yield (88.11%) was measured in the plant samples extracted with 80% ethanol, 2 h of extraction, extraction frequency of three, and solvent to material ratio of 12 mL/g [40].

HPLC-DAD-MS was formerly employed to analyze LN in the hydro-methanolic (60%) extract of *C. indicum* [41]; in addition, a method for fingerprinting analysis of its methanolic extract via HPLC-DAD was introduced by Jung et al. (2012), where it contained 14.6–15.3 μg/g [42].

#### 2.2.2. Lamiaceae

Lamiaceae, as the second richest LN natural source, has been analytically investigated by diverse groups. The occurrence of this flavone glycoside has been confirmed in various *Mentha* species. The flower methanolic fraction of *M. arvensis* was formerly analyzed and 6% of LN content was reported [46]. In a quantification measurement, the hydromethanolic (80%) extract of aerial parts of *M. arvensis* was subjected to HPLC-DAD and UPLC-ESI/Q-TOF/MS, and the LN presence was validated [47].

This compound was further detected in the *Menthahaplocalyx* (Briq.) Trautm. extract (via HPLC-MS/MS) [76]; furthermore, Erenler et al. (2018) comparatively analyzed the ethyl acetate aerial part extracts of three other *Mentha* species including *M. haplocalyx*, *Menthaspicata* L., and *Mentra* x *piperita* L. The highest and lowest LN contents were observed in *M. spicata* and *M. piperita* samples with 42.21 and 0.04 mg/g, respectively [48].

Marin et al. (2001), by using HPLC-UV, detected LN in the leaf hydro-methanolic (80%) extracts of the following plant species: *Acinos arvensis* ssp. *villosus*, *Acinos. Hungaricus* (Simonk.) Šilic, *Calamintha glandulosa* (Req.) Benth., *Micromeria albanica* (K.Malý) Šili´c, *Micromeria cristata* (Hampe) Griseb., *Micromeria dalmatica* Benth., *Micromeria juliana* (L.) Benth. ex Rchb., *Micromeria thymifolia* (Scop.) Fritsch, *Satureja cuneifolia* Ten., *Satureja kitaibelii* Wierzb. ex Heuff., and *Satureja montana* ssp. *montana*. Moreover, the leaf hydromethanolic fraction of *Calamintha officinalis* Moench was phytochemically analyzed through HPLC-UV and water–acetonitrile, and methanol as solvents, and LN with a concentration of 0.27 mg/g was identified [51].

The chemical composition of *Ziziphora clinopodioides* Lam. has further been studied analytically; UPLC-Q-TOF-MS was utilized to detect LN from its hydro-ethanolic (70%) extract [77] as well as the quantification analysis of the herb methanolic fraction, where by applying RP-RRLC (RP-rapid resolution liquid chromatography), the LN contents were detected (3.15–20.55 mg/g) [53].

#### 2.2.3. Scrophulariaceae

*Buddleja* spp. has been analytically elaborated in the case of its phytoconstituents; consequently, LN was detected as one of the main compounds. Fan et al. (2008) identified LN in the leaf methanolic fractions of *Buddleja davidii* Franch. and *Buddleja nitida* Benth., where LC-MS/MS was used. LN concentration of the ethanolic extracts (70%) was assessed in the leaf and in vitro culture samples of *Buddleja cordata* Kunth including white and green callus and root samples by using HPLC-DAD, and the highest content was detected in the leaf ethanolic extract (41.81 ± 5.21 mg/g) [56]. The hydro-ethanolic (70%) fraction of *Buddleja officinalis* Maxim. flower was analyzed via utilization of UHPLC-LTQ-Orbitrap, and LN was consequently qualified [59]. The lyophilized infusion prepared from *Linaria vulgaris* Mill. has previously been experimented through HPLC-UV and LN was quantified with a significant content of 3.84 g/kg drug [63].

#### 2.2.4. Valerianaceae

*Valeriana* spp. has been characterized for its LN content. In [78], by applying HPLC-DAD, they analytically investigated six *Valeriana* species (*Valeriana edulis* Nutt., *Valeriana officinalis* L., *Valeriana jatamansi* Jones, *Valeriana procera* Kunth, and *Valeriana sitchensis* Bong.), with the highest and lowest LN content detected in the methanolic extracts of *V. jatamansi* and *V. edulis* with 0.24 and <0.002%, respectively.

