**Figure 5.** Encapsulation of terpenoids directly affects invasomes size. (**A**) Example of a small representative section of an original cryo-electron micrograph used for measuring invasomes' size. (**B**) Control invasomes without terpenoid showing a mean radius of 40 ± 15 nm. (**C**) Thymol-comprising invasomes revealed a smaller radius of 33 ± 18 nm compared to control. (**D**) Production of invasomes with menthol resulted in an increased invasome radius of 59 ± 22 nm. (**E**) Like thymol, camphor-invasomes also shower a smaller invasome size (30 ± 16 nm radius) compared to control. (**F**) With a mean radius of 43 ± 17 nm, the size of invasomes with cineol was similar to control. Frequency plots of the radius distribution of the invasomes. A fit with the Gaussian function is displayed as a red line.

As a third parameter for characterization of our newly produced invasomes, thickness of the liposomal bilayer was measured by evaluating cryo-electron micrographs (Figure 6A). We observed no significant differences (Mann–Whitney test, one-tailed) in the liposomal bilayer thickness of control invasomes (4 ± 0.5 nm, Figure 6B) compared to menthol-invasomes (4 ± 0.3 nm, *p* = 0.2482, Figure 6D) and invasomes encapsulating camphor (4 ± 0.5 nm, *p* = 0.2987, Figure 6E). Invasomes produced with thymol revealed a slightly but significantly increased bilayer thickness of 5 ± 1 nm (Figure 6C) compared to control (\*\* *p* < 0.01), camphor-invasomes (\* *p* < 0.05) and cineol-invasomes (\*\* *p* < 0.01, Mann–Whitney test, one-tailed). In contrast, cineol-comprising invasomes showed a minor decrease in bilayer thickness (4 ± 0.5 nm, Figure 6F) in comparison to the other approaches.

**Figure 6.** Encapsulation of terpenoids does not affect the bilayer thickness of invasomes. (**A**) Example of a small representative section of an original cryo-electron micrograph used for measurement of the bilayer thickness. (**B**–**F**) A similar liposomal bilayer thickness was observable for control invasomes (4 ± 0.5 nm), thymol-invasomes (5 ± 1 nm) menthol-invasomes (4 ± 0.3 nm), camphor-invasomes (4 ± 0.5 nm) or cineol-invasomes (4 ± 0.5 nm). Frequency plots of the bilayer thickness distribution of invasomes. A fit with the Gaussian function is displayed as a red line.

Taken together, invasomes with terpenoids showed a shift towards unilamellar vesicles, except for cineol with a similar distribution between unilamellar and bilamellar vesicles comparable to control. While the bilayer thickness of invasomes was comparable in all approaches, preparation of invasomes with thymol and camphor led to significantly smaller invasomes compared control. On the contrary, menthol-comprising invasomes were significantly enlarged and we observed the radius of cineol-invasomes to be comparable to control.

#### *3.3. Invasomes Encapsulating Thymol, Camphor and Cineol Show Bactericidal Activity against S. aureus in a Growth-Inhibition Zone Assay*

After successfully producing and characterizing terpenoid-comprising invasomes, we assessed their potential bactericidal activity against *S. aureus* in a growth-inhibition zone assay. After exposure of *S. aureus* to different terpenoid-invasomes overnight, we determined the size of the inhibitory zones. Control invasomes without terpenoids did not result in growth inhibition of *S. aureus* (Figure 7A). Compared to unaffected control, we observed a clearly visible growth inhibition of *S. aureus* exposed to invasomes comprising thymol, camphor or cineol (Figure 7B,D,E). Interestingly, invasomes encapsulating menthol did not affect growth of *S. aureus* (Figure 7C). Statistical evaluation

of the measured areas of inhibition validated a strong and significant inhibition of bacterial growth by thymol-containing invasomes compared to all other terpenoid-invasomes and control (Figure 7F). However, invasomes comprising camphor or cineol still caused a significant increase in the zone of inhibition compared to control and menthol-invasomes, which revealed no zone of inhibition (Figure 7F).

**Figure 7.** Invasomes encapsulating thymol, camphor and cineol show bactericidal activity against *S. aureus* in a growth-inhibition zone assay. (**A**) Control invasomes without terpenoids did not affect bacterial growth. (**B**) Strong growth inhibition of *S. aureus* exposed to invasomes comprising thymol. (**C**) Invasomes encapsulating menthol did not affect growth of *S. aureus.* (**D**–**E**) Growth inhibition of *S. aureus* exposed to invasomes comprising camphor and cineol. (**F**) Statistical evaluation of the measured zones of inhibition validated the significant inhibition of *S. aureus* growth by thymol-containing invasomes compared to all other terpenoid-invasomes and control. \* *p* 0.05 was considered significant (Mann–Whitney test, one-tailed). (n.s. means not significant)

In Table 1, the particle size of different terpenoid formulations is depicted as measured in Figure 5. When the formulations are sorted from the highest antibacterial activity (thymol) to the lowest (cineol), as measured in Figure 7, it becomes evident that the terpenoids with the highest antibacterial activity have the highest polydispersity index also (Table 1).

**Table 1.** Invasome Particle Size and the Polydispersity Index.

#### *3.4. Flow Cytometric Analysis of Cell Death Validate High Bactericidal Activity of Thymol-Loaded Invasomes against Gram-Positive S. aureus*

To validate the strong bactericidal activity of thymol-invasomes against *S. aureus* in the growth-inhibition zone assay, we also assessed the anti-bacterial activity of thymol-invasomes quantitatively using flow cytometry. Bacterial cell death was measured by the DNA-intercalating dye Propidum Iodide (PI), which is incorporated only by dead cells. Prior to this analysis, we determined the encapsulation efficiency and loading capacity of thymol-invasomes to ensure proper encapsulation of the terpenoid. Here, we observed an encapsulation efficiency of 47 ± 13% as well as a loading capacity of 0.5 ± 0.1% for invasomes encapsulating thymol. Compared to untreated negative control (4–9% cell death), 0.5 mg/mL thymol packaged in invasomes resulted in a profound cell death of 70% (Figure 8A). Exposure of 1 mg/mL invasome-encapsulated thymol even resulted in 75% bacterial cell death (Figure 8B). Since 2 mg/mL thymol packaged in invasomes resulted in only 9% PI-stained *S. aureus* (Figure 8C), we additionally assessed the cell count per second during the flow cytometric measurement. Here, only around 1000 cells/second were observed in the *S. aureus* population treated with 2 mg/mL thymol-comprising invasomes, whereas the cell count for control conditions ranged around 40,000 cells/second (Figure 8D). Treatment of *S. aureus* with 0.5 mg/mL or 1 mg/mL invasome-encapsulated thymol resulted in cell flow of around 33,000 cells/second (Figure 8D). Thus, we suggest that 2 mg/mL thymol packaged in invasomes already resulted in a nearly complete cell death of *S. aureus* prior to PI-staining and following flow cytometric measurements. We conclude invasomes encapsulating thymol to be strongly bactericidal against Gram-positive bacteria such as *S. aureus*.

**Figure 8.** Flow cytometric analysis of cell death validate invasomes encapsulating thymol to be strongly bactericidal against Gram-positive bacteria like *S. aureus*. (**A**,**B**) Compared to untreated negative control, 0.5 mg/mL or 1 mg/mL thymol packaged in invasomes resulted in a profound cell death depicted by PI-staining. (**C**) 2 mg/mL thymol packaged in invasomes resulted in only 9% PI-stained *S. aureus*. (**D**) Assessment of cell flow revealed only around 1000 cells/second in the *S. aureus* population treated with 2 mg/mL thymol-comprising invasomes, suggesting a nearly complete cell death prior to following flow cytometric analysis. PI: Propidium iodide.