#### 2.2.5. Miscellaneous Plants

The methanolic extracts of the *Lobelia chinensis* Lour. herb belonging to the Campanulaceae family were characterized for its phytochemicals by two analytical tools (LC-MS and HPLC-DAD-MS), and LN was qualified [66]. The hydro-ethanolic (70%) fractions extracted from the inflorescence part of *Coptis chinensis* Franch. (Ranunculaceae family) were assessed via HPLC-MS and LN was identified as the main compounds [71]. Moreover, Rios et al. (2018) identified LN in the hexane, acetone, and methanolic extracts of *Zanthoxylum affine* Kunth (Rutaceae) aerial parts, whilst HPLC−Q-TOF-MS was employed with water and methanol as the solvent systems [72].

#### **3. Biological Properties of LN**

Generally, LN is still a relatively un-investigated drug resource. As a result, in this section, the therapeutic potential of LN and LN containing plants is summarized in Figure 2 and are classified according to which could be useful for potential clinical applications (Table S3).

**Figure 2.** Summary of the biological activities of LN.

#### *3.1. Anti-Alzheimer Properties*

One of the most successful strategies to target Alzheimer's disease is the development of agents that effectively interact with key enzymes involved in cholinergic dysfunction, especially acetylcholinesterase (AChE). This enzyme terminates the action of acetylcholine neurotransmitters and reduces the information transfer across the synapse [79]. Inhibitory potential of LN against AChE extracted from *B. davidii* was evaluated. Bioautographic assessment on LN and related flavonoids showed that the 4 -OMe group as well as the 7-substituted on the B-ring increased the inhibitory potency [54].

Feng et al. (2017) evaluated the AChE inhibitory potential of LN both in vitro and in vivo. In vitro assays using Ellman's colorimetric method exhibited an IC50 of 3.801 ± 1.149 μM [9]. A molecular docking study showed that the 4 -methoxyl group and the 7-O-sugar moiety of LN might be essential for AChE inhibition. Furthermore, ex-vivo study on mice showed that intraperitoneal administration of LN at doses of 35, 70, and 140 mg/kg decreased the AChE activity on the cortex and hippocampus of mice, where the inhibition effects of LN at the high dose were similar to huperzine A as the positive control (0.5 mg/kg) [80].

Pan et al. (2019) reported that 16.7 μg/mL and 50 μg/mL of LN (92% pure) had prominent AChE inhibition in zebrafish [81]. In addition, this compound could significantly improve the recovery of dyskinesia in Alzheimer's disease (animal model). The hydroxyl groups of LN showed strong hydrogen bond interactions with residues Tyr130, Asn85, Trp84, and Asp72 at the anionic subsite of AChE; however, the methoxy flavone segment of LN exhibited π–π interactions with residues Phe331, Trp279, and Phe290 of the peripheral anionic site [81]. The summary of the structure–activity relationship (SAR) of LN against AChE is presented in Figure 3.

**Figure 3.** Structure–activity relationship of LN against AChE.

#### *3.2. Antioxidant Properties*

It is well-documented that oxidative stress and neurodegeneration are destructive in central nervous system (CNS) disorders such as Parkinson's disease and Alzheimer's disease, and protection of cells from oxidative stress toxicity might be beneficial in the abovementioned diseases. In this regard, Santos et al. exhibited the neuroprotective action of the *V. officinalis* extract in neuroblastoma SH-SY5Y of Parkinson's disease. To determine the mechanism of action, in silico molecular docking and molecular dynamics evaluations on apigenin, LN, hesperidin, and valerenic acid as the main compounds of *Valeriana* against hub gene transcripts were performed. Specifically, LN fitted strongly to sulfonylurea receptor-1 (SUR1). The ligand mainly interacted with SER 857, accepting one hydrogen bond and donating two. Most likely, LN can relieve the effects of oxidative stress during ATP depletion due to its ability to binding to SUR1 [82].

The high-performance liquid chromatography-electrospray ionization–mass spectrometry (HPLC–ESI–MS) analysis of *C. japonicum* exhibited chlorogenic acid, LN, and pectolinarin as the main compounds. Furthermore, the protective effect of *C. japonicum* on adrenal pheochromocytoma (PC12) cells in vitro and *Caenorhabditis elegans* (in vivo) were also assessed. The cell viability showed a steady increase until 50 μg/mL and then decreased. Pre-treatment of extracts in PC12 cells significantly prevented intracellular ROS accumulation in comparison to the H2O2 treated control (*p* < 0.05). Under normal growth conditions, treatment with 50 and 100 μg/mL *C. japonicum* extract for 96 h greatly reduced intracellular ROS levels by 37% and 39%, respectively, compared to the control [83].

In the other study, the neuroprotective effect of LN against H2O2-induced oxidative stress in rat hippocampal neurons was assessed. The results showed that H2O2 at 400 μM markedly increased the number of apoptotic neurons, while treatment of the neurons with LN significantly reduced the cell death induced by H2O2 [84].

#### *3.3. Sleep Enhancing and Sedative Effect*

A set of flavonoid glycosides was evaluated for the sedative, sleeping, and locomotor activity. The following potencies were consequently reported 2S-hesperidin > LN > rutin > diosmin\cong 2S-neohesperidin > gossypin ~ 2S-naringin. The SAR proposed the important role of the 1→6 bond between rhamnose and glucose while changing the bond to 1→2, a remarkable decrease in the activity [85].

Nugroho et al. (2013) reported that LN isolated from the *C. boreale* methanolic extract possessed sedative and sleep-enhancing properties [86]. In detail, 10 and 20 mg/kg LN reduced the latency time for the loss of righting reflex caused by pentobarbital injection and delayed the total duration of sleeping time to around 100 min in mice [86].

#### *3.4. Anti-Osteoporosis Activity*

The potential application of LN (isolated from *B. officinalis*) in the response against oxidative stress on osteoblastic MC3T3-E1 cells exposed to H2O2 was evaluated. LN (0.2 μg/mL) significantly increased cell survival, alkaline phosphatase (ALP) activity, collagen content, calcium deposition, and osteocalcin secretion, whereas it decreased the production of the receptor activator of nuclear factor-kB ligand (RANKL), protein carbonyl (PCO), and malondialdehyde (MDA) of osteoblastic MC3T3-E1 cells in the presence of hydrogen peroxide. It was shown that LN exerts antiresorptive actions through the reduction of RANKL and oxidative damage [58]. With more focus toward the antioxidant potential

of LN, in another study, the antiosteoporosis activity of *Flos Chrysanthemi indici* on bone loss in ovariectomized mice was evaluated. All isolated compounds including acacetin, apigenin, luteolin, and LN enhanced the differentiation and proliferation of osteoblasts in MC3T3-E1 cells. They also improved the mRNA levels of runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN), and type I collagen. The AKT signaling pathway was also activated in MC3T3-E1 cells by the four compounds [39].

Li et al. (2016) comprehensively evaluated the molecular mechanism pathway of LN on osteoblast differentiation. First, extracted LN from *Flos Chrysanthemi indici* was assessed on MC3T3-E1 cells (a mouse osteoblastic cell line), and next, the osteoprotective effect of LN in mice was evaluated. LN upregulated osteogenesis-related gene expression including that of ALP, OCN, RUNX2, bone sialoprotein (BSP), and type I collagen. Additionally, it was shown that LN enhanced osteoblast proliferation and differentiation in MC3T3-E1 cells dose-dependently through enhanced ALP activity and mineralization of the extracellular matrix by activating the BMP-2/RUNX2 pathway through protein kinase A signaling in vitro, promoting osteoid gene expression and protecting against OVX-induced bone loss in vivo [87].

In addition, a reducing impact of LN on the RANKL-induced macrophage differentiation into multinucleated osteoclasts and osteoclastic bone resorption through reducing lacunar acidification and bone matrix degradation has been demonstrated. Moreover, LN reduced the transmigration and focal contact of osteoclasts to bone matrix-mimicking RGD peptide, which was accomplished by inhibiting the induction of integrins, integrinassociated proteins of paxillin, and gelsolin, cdc42, and CD44 involved in the formation of actin rings [88].

#### *3.5. Osteoarthritis Treatment*

Osteoarthritis is an age-related joint disease characterized by the degeneration of articular cartilage and chronic pain. Recent studies have confirmed the potential role of antiinflammatory agents to target osteoarthritis. The LN treatment suppressed lipopolysaccharide (LPS), causing the overproduction of nitric oxide (NO), prostaglandin E2 (PGE2), IL-6, and TNF-α in chondrocyte. In addition, the LPS-stimulated expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide nitrate (iNOS) was decreased by LN pre-treatment. The mechanism of action showed the suppression of Toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2) dipolymer complex formation and subsequently intervened in nuclear factor kappa-B (NF-κB) activation [89].