#### *3.5. Thymol-Loaded Invasomes Do Not A*ff*ect Survival of Gram-Negative E. coli*

Next to Gram-positive bacteria such as *S. aureus,* we assessed the potential anti-bacterial activity of thymol-invasomes against Gram-negative species such as *E. coli*. Notably, 0.5 mg/mL thymol packaged in invasomes resulted in only 0.2% PI-stained dead cells (Figure 9A). Treatment of *E. coli* with 1 mg/mL or 2 mg/mL invasome-encapsulated thymol only led to 6% or 5% cell death (Figure 9B,C). With regards to the low amount of PI-stained cells, we also assessed the cell count per second during the flow cytometric measurement. Here, we observed no relevant effects of the different concentrations of invasome-encapsulated thymol on the growth of *E. coli (*cell count per second, Figure 9D). In summary, the invasomes comprising thymol produced in this study are highly bactericidal to Gram-positive *S. aureus*, but do not affect survival of Gram-negative *E. coli*.

**Figure 9.** Invasomes encapsulating thymol are not bactericidal against Gram-negative bacteria such as *E. coli*. (**A**–**C**) Compared to untreated negative control, treatment of *E. coli*. with 0.5 mg/mL, 1 mg/mL or 2 mg/mL thymol packaged in invasomes did not result in elevated amounts of cell death. (**D**) Assessment of cell flow revealed no relevant effects of the different concentrations of invasome-encapsulated thymol on the growth of *E. coli*.

#### *3.6. Growth-Inhibition Zone Assay Shows Strong Bactericidal Activity of Cineol Invasomes against E. coli*

In addition to thymol-invasomes, we assessed the potential bactericidal activity of invasomes encapsulating menthol, camphor and cineol against *E. coli.* In line with our flow cytometric analysis of cell death, thymol-invasomes revealed no elevated growth inhibition of *E. coli*, similarly to control-invasomes without encapsulated terpenoids (Figure 10A,B). While menthol-invasomes led to a slight growth inhibition of *E. coli* (Figure 10C)*,* invasomes encapsulating camphor showed no bactericidal activity against *E. coli* (Figure 10D). Notably, exposure of *E. coli* to invasomes loaded with cineol resulted in a strong and significant growth inhibition (Figure 10E,F).

**Figure 10.** Invasomes encapsulating cineol reveal bactericidal activity against *E. coli* in a growth-inhibition zone assay. (**A**,**B**) Control invasomes without terpenoids or thymol invasomes did not affect bacterial growth. (**C**,**D**) While menthol-invasomes led to a slight inhibition of *E. coli* growth, camphor-invasomes did not affect growth of *S. aureus.* (**E**) Strong growth inhibition of *E. coli* exposed to invasomes comprising cineol. (**F**) Statistical evaluation of the measured zones of inhibition validated the significant inhibition of *E. coli* growth by cineol-containing invasomes compared to all other terpenoid-invasomes and control. \* *p* 0.05 was considered significant (Mann–Whitney test, one-tailed). (n.s. means not significant)

We next determined the potential bactericidal activity of non-extruded terpenoids as a control to our encapsulation approach. In contrast to terpenoids encapsulated in invasomes (Figures 7–10), application of non-extruded terpenoids did not result in growth inhibition of *E. coli* or *S. aureus* (data not shown). In summary, the invasomes comprising thymol produced here are highly bactericidal to Gram-positive *S. aureus,* while cineol-invasomes affect the survival of Gram-negative *E. coli* (Figures 7–10).

Potential antibacterial mechanisms of invasome formulations with terpenoids are depicted in Figure 11.

Although terpenoids such as limonene, cineole or beta-citronellene have been widely used for formulation of invasomes [26,27], they were mostly applied as permeation enhancer for transdermal delivery and bioavailability and not as bioactive components [2]. In the present study, we focused on the production of invasomes with terpenoids as the bioactive components to inhibit bacterial cell growth. We found our terpenoids-invasomes to be bactericidal against Gram-positive *S. aureus*, with increasing efficiency from cineol- and camphor-invasomes (moderate bactericidal activity) to thymol-invasomes showing the strongest bactericidal effects. These findings are in line with the commonly reported bactericidal activity of thymol, camphor and cineol [10,12,13]. Interestingly, Mulyaningsih and colleagues reported that exposure of MRSA even to high concentrations of cineol does not inhibit multi-resistant *S. aureus*. However, a combination of the terpene aromadendrene with cineol resulted in reduced bacterial cell growth [34]. Extending these findings, we show that encapsulation of cineol into invasomes alone is sufficient for inhibiting growth of *S. aureus* without the application of additional terpenes. In accordance to the strong bactericidal effects of thymol-invasomes observed here, encapsulation of thymol into other nanocarriers such as ethylcellulose/methylcellulose nanospheres

was also reported to preserve its anti-bacterial activity against *S. aureus* [35]. In contrast to invasomes encapsulating thymol, camphor and cineol, we observed no anti-bacterial effects for menthol-invasomes against *S. aureus*, which is contrary to the already described bactericidal activity of menthol [11,12]. With regard to the very low water-solubility of menthol, we suggest the invasomal packaging of menthol to be challenging, in turn, affecting its bactericidal activity. In this line, we observed an increased average size of menthol-invasomes compared to all other terpenoid-invasome preparations and control-invasomes lacking terpenoids. In addition polydispersity index as a measurement of the uniformity of invasome size distribution, with a higher value resulting in a broader distribution, was highest with thymol (0.3) and camphor (0.3), suggesting a correlation to antibacterial activity.

**Figure 11.** Schematic view on invasomal packaging of thymol and its selective bactericidal activity against Gram-positive *S. aureus*.

Next to Gram-positive bacterial species such as *S. aureus*, we also investigated potential anti-bacterial properties of our invasomes and particularly, thymol-invasomes on Gram-negative *E. coli*. Here, cineol-comprising invasomes led to a strong inhibition of *E. coli* growth, which is in line with our previous observations [13]. In contrast to the strong bactericidal effects against *S. aureus*, cell growth of Gram-negative *E. coli* was not affected by thymol-invasomes. These observations are contrary to the findings by Salvia-Trujillo and colleagues reporting a bactericidal activity of essential oils of thyme (containing thymol) after incorporation into nano-emulsions [35]. In particular, nano-emulsions comprising essential oils of thyme with heterogeneous droplets sizes between 10 nm to 500 nm were shown to reduce growth of *E. coli* [36]. However, the authors applied unfractionated essential oils, suggesting a synergistic action of many terpenes to be necessary for bactericidal activity against Gram-negative species. Interestingly, Trombetta and coworkers demonstrated that *S. aureus* appears to be far more sensitive to thymol than *E. coli* [37], which is in line with our present data. The authors reported a minimal inhibiting concentration of 5.00 mg/mL thymol for *E. coli* [37], suggesting the concentration of up to 2 mg/mL thymol in the invasomes applied here to be not sufficient for inhibition of *E. coli* growth. Furthermore, *S. aureus* is known to secrete pore-forming toxins (PFTs), which were shown to mediate the release of encapsulated clove oil from liposomes. In particular, Cui and coworkers reported that PFTs form pores within the liposome membranes, allowing release of the encapsulated clove oil and facilitating its antibacterial activity. On the contrary, liposomal packaged clove oil had no bactericidal effects on *E. coli*, which does not secrete PFTs and thus prevents the release of antibacterial essential oil from the invasome [38]. Our present observations may suggest a similar mechanism for thymol-invasomes leading to its selective activity against *S. aureus* (Figure 11). In addition, electrophoretic mobility measurement revealed a harder surface of *E. coli* compared to *S. aureus* [39]. The softer surface of *S. aureus* mainly comprising peptidoglycan may facilitate entry of thymol-invasomes into the bacterial cells more easily compared to Gram-negative *E. coli* (Figure 11). Accordingly, we achieved a highly efficient killing of *S. aureus* with only 0.5 mg/mL thymol encapsulated in invasomes in the present study (Figure 11).

In summary, we demonstrate the successful production of invasomes encapsulating thymol, menthol, camphor or cineol and show a strong selective activity of thymol-invasomes against Gram-positive *S. aureus*. As a further benefit of our approach, encapsulation of terpenoids into nanocarrier systems such as invasomes is suggested to increase stability and protect against environmental factors causing degradation [2,33,40]. Here, liposomes composed of lipoid S100 and cholesterol were reported to retain considerable concentrations of isoeugenol, pulegone, terpineol, and thymol liposomes even after 10 months [29]. The application of soy lecithin liposomes comprising cinnamon oil was further shown to improve stability of the essential oil and extend the bactericidal action time [20]. In addition, the application of invasomes was particularly found to elevate the stability of the encapsulated compounds (reviewed in [24]).