The osteoarthritis mechanism of action of *C. zawadskii* var. *latilobum* extract revealed that the matrix metalloproteinases-1 (MMP-1), MMP-3, MMP-9 and MMP-13 expressions were inhibited by the dose-dependent extract, while expressions of the ECM synthetic genes, COL2A1 and ACAN, and the transcription factor SOX9 were increased to normal condition by the extract treatment dose-dependently. It would be interesting to note that SOX9 is a repressor of ECM-degrading aggrecanases, disintegrin, and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) and ADAMTS-5, and this extract considerably reduced the levels of these enzymes; it is worth mentioning that these potencies can remarkably be correlated to the LN content of the extract possessing 22.8 mg/g [75].

#### *3.6. Ischemia Protection*

In the other study, the effect of LN to inhibit ischemia-reperfusion injury was also evaluated. The primary study confirmed the low toxicity of LN (≤30 μM) against normal H9C2 cells. Further assessments showed that LN could protect myocardial tissue from the injury of ischemia-reperfusion related to activation of the Nrf-2 and PI3 K/Akt signaling pathway. Meanwhile, the antioxidative enzymes, regulated by Nrf-2, were enhanced against the oxidative stress caused by hypoxia-reoxygenation. Importantly, with the inhibition of oxidative stress, some proliferation and apoptosis-related proteins such as NF-κB and cytochrome C were adjusted to support the viability of cells [90].

Furthermore, the anti-inflammatory effect of LN during ischemia-reperfusion-acute kidney injuries was assessed. LN inhibited the acute kidney injury in an in vivo ischemiareperfusion injury model and decreased the expression of interleukin-12 (IL-12) p40 in in vivo and in vitro models. Evaluation on the mechanism of action of LN identified E26 oncogene homolog 2 (ETS2) protein transcription factor for its regulatory action on IL-12 p40 according to microarray analysis and protein–protein interaction. In addition, in silico study showed that the contact area ETS2 is highly conserved and located on a PPI domain of ETS2, which designates that LN may alter the interaction with synergistic proteins in the regulation of IL-12 p40 expression [91].

#### *3.7. Anti-Inflammation Activity*

Anti-inflammatory assessment of forty-two identified compounds from *Chrysanthemi indici* showed that LN, 3,5-dicaffeoylquinic acid, and luteolin with good biocompatibility could be considered as the important contributors to the anti-inflammatory effect of this plant, which decreased levels of NO, TNF-α, IL-6, and PGE2 in RAW264.7 macrophage cells treated with LPS [92].

In another study, the pelvic inflammatory disease with dampness-heat stasis syndrome was investigated and showed that LN at 8–32 μM can significantly inhibit the NO release in a concentration-dependent manner. Results also confirmed that the inhibitory effects on NO production were not due to the cytotoxicity but strong inhibition of NO production. However, the rapid response of LN on the release of TNF-α upon LPS stimulation for 2 h was not significant [93].

#### *3.8. Photoprotective Properties*

Acevedo et al. (2005) studied the photoprotective properties of the methanolic extract of *Buddleja scordioides* as well as verbascoside, LN, and linarin peracetate against UV-B induced cell death using *E. coli* as a cell model. Linarin peracetate (2 mg/mL) protected bacteria efficiently with cell death after 125–250 min, while LN reached cell death until 40–80 min. Interestingly, the sun protection factor (SPF) in guinea pigs was 9 ± 0.3 in the LN (2 mg/cm2) receiving group, while linarin acetate showed a SPF of 5 ± 0.2. The methanolic extract had the smallest SPF (3 ± 0.09), probably due to the low concentration of the photoprotective compound [61].

Examination of the photoprotective properties of *Buddleja cordata* against UVB-induced skin damage in SKH-1 hairless mice showed that 200 μL of 2 mg/mL extract successfully reduced the redness of UVB irradiation to around 120 within 24 h of UV exposure compared to the untreated group with a redness of 300 [94].

#### *3.9. Radioprotection*

In another study, LN isolated from *Chrysanthemum morifolium* flowers significantly decreased the IR-induced cell migration and invasion at a concentration of 5 μM in A549 (human lung cancer cells). LN affected cell viability with an IC50 value of 282 μM. The mechanism was confirmed via inhibiting NF-κB and IκB-α phosphorylation as well as MMP-9 downregulation [95].

#### *3.10. Anti-Apoptosis Potential*

The liver injury and hepatic fibrosis caused by the co-treatment with D-galactosamine (GalN)/lipopolysaccharide (LPS) have been extensively approved. Apoptosis is an important cellular pathological process in GalN/LPS-induced liver injury.

In a study conducted by JooKim et al., the cytoprotective mechanisms of LN against GalN/LPS-induced hepatic failure in mice were evaluated. After 6 h of GalN/LPS injection, the serum levels of alanine aminotransferase, aspartate aminotransferase, TNF-α, IL-6, and interferon-γ as well as TLR4 and interleukin-1 receptor-associated kinase (IRAK) expression were significantly elevated.

LN (50 mg/kg) treatment reversed the lethality induced by GalN/LPS via decreasing the levels of TLR4, IRAK, and suppressing the serum release and hepatic mRNA expression of TNF-α, IL-6, and IFN-γ. In the TUNEL assay, in which the apoptotic cells were monitored, LN also suppressed the increase in the number of apoptotic cells and reduced the cytosolic release of cytochrome c and caspase-3 cleavage.

LN administration increased the level of anti-apoptotic Bcl-xL and ratio of p-STAT3/ STAT3 protein. Furthermore, LN attenuated the expression of FAS-associated death domain and caspase-8, and reduced the pro-apoptotic Bim phosphorylation induced by GalN/LPS.

These results confirmed the potential properties of LN to suppress TNF-α-mediated apoptotic pathways and pro-apoptotic Bim phosphorylation as well as enhance STAT3 activity and increase anti-apoptotic Bcl-xL levels [33].

#### *3.11. Hepatoprotective Function*

HPLC-MS analysis of the *Coptis chinensis* inflorescence extract detected 18 flavonoids and alkaloids derivatives including magnoflorine, thebaine, anonarine 5-OH berberine, jateorhizine, columbamine, coptisine, epiberberine, palmatine, berberine, worenine, and LN. Cell viability assessment of *Coptis chinensis* inflorescence extract and LN in HepG2 cells exhibited IC50 values of 291.15 and 83.88 μg/mL, respectively. Next, the hepatoprotective function of *C. chinensis* and LN showed the reduction in reactive oxygen species (ROS) generation induced by CCl4 in HepG2 cells. LN could also phosphorylate mitogenactivated protein kinases (MAPKs) and upregulate Kelth-like ECH-associated protein (Keap1). The pathways of MAPKs and Keap1 lead to the separation of Keap1 and nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Note that the free Nrf2 transferred to the nucleus and enhanced the expression of phase II detoxification enzymes [71].

#### *3.12. Non-Alcoholic Steatohepatitis Effect*

Nonalcoholic steatohepatitis (NASH), known as liver inflammation and damage caused by a buildup of fat in the liver, is recognized as a common cause of elevated liver enzymes [96]. Investigations of high-fat high-cholesterol diet in rats showed that LN could suppress the expression of mRNA levels of hepatic inflammation cytokines including monocyte chemotactic protein and TNF-α as well as chemokine ligand 1 (CXCL1). A high dose of LN-extract (60 mg/kg) significantly lowered the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and inhibited the activation of the c-Jun N-terminal kinase (JNK) induced by a high-fat high-cholesterol diet [97].

#### *3.13. Anti-Diabetic Effects*

The anti-diabetic effects of the *Chrysanthemum zawadskii* extract at different doses (125, 250, and 500 mg/kg body weight) were investigated every day for five or six weeks. The extraction was standardized and showed 1.32 ± 0.22 mg LN/g extract. Subsequently, the extract significantly decreased fasting blood glucose levels in streptozotocin and streptozotocin and high fat diet-induced diabetic models, even at low doses. In addition, glucose tolerance and insulin tolerance were improved by increasing insulin levels and decreasing hemoglobin A1c (HbA1c) levels in serum [98].

Yang-Ji et al. (2016) also demonstrated that the *Chrysanthemum zawadskii* extract could effectively inhibit the lipase and α-glucosidase enzymes to target the diabetic. This potency might well be correlated with the LN content [99].

Similarly, molecular docking, molecular dynamic, conceptual DFT, and pharmacophore mapping studies against α-amylase and α-glucosidase illustrated that LN could be a beneficial preventative and possibly therapeutic agent against diabetes [100].

#### *3.14. Analgesic and Anti-Pyretic Properties*

MartInez-Vázquez et al. (1996) evaluated the potential analgesic and antipyretic activities of aqueous extract of leaves of *Buddleia cordata* as well as its main compound LN in animal models [101]. The oral administration of an aqueous extract of *B. cordata* and

LN showed a dose-dependent antipyretic activity. Aqueous extract and LN (100 mg/kg) remarkably increased the reaction time of mice by 70% and 55% on heat-induced pain, respectively. Similarly, the antipyretic effect of LN was better than that of the aqueous extract in the yeast-induced hyperthermia test. Three hours after the treatment, LN displayed maximal inhibitory effect with the average temperature being reduced by 1.8 ◦C (50 mg/kg) and 2.0 ◦C (100 mg/kg), whilst the extract reduced hyperthermia by 1.4 and 1.9 ◦C at 100 and 200 mg/kg, respectively [101].