There are several nanocarrier systems, encapsulating terpenoids, which are systematically reviewed in [2]. The formulation systems encapsulating terpenoids include polymer-based systems such as nano-capsules, nano-particles, nano-fibers and nano-gels. Furthermore, lipid-based systems are frequently used (67% of the formulations), presumably due to the low toxicity. A subgroup of lipid systems are the vesicular systems, which include invasomes. The most investigated biological activity of terpenoids in nano carrier systems is the anti-inflammatory action. Invasomes were used as anti-acne treatments, hypertension treatment and photosensation therapy [2].

Antimicrobial activity was reported with nano capsules with essential oils from lemon grass, nano emulsions with tea tree oil and penetration-enhancing vehicles with essential oil from Santolina insularis. Here, we report a novel antimicrobial application of invasome formulations with terpenoids.

We conclude that our findings might provide a promising approach to increase the bioavailability of terpenoid-based drugs and might be applicable for treating severe bacterial infections such as MRSA in the future. In this regard, the major treatment aims of our formulations include a broad spectrum of applications, ranging from mucosal infections in airway diseases to systemic infections such as sepsis. In this direction, we have previously shown that patients with chronic rhinosinusitis have increased levels of S. aureus-containing biofilms in the nose [13]. Growth of *S. aureus* biofilms on the nasal mucosa could be inhibited by 1,8-cineol. Here, we extend these findings to thymol-containing invasomes, which have superior antibacterial activity than formulations with 1,8-cineol (see Figure 7). Taken together, an invasome formulation as described here, containing thymol might be useful as an aerosol spray for pre-operative nose cleaning and might have fewer side effects in comparison to disinfectants directly applied on the mucosa. As a general use, it might be envisaged that invasomes containing thymol or other terpenoids could be employed to treat infected surfaces as in nose, lung and skin wounds. Finally, invasomes containing terpenoids might be used in addition or as an alternative to antibiotics.

**Author Contributions:** Conceptualization, A.H., C.K. and B.K.; validation, A.H., C.K. and B.K.; formal analysis, B.P.K., J.F.W.G., I.E. and A.H.; investigation, B.P.K., I.E., J.F.W.G. and R.D.; resources, A.H., C.K., B.K. and A.P.; data curation, A.H., C.K., B.K. and J.F.W.G.; writing—original draft preparation, B.P.K. and J.F.W.G.; writing—review and editing, A.H., C.K., B.K., I.E., R.D. and A.P.; visualization, B.P.K. and J.F.W.G.; supervision, A.H., C.K. and B.K.; project administration, A.H., C.K. and B.K.; funding acquisition, A.H., C.K., B.K. and A.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the University of Bielefeld and received no external funding.

**Acknowledgments:** The excellent technical help of Angela Kralemann-Köhler is gratefully acknowledged.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

© 2020 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 (http://creativecommons.org/licenses/by/4.0/).

## *Article Aristolochia trilobata***: Identification of the Anti-Inflammatory and Antinociceptive E**ff**ects**

**Dayana da Costa Salomé 1, Natália de Morais Cordeiro 1, Tayná Sequeira Valério 1, Darlisson de Alexandria Santos 2,3, Péricles Barreto Alves 2, Celuta Sales Alviano 4, Daniela Sales Alviano Moreno <sup>4</sup> and Patricia Dias Fernandes 1,\***

Received: 30 March 2020; Accepted: 21 April 2020; Published: 6 May 2020

**Abstract:** *Aristolochia trilobata*, popularly known as "mil-homens," is widely used for treatment of stomach aches, colic, asthma, pulmonary diseases, diabetes, and skin affection. We evaluated the antinociceptive and anti-inflammatory activities of the essential oil (EO) and the main constituent, 6-methyl-5-hepten-2-yl acetate (sulcatyl acetate, SA). EO and SA (1, 10, and 100 mg/kg, p.o.) were evaluated using chemical (formalin-induced licking) and thermal (hot-plate) models of nociception or inflammation (carrageenan-induced cell migration into the subcutaneous air pouch, SAP). The mechanism of antinociceptive activity was evaluated using opioid, cholinergic receptor antagonists (naloxone and atropine), or nitric oxide synthase inhibitor (L-NAME). EO and SA presented a central antinociceptive effect (the hot-plate model). In formalin-induced licking response, higher doses of EO and SA also reduced 1st and 2nd phases. None of the antagonists and enzyme inhibitor reversed antinociceptive effects. EO and SA reduced the leukocyte migration into the SAP, and the cytokines tumor necrosis factor and interleukin-1 (TNF-α and IL-1β, respectively) produced in the exudate. Our results are indicative that EO and SA present peripheral and central antinociceptive and anti-inflammatory effects.

**Keywords:** *Aristolochia trilobata*; sulcatyl acetate; antinociceptive effect; anti-inflammatory activity

#### **1. Introduction**

Inflammation and pain continue to be major problems in individuals. Both of them are a normal response of the body against invasion and/or damage. Inflammation can develop in response to an invasion by a microorganism or by physical damage and is a critical protective action to injury or infection. This phenomenon presents the five cardinal signs (i.e., redness, heat, swelling, pain, and loss of function) [1]. Pain, one sign of inflammation, is an international health problem, affecting about one in five individuals. Both situations affect almost 20% of worldwide population. Drugs used to treat the symptoms can be the non-steroidal anti-inflammatory drugs (NSAIDs) and/or opioids (specifically to pain treatment). However, both groups present a large variety of side effects. For example, opioid drugs are responsible for abuse and dependence affecting 2.1 million people, and NSAIDs also increase the risk of gastrointestinal bleeding and cardiac events [2].

In Central and South America, *Aristolochia trilobata* L. (Aristolochiaceae), popularly known as "mil-homens", is widely used in folk medicine and is an important medicinal plant [3,4]. Conditions such as stomach ache, colic, poisoning, asthma, pulmonary diseases, diabetes, and skin affections have been treated with different species from *Aristolochia* genus [5,6]. Previous studies have shown that a chloroform extract of *A. trilobata* leaves had antiphlogistic potency similar to that of indomethacin (a non-steroidal anti-inflammatory drug) [7].

The present work aims to demonstrate the traditional use of *A. trilobata* for treatment of inflammation and pain. For these purposes, we decided to obtain the essential oil of *A. trilobata* stems from enriching volatile substances, enabling a scientific study to evaluate their possible involvement in the anti-inflammatory and antinociceptive effects testing it in a well-known thermal (hot-plate) model of nociception and carrageenan-induced cell migration as a model of inflammation. We also tried to identify the mechanism by which *A. trilobata* presents its effect.

#### **2. Experimental Section**

#### *2.1. Animals*

All protocols used Swiss Webster mice (male, 20–25 g, 8–10 weeks) donated by the Instituto Vital Brazil (Niterói, Rio de Janeiro, Brazil). National Council for the Control of Animal Experimentation (CONCEA), the Biomedical Science Institute/UFRJ, and Ethical Committee for Animal Research approved the protocols used (DFBCICB015–04/16). The animals were maintained under standard conditions (a room with a 12 h light–dark cycle at 22 ± 2 ◦C, 60% to 80% humidity, and with food and water provided ad libitum).

#### *2.2. Plant Material*

Stems of *Aristolochia trilobata* were collected in Estância/SE, Brazil, in October/2011 (Geographic coordinates: S 11◦ 14 22.4 and W 037◦ 25 00.5) and received a voucher # ASE 23.161 deposited in the Herbarium of the Federal University of Sergipe. Stems of *A. trilobata* were cut in small pieces and crushed in a fourknife mill (Marconi, model MA680). The essential oil (EO) was obtained after hydrodistillation of 200 g of stem (in 1500 mL of distilled water) along 3 h and with help of a Clevenger-type apparatus. After physically separating oil and water, the first one was dried over anhydrous sodium sulfate and filtered. EO was stored in a freezer until further analyses and assays. Identification of constituents of EO was performed as Santos and collaborators [8].

#### *2.3. Drugs and Treatments*

Acetylsalicylic acid (ASA), atropine sulfate monohydrate, dexamethasone, and L-nitro arginine methyl ester (L-NAME) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Morphine sulfate and naloxone hydrochloride were kindly provided by Cristália (São Paulo, Brazil) and formalin was purchased from Isofar (Rio de Janeiro, Brazil). EO was dissolved in pure oil in order to prepare a stock solution (100 mg/mL). From this stock solution, intermediate solutions were prepared and administered by oral gavage at doses varying from 1 to 100 mg/kg, in a final volume of 0.1 mL of pure oil per animal. Essential oil, as well as all drugs, were diluted just before use and the pure oil used as vehicle did not present any effect per se.

#### *2.4. Formalin-Induced Licking Behavior*

Formalin (2.5%, μL *v*/*v*) was injected into the dorsal surface of the left hind paw of mice. The time in which animals remained licking the formalin-injected paw was recorded according to Reference [9] with some adaptations done by Matheus et al. [10]. The response was divided into two phases: the first one (neurogenic phase) occurs in the first 5 min post-formalin injection and the second one (inflammatory phase) occurs between 15 and 30 min post-formalin injection.

#### *2.5. Thermal Nociception Model (Hot-Plate)*

The reaction time (licked fore and hind paws) that mice remained on a hot plate (Insight Equipment, Brazil) set at 55 ± 1 ◦C was recorded at several intervals of 30 min post-oral administration of EO or sulcatyl acetate (1, 10, or 100 mg/kg), vehicle, morphine, or antagonists. Baseline was calculated by the mean of two reaction time measurements at 60 and 30 min before oral administration [9] adapted by Matheus et al. [10]. Area under the curve (AUC) graphs were calculated from time–course graphs. The following formula, which is based on the trapezoid rule, was used to calculate the AUC: AUC = 30 × IB [(min 30) + (min 60) + ... + (min 180)]/2, where IB is the increase from the baseline (in %).

#### *2.6. Evaluation of the Possible Mechanisms of Antinociception of A. Trilobata EO and Sulcatyl Acetate*

The participation of nitrergic, opioid, and cholinergic pathways in the antinociception caused by EO and sulcatyl acetate was evaluated 15 min after intraperitoneal injection of antagonists and prior to oral administration of EO or sulcatyl acetate (at 100 mg/kg dose each). In assays conducted in our laboratory, dose–response curves for each antagonist against the respective agonist were previously constructed and the dose that reduced the agonist effect by 50% was chosen for the assays. Based on these data the doses used were: naloxone (opioid receptor antagonist) 1 mg/kg, atropine (cholinergic receptor antagonist) 1 mg/kg, L-NAME (nitric oxide synthase inhibitor) 3 mg/kg. The antinociceptive effect was evaluated via the hot plate test as described above.

#### *2.7. Carrageenan-Induced Leukocyte Migration into the Subcutaneous Air Pouch (SAP)*

Mice back received a subcutaneous injection of sterile air (10 mL) with a replacement of another 7 mL after 3 days. Twenty-four hours after the last injection of air, a solution of carrageenan (1%, 0.5 mL) was injected in subcutaneous air pouches [11,12] with modifications described in Raymundo et al. [13]. Treated groups were composed of mice that received oral administration of vehicle, EO (1, 10, or 100 mg/kg), sulcatyl acetate (1, 10, or 100 mg/kg), or dexamethasone (0.3 mg/kg, i.p.) 1 h before carrageenan injection in the SAP. The negative control group was composed of animals that received oral administration of pure oil and phosphate buffer saline (PBS, 1 mL) in SAP. After 24 h, animals were euthanized, the pouches were washed with 1 mL of sterile PBS, and exudates were collected. The total number of leukocytes was determined using a CellPocH-100Iv Diff (Sysmex) hematology analyzer. Exudates were centrifuged at 170× *g* for 10 min at 4 ◦C, and the supernatants were collected and stored at −20 ◦C until use.

To rule out a possible toxic effect of EO and sulcatyl acetate, mice treated with the highest dose of each one had their bone marrow cells collected through flushing the femur with 1 mL of PBS. Peripheral blood was also collected in heparinized tubes. The counting of the cells in the femoral lavage or in the blood was performed with the aid of a CellPocH-100Iv Diff (Sysmex) hematology analyzer.

#### *2.8. Cell Culture*

All cell culture reagents were purchased from Sigma-Aldrich (USA). RAW 264.7 (TIB-71) was obtained from the American Type Culture Collection. Cells were routinely grown in Roswell Park Memorial Institute (RPMI) medium containing 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin-streptomycin (henceforth called RPMI) in a humidified 5% CO2 atmosphere at 37 ◦C. Cells were cultured up to confluence and used in the assays.

#### *2.9. Cell Viability Assay*

Cell viability was determined using 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2*H*-tetrazolium bromide (MTT) reagent (Sigma-Aldrich, USA) using method described by Denizot and Lang [14]. Briefly, cells were plated at an initial density of 5 <sup>×</sup> 10<sup>4</sup> cells per well in 96-well plates and incubated for 24 h at 37 ◦C and 5% CO2. After 24 h, cultures were treated with EO or SA at a final concentration of 10, 30, or 100 μg/mL and further incubated for 24 h. The supernatants were removed and then 10 μL

of MTT solution (5 mg/mL in RPMI)/100 μL of medium were added to each well and incubated for 4 h at 37 ◦C, 5% CO2. The resultant formazan crystals were dissolved in dimethylsulfoxide (100 μL) and absorbance was measured in a microplate reader (FlexStation Reader, Molecular Devices, San Jose, CA, USA) at 570 nm. All experiments were performed in triplicate, and cell viability was expressed as a percentage relative to the untreated control cells.

#### *2.10. Quantification of TNF-*α *and IL-1*β

Supernatants from the exudates collected from the SAP were used to measure the levels of the cytokines tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β) by enzyme-linked immunosorbent assay (ELISA) using the protocol supplied by the manufacturer (B&D, Franklin Lakes, NJ, USA).

#### *2.11. Nitrate and Nitrite Measurement*

To evaluate the nitrate accumulated in SAP, exudates were measured according to the method described by Bartholomew [15] and adapted by Raymundo et al. [13], followed by measurement of nitrite according to the Griess reaction [16].

#### *2.12. Detection of Enzymes Expression*

Immunoblots were carried out as described previously [17]. Briefly, RAW 264.7 cells (4 × 106/mL) were plated in 12-well plate, incubated for 1 h with EO or SA, activated with lipopolysaccharide (LPS) (1 μg/mL), and further incubated for 10 min, 1 h, or 8 h. Cells were lysed with cold lysis buffer (10% NP40, 150 Mm NaCl, 10 Mm Tris HCl pH 7.6, 2 Mm PMSF, and 5 μM leupeptin). After determination of the protein concentration in the suspensions by the BCA method (BCA™ Protein Assay Kit, Pierce, Waltham, MA, USA), the suspensions were boiled in application buffer (DTT 100 Mm, Bromophenol Blue 0.1%). Aliquots of 30 μg of protein were submitted to electrophoresis in 10% polyacrylamide gel. Proteins were electrophoretically transferred onto nitrocellulose membranes. Membranes were incubated with primary antibodies (Cell Signaling, Danvers, MA, USA) and further with secondary antibodies (anti-mouse IgG antibody conjugated to horseradish peroxidase). Proteins were detected using enhanced chemiluminescence (ECL) reagents and quantified using a ChemiDoc system (BioRad, Hercules, CA, USA).

#### *2.13. Statistical Analysis*

Each experimental group consisted of 6 to 8 mice, and the results are expressed as the mean ± S.D. The area under the curve (AUC) was calculated using Prism Software 5.0 (GraphPad Software, La Jolla, CA, USA). Significant differences between the groups were established using Bonferroni's test for multiple comparisons after analysis of variance (ANOVA) testing. *p* values less than 0.05 were considered significant.