#### *3.15. Spasmolytic Properties*

So far, many studies have approved the remarkable antispasmodic effects of the flavonoids presented in diverse plant species [102–104]. LN also showed an acceptable effect investigated by one study. Phytochemical investigation of the hydro-ethanolic extract of *L. japonicus* resulted in the extraction of three flavonoid glycosides named spinosin, LN, and apigenin-7-O-β-D-glucopyranoside as well as four cyclopeptides and nine alkaloids. These compounds were used in the uterine contraction assay. The findings demonstrated that the flavonoid glycosides (spinosin, LN, and apigenin-7-O-β-D-glucopyranoside) at 50 μM inhibited the contraction of the uterine smooth muscle strips significantly; viscerally, cyclopeptides and alkaloids increased contraction of uterine smooth muscle [50].

#### *3.16. Treatment of Chronic Venous Hypertension*

In a previous experiment, 100 mg/kg/day MPFF (diosmetin, hesperidin, LN, and isorhoifolin) in a chronic venous hypertension animal model showed significant prevention of capillary rarefaction and inflammatory cascade by decreasing the number of sticking leukocytes. MPFF reduced the enlargement of venular diameter as well as maintained venous tone [105].

#### *3.17. Anti-Bacterial Activity*

Corn mint (*Mentha arvensis*) provides a good source of LN and rosmarinic acid. The methanolic extract inhibited the growth of *Chlamydia pneumoniae* CWL-029 in vitro in a dose-dependent manner. The antichlamydial effect of LN showed complete growth inhibition of strain bacterium *Chlamydia pneumoniae*, and inhibited the growth of strain K7 by >60% at 100 μM. Administration of *M. arvensis* extract (20 mg/kg, 3 days) was able to significantly diminish the inflammatory parameters related to *C. pneumoniae* infection in mice (*p* = 0.019) [47].

#### *3.18. Anti-Viral Activity*

Virus is a threat to public health due to its high mutation rate and resistance to existing drugs. Recently, the antiviral activity of LN was investigated to develop new antiviral agents. Evaluation of the flavonoid prescription drug baicalin-linarin-icariinnotoginsenoside R1 was assessed on duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1). The mentioned drug showed an anti-DHAV-1 ability with T and B lymphocytepromoting effects. It also inhibited DHAV-1 reproduction by suppressing its adsorption and release. The mechanism of this antiviral effect showed that the drug at 5 μg/mL increased T and B lymphocyte proliferation. Moreover, according to the in vivo study, the drug stimulated total anti-DHAV-1 antibody secretion in ducklings at the dosage of 4 mg per duckling, but had no significant stimulation impact on the IL-2 and IFN-c secretion [106].

In another study, Chen et al. (2017) assessed the baicalin-LN-icariin-notoginsenoside R1 on DHAV-1 as well as its hepatoprotective and antioxidative potencies. Results showed that the DHAV-1 inhibitory rate of this multi-therapy was 69.3% at 20 μg/mL. The survival rate of ducklings treated by 3 mg drug per duckling (once a day for five days) was about 35.5%, which was significantly higher than that of the virus control (0.0%). Additionally, the degree of oxidative stress, the serum MDA, SOD, CAT, and GSH-Px levels at 8 and 54 h were measured and demonstrated a significant reduction compared to the blank and virus groups, which showed the reduction of oxidative stress in the infected duck [107].

Human immunodeficiency virus (HIV) is an infection that attacks the body's immune system, specifically the white blood cells called CD4. The development of anti-HIV-1 drugs has gained much attention nowadays [108]. It has been shown that human γδ T cells (lymphocytes) consist of Vδ1-TCR-expressing Vδ1+ T cells and Vδ2-TCR-expressing Vδ2+ T cells, which play pivotal roles in bridging innate and adaptive immunity. It was proposed that stimulation Vδ1+ T cells may constitute a new class of anti-HIV drugs, targeting the mucosal compartment to suppress the R5-type of HIV-1. Yonekawa et al. (2019) reported that LN at 100 μg/mL and some flavonoid glycosides, which have both rutinose at the A ring and methoxy substitution at the B ring, can activate host Vδ1+ T cells in HIV patients and can contribute to limiting the R5-type of HIV-1 replication. LN stimulated PBMCderived Vδ1+ T cells to secrete chemokines MIP-1α, MIP-1β, and RANTES and cytokines such as IL-5 and IL-13, which may improve the immune system [109]. Figure 4 exhibits the structure–activity relationship of LN against HIV.