#### **3. Results**

#### *3.1. Antinociceptive E*ff*ect*

#### 3.1.1. Formalin-Induced Licking Behavior

We further decided to evaluate a possible antinociceptive activity from the EO and its major component, sulcatyl acetate. Doses of 1, 10, or 100 mg/kg given orally significantly reduced both phases of formalin-induced licking behavior (1st and 2nd). While first phase was inhibited by 10 and 100 mg/kg, the second phase was only inhibited by the highest dose (of 100 mg/kg) of EO. When studying sulcatyl acetate it could be observed that even 1 mg/kg dose significantly reduced the first phase, while only the highest dose reduced the second phase (Figure 1).

**Figure 1.** Effect of essential oil from *Aristolochia trilobata* and sulcatyl acetate on formalin-induced licking response in mice. Animals were orally pretreated with essential oil of sulcatyl acetate (1, 10, or 100 mg/kg), acetylsalicylic acid (ASA, 200 mg/kg), morphine (2.5 mg/kg), or vehicle (DMSO + phosphate buffer saline (PBS) 60 min before intraplantar injection of formalin (2.5%). The results are expressed as the mean ± S.D. (*n* = 6) of the time the animals spent licking the formalin-injected paw. Statistical significance was calculated by one-way ANOVA with Bonferroni's as post-test. \* indicates *p* < 0.05 when compared to the vehicle-treated mice.

#### 3.1.2. Hot-Plate Model

The significant effect observed in the first phase of the formalin-induced licking behavior is suggestive of an antinociceptive activity. Previous data from literature had shown that EO from *A. trilobata* presented antinociceptive effect in models of chemical nociception (acetic acid-induced writhings and formalin-induced licking response). However, in such paper, authors used high doses (25, 50, and 100 mg/kg) [6]. We further decided to evaluate if lower doses of EO and its majority component (sulcatyl acetate) present antinociceptive effect in thermal model of nociception (the hot plate). We also tried to evaluate the possible mechanism of action.

Data showed in Figure 2 indicate that both EO and sulcatyl acetate presented central antinociceptive effect. Even 30 min after oral administration of EO a significant effect was observed with all three doses tested (1, 10, and 100 mg/kg). This effect was maintained until 90 min post-oral administration and being gradually reduced in later times. The conversion of data from line graphs to area under the curve graph demonstrated that all three doses tested developed a significant central antinociceptive effect when comparing to the vehicle-treated group.

**Figure 2.** Effect of essential oil from *A. trilobata* and sulcatyl acetate on thermal nociception model (the hot plate) in mice. Animals were orally pretreated with essential oil of sulcatyl acetate (1, 10, or 100 mg/kg), morphine (2.5 mg/kg), or vehicle (DMSO + PBS). The results are presented as the mean ± S.D. (*n* = 6–8) of the increase in the response time relative to baseline levels (left graphs) or area under the curve (right graphs) calculated with the Prism Software 5.0. Statistical significance was calculated by one-way ANOVA with Bonferroni's as post-test. \* indicates *p* < 0.05 when compared to the vehicle-treated mice. Where no error bars are shown is because they are smaller than the symbol.

When mice were orally treated with sulcatyl acetate, we could observe a significant effect after 30 min of administration. Differently to that observed with EO, sulcatyl acetate antinociception was maintained until 150 min. At this time point, antinociceptive effect was even higher than morphine-treated mice. The graph of the area under the curve demonstrated that all 3 doses presented a significant antinociceptive effect.

#### 3.1.3. Mechanism of Antinociceptive Action

In the next step, we decided to investigate which pathways could be involved in the central antinociceptive effect of EO and sulcatyl acetate. Mice were pretreated with an opioid antagonist (naloxone), a cholinergic antagonist (atropine), or with an inhibitor of nitric oxide synthase enzyme (L-NAME). Data obtained showed that none of the antagonists of inhibitor significantly reversed the antinociceptive effect of either EO or sulcatyl acetate (Figure 3).

**Figure 3.** Effect of the different antagonists on the antinociceptive activity of essential oil from *A. trilobata* and sulcatyl acetate in the hot-plate model. The animals were pretreated with naloxone (1 mg/kg, i.p.), atropine (1 mg/kg, i.p.), or L-NAME (3 mg/kg, i.p.) 15 min before oral administration of essential oil or sulcatyl acetate (100 mg/kg) or vehicle. The results are expressed as the mean ± S.D. of the area under the curve calculated with Prism Software 5.0 (*n* = 6–8). One-way ANOVA followed by Bonferroni's test was used to calculate the statistical significance. \* indicates *p* < 0.05 when compared to the vehicle-treated group.

#### *3.2. Anti-Inflammatory E*ff*ect*

The observation that EO and sulcatyl acetate inhibited the second phase of formalin-induced licking, a well-known inflammatory phase, led us to investigate if both substances could present an anti-inflammatory effect. In this regard, EO and sulcatyl acetate were evaluated in their abilities in reducing carrageenan-induced leukocyte migration into a subcutaneous air pouch (SAP) and cytokines production.

#### 3.2.1. Leukocyte Migration

Injection of carrageenan into the SAP led to a 76-fold increase in leukocyte number (2.14 ± 1.65 <sup>×</sup> <sup>10</sup><sup>6</sup> cells/mL in the group that received saline in SAP versus 162.6 <sup>±</sup> 31.17 <sup>×</sup> 106 cells/mL in the group that received carrageenan in SAP). Pretreatment of mice with the steroidal anti-inflammatory drug (SAID), dexamethasone (Dex) resulted in a reduction of 50% in leukocyte number present in SAP. The crescent doses of EO (1, 10, and 100 mg/kg) significantly reduced the cell migration with

values similar to the SAID. Sulcatyl acetate also reduced the number of cells that migrated to the pouch. However, this effect was less prominent when compared with that obtained with EO (Figure 4).

**Figure 4.** Effect of essential oil from *A. trilobata* and sulcatyl acetate in leukocyte migration into the subcutaneous air pouch (SAP). The animals were pretreated with dexamethasone (Dex, 1 mg/kg, i.p.), essential oil, or sulcatyl acetate (1, 10, or 100 mg/kg) or vehicle, 1 h before carrageenan injection into the SAP. The results are expressed as the mean ± S.D. calculated with Prism Software 5.0 (*n* = 6–8). One-way ANOVA followed by Bonferroni's test was used to calculate the statistical significance. \* indicates *p* < 0.05 when compared to the vehicle-treated group (animals that received carrageenan in the SAP) and # indicates *p* < 0.05 when compared to the vehicle-treated group (animals that received saline in the SAP).

#### 3.2.2. Protein Extravasation

As EO and SA presented a significative effect in reducing leukocyte migration into SAP similarly to that observed with the positive control group (SAID), we decided to further investigate if they could reduce other parameters of the inflammatory process in a tentative to investigate the possible mechanism of anti-inflammatory action. The exudate collected in the SAP protein was measured and results demonstrated that pretreatment of mice with 10 or 100 mg/kg doses of EO significantly reduced the amount of protein in exudate. It is interesting to note that none of the doses of SA inhibit protein extravasation even at a higher dose (100 mg/kg) (Figure 5).

**Figure 5.** Effect of essential oil from *A. trilobata* and sulcatyl acetate in protein extravasation into the subcutaneous air pouch (SAP). The animals were pretreated with dexamethasone (Dex, 1 mg/kg, i.p.), essential oil, or sulcatyl acetate (1, 10, or 100 mg/kg) or vehicle 1 h before carrageenan injection into the SAP. The results are expressed as the mean ± S.D. calculated with Prism Software 5.0 (*n* = 6–8). One-way ANOVA followed by Bonferroni's test was used to calculate the statistical significance. \* indicates *p* < 0.05 when compared to the vehicle-treated group (animals that received carrageenan in the SAP) and # indicates *p* < 0.05 when compared to the vehicle-treated group (animals that received saline in the SAP).

#### 3.2.3. Nitric Oxide Production

We also decided to quantify the amount of nitric oxide (NO) produced in the exudate. When NO is produced in biological fluids it decays to the stable metabolite nitrate. Figure 6 shows that injection of carrageenan in the SAP lead to an increase in the amount of NO accumulated in the pouch when compared with mice that received saline. EO almost completely abolished NO production resulting in NO levels similar to those observed in saline-treated mice. Although SA did not completely inhibit the NO production, the reduction observed vary between 50% and 80% (Figure 6).

**Figure 6.** Effect of essential oil from *A. trilobata* and sulcatyl acetate in nitric oxide production into the subcutaneous air pouch (SAP). The animals were pretreated with dexamethasone (Dex, 1 mg/kg, i.p.), essential oil, or sulcatyl acetate (1, 10, or 100 mg/kg) or vehicle 1 h before carrageenan injection into the SAP. The results are expressed as the mean ± S.D. calculated with Prism Software 5.0 (*n* = 6–8). One-way ANOVA followed by Bonferroni's test was used to calculate the statistical significance. \* indicates *p* < 0.05 when compared to the vehicle-treated group (animals that received carrageenan in the SAP) and # indicates *p* < 0.05 when compared to the vehicle-treated group (animals that received saline in the SAP).

#### 3.2.4. Cytokine Production

The measurement of cytokines TNF-α and IL-1β accumulated in the exudate obtained from SAP showed that highest doses of EO (10 and 100 mg/kg) significantly reduced levels of both cytokines. However, pretreatment of mice with the majority component of the EO, sulcatyl acetate, led to an almost 50% reduction in cytokines production even with 1 mg/kg dose (Figure 7).

**Figure 7.** Effect of essential oil from *A. trilobata* and sulcatyl acetate in TNF-α and IL-1β production in the subcutaneous air pouch (SAP). The animals were pretreated with dexamethasone (1 mg/kg, i.p.), essential oil, or sulcatyl acetate (1, 10 or 100 mg/kg) or vehicle 1 h before carrageenan injection into the SAP. The results are expressed as the mean ± S.D. calculated with Prism Software 5.0 (*n* = 6–8). One-way ANOVA followed by Bonferroni's test was used to calculate the statistical significance. \* indicates *p* < 0.05 when compared to the vehicle-treated group (animals that received carrageenan in the SAP) and # indicates *p* < 0.05 when compared to the vehicle-treated group (animals that received saline in the SAP).

#### 3.2.5. In Vitro Cell Viability and Nitric Oxide Production

To further evaluate the possible anti-inflammatory mechanism of action, we decided to study the effects of EO and SA in vitro using macrophage cell line RAW 264.7. Data obtained showed that none of the concentrations used (1, 10, or 30 μg/mL) significantly affected the cell viability (data not shown). We next measure NO production by LPS-activated cells incubated or not with the three concentrations of EO and SA. Neither EO nor SA did induce NO production *per se* (data not shown). Figure 8 shows that there is an inhibitory effect on NO production when LPS-activated cells were pre-incubated with SA for 1 h (Figure 8D). In another set of experiments, cells were activated with LPS and after 8 h different concentrations of OE or SA were added to each group. As can be observed in Figure 8C,D, we do not observe inhibitory effect in NO production when EO or SA was added 8 h post-LPS.

**Figure 8.** Effect of essential oil from *A. trilobata* (EO) and sulcatyl acetate (SA) in nitric oxide production by RAW 264.7 macrophage. Cells (2 <sup>×</sup> 106/mL) were incubated with lipopolysaccharide (LPS, 1 <sup>μ</sup>g/mL) and EO or SA (1, 10, or 30 μg/mL). Graphs **A** and **B** show the results of pre-incubation of cells for 1 h before LPS addition to cells. Graphs **C** and **D** show the results of NO production when EO or SA was added 8 h post-LPS activation. The results are expressed as the mean ± S.D. calculated with Prism Software 5.0 (*n* = 6). One-way ANOVA followed by Bonferroni's test was used to calculate the statistical significance. # indicates *p* < 0.05 when compared to the vehicle (non-LPS group). \* indicates *p* < 0.05 when compared to the group that was activated with LPS and received vehicle.

In a tentative to demonstrate the possible mechanism of anti-inflammatory effect of EO and SA, we decided to evaluate their effects on some inflammatory pathways. For this purpose, expression of inducible nitric oxide synthase (iNOS), p38 mitogen-activated protein kinase (p38 MAPK), its activated form (the phosphorylated p38), and spleen tyrosine kinase (Syk) was evaluated after incubation of RAW 264.7 cells with EO or SA (30 μg/mL) and LPS. As expected, non-activated cells did not express those inflammatory enzymes, while LPS incubation induced an increase in levels of all of them. As illustrated in Figure 9, preincubation with EO significantly reduced the expression of iNOS. To ascertain whether the mechanism by which both substances were reducing the expression of the enzyme, we evaluated the involvement of enzymes present in the early stages of the activation pathways triggered by LPS in the toll receptor 4. In this regard, we evaluated expression of p38 MAPK and Syk and the corresponding increase in levels of phosphorylated p38, a consequence of cell activation and p38 activation. It can be observed that EO reduced expression of Syk enzyme. After an activation induced by LPS we can observe the activation/phosphorylation of p38 MAPK. In this context, it could be noted an increase in levels of this enzyme (p-p38). However, preincubation of activated cells with EO or SA did not affect phosphorylation levels of p38 MAPK. In summary, EO and SA did not affect the levels of p-p38 expressed in cells after activation with LPS (Figure 9).

**Figure 9.** Effect of essential oil from *A. trilobata* (EO) and sulcatyl acetate (SA) in expression of Syk, p38 MAPK, or inducible nitric oxide synthase (iNOS) by RAW 264.7 cells. Cells (2 <sup>×</sup> <sup>10</sup>6/mL) were incubated with dexamethasone (350 μg/mL), EO, or SA (30 μg/mL) 1 h before lipopolysaccharide (LPS, 1 μg/mL). After 10 min (for Syk and p38 MAPK) or 8 h (for iNOS), cell lysates were collected for Western blot analyses. The results are expressed as the mean ± S.D. calculated with Prism Software 5.0 (*n* = 3). One-way ANOVA followed by Newman's test was used to calculate the statistical significance. # indicates *p* < 0.05 when compared to the vehicle (non-LPS group). \* indicates *p* < 0.05 when compared to the group that was activated with LPS and received vehicle.

#### **4. Discussion**

The present work demonstrated that the essential oil (EO) of *A. trilobata* and its majoritarian substance, sulcatyl acetate (SA), present significant anti-inflammatory and antinociceptive effects. We also demonstrated that opioidergic, nitrergic, and cholinergic pathways do not participate in the antinociceptive effect. Contributing to the knowledge about this species, we also demonstrated that both EO and SA presented anti-inflammatory effect reducing leukocyte migration, production of cytokines, nitric oxide (NO), and protein extravasation.

The evaluation of *A. trilobata* EO chemical constituents highlighted the presence of SA as the major substance. This characteristic is not usual in essential oils in general. However, this finding confirms previous works, which states *A. trilobata* EO as a rich source of SA [4,7].

Quintas et al. [7] demonstrated that EO from *A. trilobata* and SA presented antinociceptive effect in two models of chemical nociception (i.e., acetic acid-induced writhing and formalin-induced paw licking). Although the essential oil tested in that work was very similar to that used by us, there are several differences among assays and results. In that work, authors used doses varying from 25 to 100 mg/kg, administered by intraperitoneal route. EO and SA were evaluated only in two models of chemical nociception. Differently, in the present work, we evaluated EO and SA administered by oral route and doses of 1, 10, and 100 mg/kg. The effect observed in this model could be explained, at least in part, due to the presence of other substances with antinociceptive activity. It was demonstrated by Amaral and collaborators [18] that the monoterpene limonene presented antinociceptive activity more related to peripheral analgesia. Kaiamoto and collaborators [19] also suggested that limonene may act as a transient receptor potential cation channel 1 (TRPA1) agonist when applied topically. However, when systemically administered, it presents an antinociceptive effect. In our work, we did

not test isolated limonene. We used only the majoritarian substance (SA). It is also important to note that formalin-induced licking is a multicomplex phenomenon with the involvement and activation of nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Nrf2 is a transcriptional factor related to activation of heme-oxygenase 1 (HO-1) [20], leading to an antinociceptive effect in formalin-induced licking [21]. It may be that in such a way, by oral administration, all the substances presented in the EO may acting together to present an anti-inflammatory effect.