**Figure 4.** Structure–activity relationship of LN against HIV.

In another study, virtual screening on Chinese medicinal compounds was applied to discover novel natural drugs against the influenza A virus using Naïve Bayesian classifiers, and mt-QSAR models. In the selected set, LN exhibited a significant reduction in TNF-α expression to around 40 pg/mL compared to the control group with ~80 pg/mL, whereas it may regulate the expression of cytokines and chemokines, which represent direct and indirect suppression of influenza A [110].

#### *3.19. Anti-Cancer and Anti-Proliferative Activity*

Cancer is one of the major causes of death worldwide, affecting more than 14.1 million people worldwide [111]. Over the past few years, attention has been paid to find potent natural products as anticancer therapeutic agents [79,112,113].

Flavonoids are known to be one of the most popular groups of bioactive phytochemicals with anticancer activity; however, limited study has been conducted to evaluate the activity of LN as anticancer agents [79].

The methanolic extract of *Chrysanthemum indicum* and purified LN exerted antiproliferative activity against human non-small cell lung cancer cells via suppression of Akt activation and induction of cyclin-dependent kinase inhibitor p27Kip1, as evidenced by cell cycle analysis and treatment with LY294002. These findings may indicate the anticancer potential of LN as the core functional constituent of *C. indicum* [114].

Glioma is the most common form of malignant brain cancer with a high mortality rate in humans. NF-κB activity is a common phenomenon in various cancers, resulting in abnormal cell proliferation, malignant transformation, or resistance to cell death. Previously, the anti-cancer role of LN in glioma was tested in vitro and in vivo. LN suppressed glioma cell proliferation and migration by inducing apoptosis, which was through reducing the cell cycle-related signals including survivin, p-Rb, and cyclin D1, while promoting p21, Bax, caspase-3, and poly (ADP-ribose) polymerase (PARP) activation. LN also showed an increase in P53 as an essential tumor suppressor. Moreover, it reduced cellular proliferation of glioma through p53 upregulation and NF-κB/p65-downregulation, thereby inhibiting glioma cell growth [115].

The cytotoxicity of *Jatropha pelargoniifolia* loaded chitosan nanoparticles against A549 human lung adenocarcinoma cells (IC50 = 13.17 μM) was higher than that of the free extract (IC50 = 25.16 μM) and comparable to that of methotrexate (IC50 = 11.84 μM) as an anticancer drug [116].

Oral squamous cell carcinoma is characterized by overexpression of Akt1 (RACalpha serine/threonine-protein kinase) and Akt2 (RAC-beta serine/threonine-protein kinase). It was reported that Akt1 and Akt2 inhibitors can lead to oral squamous cell carcinoma treatment with no affinity toward monoamine oxidase B (MAOB). In silico studies introduced LN as inhibitors of Akt1 and Akt2 with strong binding affinities of 11.5 kcal/mol and 11.1 kcal/mol, respectively, with no affinity toward MAOB, which can be an ideal candidate for oral squamous cell carcinoma treatment [117].

#### *3.20. Negative Biological Results of LN*

#### 3.20.1. Estrogenic Activity

The estrogenic activity of six chemical constituents (apigenin, hispidulin, cirsimaritin, cirsimarin, pectolinarin, and LN) isolated from *Cirsium japonicum* on MCF-7 cells was assessed. Among them, hispidulin and cirsimaritin showed strong estrogen receptor transactivation, while the rest of the compounds had weaker or relatively no effects. The SAR confirmed that estrogen receptor transactivation increases as the number of –OH groups in the flavonoid structure increased [19].

#### 3.20.2. Anti-Fungal Effect

Combined chromatographic techniques were implemented in the phytochemical analysis of *Lippia rubella*, leading to the isolation of several compounds such as lippiarubelloside A and lippiarubelloside B, verbascoside as well as LN. Inhibitory evaluation of LN against some fungal strains such as *Candida albicans* (ATCC 10231) and *Candida parasilopsis* (ATCC 22019) asserted no significant activity (MIC >125 μg/mL), and moderate effects against *Cryptococcus neoformans* and *Cryptococcus neoformans* (MIC: 125 μg/mL) [73].