Considering the *A. trilobata* EO results and its major substances, a synergistic effect of SA and limonene can be occurring, especially in the second phase of formalin-induced licking response. Besides evaluating the effects in the formalin-induced paw licking, we also used the thermal model of nociception, the hot-plate, and searched the mechanism of action of EO and SA using three different antagonists.

In another group of assays, we also studied the anti-inflammatory effect of EO and SA in their capacity to inhibit the leukocyte migration into the subcutaneous air pouch induced by carrageenan and production of some inflammatory mediators. Our data complement those obtained by the other group and identify the possible mechanism of action. We also demonstrated that EO and SA presented significant effects even when used by oral route at a dose as low as 1 mg/kg.

In the present study, we attempted to further characterize some of the mechanisms through which EO from *A. trilobata* and SA exerts its antinociceptive effect. The sensation of pain can be divided into four components, i.e., transduction, transmission, modulation, and perception. Several systems, such as oxidonitrergic, gamma aminobutyric acid GABAergic, glutamatergic, opioidergic, cholinergic, serotonergic, adrenergic, and others, may act in different steps of the pain turning it a complex phenomenon in such a way that alterations in one of the system cited above can alter the response in all the other systems [22]. A substance, whether natural or synthetic, can then act in one of four components to produce analgesia. Our data are suggestive that nor opioidergic, nitrergic, or cholinergic pathways appear to be involved in the antinociceptive effect of EO and SA since none of the antagonists used (naloxone, L-NAME, or atropine) reversed the effects of EO and SA. Despite the opioid system is one of the most important in pain perception and modulation, the activation of naloxone-sensitive pathway is probably not involved in the antinociception produced by EO and SA because naloxone significantly reversed morphine (data not shown) but not EO and SA antinociception.

We also demonstrated that doses as low as 1 mg/kg significantly reduced the licking response induced by formalin injection in mice paw. Nociception induced by formalin develops a biphasic pattern with an initial phase (5 to 15 min post-injection) followed by a second phase (15 to 30 min post-injection) [23–26]. First phase is due to direct activation of nociceptors, whereas the second one is due to the release of inflammatory mediators acting together in nociceptors and their own local receptors [27–29]. The involvement of serotonin and bradykinin in both phases was also described [30].

As mediators previously cited are also involved in inflammatory processes, we further decided to study if EO and SA could present an anti-inflammatory effect. In this regard, we used the leukocyte migration induced by injection of carrageenan into the subcutaneous air pouch (SAP). Carrageenan-induced inflammation is a multicomplex phenomenon with the involvement of synthesis and/or liberation of a range of mediators such as prostaglandins, histamine, bradykinin, serotonin, nitric oxide, and leukotrienes and chemotaxis of neutrophils and macrophages [31]. After 24 h of carrageenan injection, there is an intense migration of leukocytes and an increase in levels of NO and cytokines [32–36].

Results obtained in the SAP model complement those obtained in the second phase of formalin-induced licking since both models present an inflammatory profile with involvement in a diversity of inflammatory mediators. One hypothesis for the reduction observed in this model could be related to reduction in production and/or liberation of inflammatory substances involved in leukocyte chemotaxis. In this context, we can infer that the reduction in cell migration into the SAP could be due to a reduction in the levels of these cytokines. Our data are in accordance with those obtained in Reference [37], which observed that tacrolimus (an immunosuppressor) reduced neutrophil infiltration in the pancreatitis model due to a reduction in expression of mRNA of TNF-α and IL-1β. In an inflammatory event, inducible nitric oxide synthase is expressed in different cells and culminating with NO production. NO plays multiple roles in the inflammatory response, vasodilation, and regulation of leukocytes rolling, migration, cytokines production, and proliferation. It has been shown that some iNOS inhibitors demonstrated an important effect in several inflammatory models, such as the air pouch model [38,39]. The fact that EO and SA reduced NO levels in exudate also contribute to their anti-inflammatory effect. It is also interesting to note that EO reduced NO levels similar to the positive control group, dexamethasone, a well-known steroidal anti-inflammatory drug.

Data obtained using the in vivo model of SAP was corroborated by in vitro assays. Reduction in nitric oxide production in vitro and in vivo was not due to a direct effect in iNOS enzyme activity. It is well known that iNOS synthesis occurs until 6–8 h after LPS activation. After this time point, nucleus synthesis is finished and enzymes synthesized initiate NO production [39]. Since addition of EO or SA 8 h after LPS activation did not affect NO production, it can be suggested that the inhibitory effect observed was not due to reduced enzyme activity.

As a final step in our effort to delineate the inhibitory effects of EO and SA, we quantified Syk and p38 MAPK enzymes expression. Several intracellular signaling molecules are involved and activated during the inflammatory responses in macrophages. Tyrosine kinase families have been considered as the major effector molecule. Spleen tyrosine kinase (Syk) binds with Toll-like receptor-4 (TLR4) and is activated, resulting in the transduction of stimulatory signals through the activation of various downstream signaling molecules. Since Syk is one of the upstream signaling molecules, it orchestrates many downstream signaling molecules and amplifies inflammatory signals. Therefore, Syk has been considered to play critical roles in inflammatory responses [40–42]. The observation that EO partially reduced expression of Syk can explain the inhibition of expression of iNOS and justify the inhibition observed in NO production.

p38 MAPK was first recognized for its role in inflammation in regulating the biosynthesis of pro-inflammatory cytokines (i.e., IL-1 and TNFα) in LPS-stimulated cells [41] and expression of cycloxigenase-2 (COX2) [42–44]. It can be suggested that EO and SA mechanism of action do not seem to affect p38 MAPK activation into the phosphorylated form (the p-p38). We could speculate that EO effect may act through Syk pathway do not interfere with p38 MAPK since both are independent systems that may act without cross-interference with each other.

Our data also demonstrated that SA did not affect *per se* any of enzymes evaluated (Syk, p38 MAPK, and iNOS). It is important to note that as part of a complex mixture of substances it is reasonable that EO can present effect and one isolated substance cannot present the same effect and mechanism of action.

#### **5. Conclusions**

In the present work, we demonstrated that *A. trilobata* essential oil as well as the majority component, sulcatyl acetate, present antinociceptive and anti-inflammatory effects. This activity is accompanied by reduction in cell migration and production of NO and cytokines. It seems that at least part of this effect is mediated by inhibition of Syk and iNOS enzymes expression. Together, we can suggest *A. trilobata* as an anti-inflammatory and antinociceptive species.

**Author Contributions:** Data curation, P.D.F.; formal analysis, D.d.C.S. and P.D.F.; funding acquisition, P.D.F.; investigation, D.d.C.S., N.d.M.C., P.D.F., and P.D.F.; methodology, D.d.C.S., T.S.V., D.d.A.S., P.B.A., D.S.A.M., and P.D.F.; project administration, P.D.F.; resources, C.S.A. and P.D.F.; supervision, P.B.A. and P.D.F.; writing—original draft, P.D.F.; writing—review and editing, P.D.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Conselho Nacional de Pesquisa (CNPq, fellowship and grants for P.D.F. and C.S.A.), Fundação Carlos Chagas Filho de Apoio à Pesquisa (FAPERJ, fellowship and grants for P.D.F., C.S.A., D.S.A.M.), and Coordenação de Aperfeiçoamento de Pessoal (CAPES, for fellowship to NMC, D.d.C.S.).

**Acknowledgments:** We would like to thank Alan Minho for technical assistance and Instituto Vital Brazil (Niterói, Brazil) for donating the mice.