#### 3.20.3. Anti-Depressant Properties

Depression is a mental health disorder characterized by loss of interest, pleasure, with feelings of sadness, low self-worth, and tiredness, which disturbed sleep or appetite, leading to suicide in severe cases. The exact mechanism of depression is still unknown, and most of the antidepressants act as inhibitors of intracellular monoamine (exp, norepinephrine) reuptake. Additionally, it has been shown the gamma-aminobutyric acid (GABA) levels as well as cortical GABAA receptors decreased in patients with depression.

In this regard, the norepinephrine reuptake of *Cirsium japonicum* and its major constituents (linarin, pectolinarin, chlorogenic acid, luteolin) were evaluated. *Cirsium japonicum* showed an antidepressant effect by significantly reducing the immobile behavior of mice in the forced swimming test, without enhancing locomotor activity in the open-field test. In addition, the *C. japonicum* extract had no effect on monoamine uptake while significantly promoting Cl– ion influx in human neuroblastoma cells and modulating the GABAA receptor. Further evaluation showed that among the major constituents of the *C. japonicum* extract, only luteolin produced antidepressant activity as a positive modulator of the GABA-mediated Cl<sup>−</sup> ion channel complex and LN was almost inactive [118]. Results showed that the antidepressant effect of *Cirsium japonicum* could be due to the luteolin constituent.

#### **4. Perspectives**

#### *Anti-SARS-CoV-2 (COVID-19) Effect*

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a RNA airborne virus infection known as the pathogen responsible for coronavirus disease 2019 (COVID-19) [119]. Millions of COVID-19 patients have been reported thus far; however, there is no concrete evidence on the effectiveness and safety of the specific treatment against SARS-CoV-2 [120,121]. One area that has been affected immensely is the investigation of natural remedies as medications and/or supportive therapies to treat patients with

COVID-19 infection. Mostly, antiviral drugs directly target the infecting pathogen to halt its development [122]. In the case of SARS-CoV-2, the influence of active substances of medicinal plants were surveyed in inhibiting four important druggable targets including S and N proteins, 3CLpro, and RdRp. RdRp controls the replication of SARS-CoV-2 while 3CLpro is the main protease of the virus. Moreover, N and S proteins are responsible for SARS-CoV-2 assembly and attachment, respectively. Molecular docking outcomes of the study revealed that LN, amentoflavone, (-)-catechin gallate, and hypericin had an affinity for these basic proteins, which possess an effective role in SARS-CoV-2 infection [123].

#### **5. Conclusions**

Investigation of plant secondary metabolites with valuable impacts on human health is an attractive and broad research area. Flavonoids, a large family of phenolic compounds due to their pivotal therapeutic effects, have been the subject of many studies. Nowadays, their diverse derivatives are widely consumed as dietary supplements. Although the most renowned flavonoids (i.e., apigenin, luteolin, hispidulin, kaempferol, myricetin, quercetin, naringenin, etc.) are aglycosylated [124], the glycosylated forms are also of interest. It is believed that the glycosylation of flavonoids can lead to the development of their biological features by reducing the probable toxicity and increasing their bioavailability [125].

The present context overviewed a very promising but not well-investigated glycosylated flavone named LN. From the phytochemical viewpoint, the plant genus *Cirsium*, *Micromeria*, *Buddleja*, and *Chrysanthemum* are the major natural sources of LN. This compound demonstrated promising bioactivities through the studies carried out in vitro and in vivo. The encouraging properties of LN have been shown through osteoblast proliferation and differentiation with high anti-arthritis and antiosteoporosis potencies; however, its effect on the treatment of CNS disorders have also been pointed out.

Further phytochemical investigations of different natural sources leading to the isolation and identification of LN as well as exploring the optimized extraction methods can support the implementation of its bioactivity assessments. Complementary biological and pharmacological evaluations (particularly toxicity and clinical trials) of LN and its derivatives are proposed in future in order to develop potential natural-based drugs/supplements with the least side effects.

**Supplementary Materials:** The followings are available online at https://www.mdpi.com/article/ 10.3390/ph14111104/s1, Table S1: Isolation and identification of linarin from plant species; Table S2: Identification and characterization of linarin from plant species; Table S3: Biological properties of linarin. References [126–138] are cited in the supplementary materials.

**Author Contributions:** Conceptualization, methodology, writing, and supervision by J.M.; Data collection by H.T., A.B.D. and M.M.S.; Investigation and writing by K.Z., C.I. and A.I. were performed. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors wish to thank the support of the Vice-Chancellor for Research of Shiraz University of Medical Sciences (Grant No. 24425). This agency was not involved in the design of the study and collection, analysis, and interpretation of data as well as in writing the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

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