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

#### **References**

© 2020 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 (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **Phytogenic Synthesis of Nickel Oxide Nanoparticles (NiO) Using Fresh Leaves Extract of** *Rhamnus triquetra* **(Wall.) and Investigation of Its Multiple In Vitro Biological Potentials**

**Javed Iqbal 1,\*,**†**, Banzeer Ahsan Abbasi 1,**†**, Riaz Ahmad 2, Mahboobeh Mahmoodi 3, Akhtar Munir 4, Syeda Anber Zahra 1, Amir Shahbaz 1, Muzzafar Shaukat 1, Sobia Kanwal 5, Siraj Uddin 1,6, Tariq Mahmood <sup>1</sup> and Ra**ff**aele Capasso 7,\***

Received: 7 April 2020; Accepted: 3 May 2020; Published: 12 May 2020

**Abstract:** Chemically nickel oxide nanoparticles (NiONPs) involve the synthesis of toxic products, which restrict their biological applications. Hence, we developed a simple, eco-friendly, and cost-efficient green chemistry method for the fabrication of NiONPs using fresh leaf broth of *Rhamnus triquetra* (RT). The RT leaves broth was used as a strong reducing, capping, and stabilizing agent in the formation of RT-NiONPs. The color change in solution from brown to greenish black suggests the fabrication of RT-NiONPs which was further confirmed by absorption band at 333 nm. The synthesis and different physicochemical properties of RT-NiONPs were investigated using different analytical techniques such as UV-Vis (ultraviolet−visible spectroscopy), XRD (X-ray powder diffraction), FT-IR (Fourier-transform infrared spectroscopy), SEM (scanning electron microscopy), TEM (transmission electron microscopy), EDS (energy-dispersive X-ray spectroscopy), DLS (dynamic light scattering) and Raman. Further, RT-NiONPs were subjected to different in vitro biological activities and revealed distinctive biosafe and biocompatibility potentials using erythrocytes and macrophages. RT-NiONPs exhibited potential anticancer activity against liver cancer cell lines HUH7 (IC50: 11.3 μg/mL) and HepG2 (IC50: 20.73 μg/mL). Cytotoxicity potential was confirmed using Leishmanial parasites promastigotes (IC50: 27.32 μg/mL) and amastigotes (IC50: 37.4 μg/mL). RT-NiONPs are capable of rendering significant antimicrobial efficacy using various bacterial and fungal strains. NiONPs determined potent radical scavenging and moderate enzyme inhibition potencies. Overall, this study suggested that RT-NiONPs can be an attractive and eco-friendly candidate. In conclusion, current study showed potential in vitro biological activities and further necessitate different in vivo studies in various animal models to develop leads for new drugs to treat several chronic diseases.

**Keywords:** *Rhamnus triquetra*; NiONPs; cytotoxicity; biocompatibility; antimicrobial; enzyme inhibition

#### **1. Introduction**

Nanotechnology deals with different approaches to synthesize materials ranging from 1 to 100 nm, at least in one dimension, and have unique properties, such as small size, surface charge, porosity, high surface energy, and high surface area/volume (S/V) ratio, which enhance their catalytic properties and interaction with other molecules. The main reason why metal nanoparticles (MNPs) have gained the specific attention of researchers is due to their unique properties, namely particle size, shape, crystal structure, surface effect, magnetic, catalytic, optical, as well as chemical and mechanical characteristics from their bulk counterpart [1,2]. Until now, different multifunctional metals and metal-oxide nanoparticles have been synthesized [3,4]. Among the different nanoparticles(NPs), NiONPs have gained the specific attention of biologists and chemists due to their numerous applications in battery electrodes, magnetic materials, heterogeneous catalysts, gas sensors, electrochromic films, and solid-oxide fuel cells and help in the adsorption of inorganic pollutants and dyes [5,6]. Further, NiONPs have shown significant antibacterial, antifungal, antioxidants, anti-inflammatory, anticancer, and enzyme inhibition potentials [6,7]. NiONPs have shown toxicity towards different microbial agents and microalgae by producing ROS, inducing oxidative stress and releasing (Ni2+) inside the cell [8].

Currently, NiONPs are fabricated via different physical and chemical approaches. However, these synthesis routes face several challenges as they utilize costly metal salts, organic solvents, toxic reducing agents (sodium borohydrides, hydrazine hydrate, sodium citrate and Gallic acid), stabilizing and capping agents (thiols, amines, sodium citrate), and demand expensive equipment. These approaches are not only expensive at the industrial scale, but also cause some undesired effects on human life and the surrounding environment, and may result in cytotoxicity, carcinogenicity, and genotoxicity, thus restricting their utilization in biomedical purposes [9]. Hence, these problems must be solved, and actions are required to develop an alternative solution for the fabrication of NPs.

Therefore, scientists have developed green chemistry methods which are more sustainable, cleaner and eco-friendly. Presently, new developments have been made in the synthesis of nanomaterials using different biological sources (microbes, algae, fungi, various lower and higher plants). This method is relatively simple, ecofriendly, energy-efficient, nontoxic, eliminates the need for high energy, temperature, and pressure and needs no reducing, stabilizing, and capping agents from outside. The major disadvantages associated with microbial synthesis is the maintenance of an aseptic environment, culturing in media, high isolation cost, high incubation time, difficulty in handling, pathogenicity in nature, and the requirement of comprehensive biological knowledge [10].

Phytofabrication has flourished for the formation of several nanomaterials and has attracted the attention of the nano task force due to its sample, environmentally benign, and cost-effective nature [11–14]. In green synthesis, phytoconstituents (alkaloids, terpenoids, polyphenols, glycosides, flavanoids, proteins, vitamins, polysaccharides) function as a capping and reducing agent like different chemical substitutes used in the chemical synthesis of nanoparticles [15,16]. There are multiple factors that influences green synthesis of nanoparticles, such as nature of plant extracts, concentration of extracts, metal salt, pH, and synthesis protocol used. Thus, for the green synthesis of MNPs, 12 basic principles of green chemistry are now becoming a reference guideline for researchers, chemical technologists, and chemists worldwide to develop less dangerous chemical products and byproducts [17–19]. Therefore, green nanotechnology is an alternate route for the formation of safe and stable materials using different medicinal plants and thus has experienced for rapid rise [20–23].

In the present study, fresh leaves extract of *R. triquetra* were used to synthesize NiONPs. The plant is found in abundance in Pakistan (Kashmir, Margalla Hills), Nepal, and India during the summer season between July and August. The bark, leaves, and fruits of *R. triquetra* are used to treat hemorrhagic septicemia in livestock, intestinal worms, and malarial fevers, possessing significant antimicrobial, deobstruent, anti-inflammatory, astringent, and antioxidant properties. This plant contains several ecofriendly phytoconstituents such as emodin, Kaempferol-7-O-CH3 ether, Kaempferol-4-O-CH3 ether, gluside, quercetins, and physcion [24,25] which help in the phytofabrication of NiONPs. As per the available literature and knowledge, this is perhaps the first study reported on the green synthesis of NiONPs employing *R. triquetra* leaves broth. NiONPs were characterized using different characterization techniques. Further, considering biological and therapeutic potential of *R. triquetra*-NiONPs, different biological activities; anticancer, antimicrobial, antileishmanial, antioxidant, and enzyme inhibitory assays were performed.

#### **2. Material and Methods**

#### *2.1. Preparation of R. Triquetra Leaf Extract*

The preparation of *R*. *triquetra* leaves extract was achieved using previously optimized protocol [26]. Precisely, *R. triquetra* leaves were collected from Pir Suhawa Margalla hills Pakistan (33.7870◦ N, 73.1084◦ E). The sample was identified by senior taxonomist Dr. Syed Afzal Shah, Department of Plant Sciences, QAU Islamabad, Pakistan. The leaves were thoroughly washed with running tap water followed by washing with distill water. The leaves were shade dried and crushed into fine powder. Then, twenty gram leaves powder was added into 100 mL distill water and heated at 80 ◦C for 1 h. The resultant extract was filtered three time using Whattman filter paper No.1 (cone shaped), centrifuged at 5000 rpm for 20 min to remove all unwanted aggregates. Finally, the plant extract was stored at 4 ◦C till further use.

#### *2.2. Green Fabrication of NiONPs*

Synthesis of NiONPs was performed by reducing nickel nitrate using *R*. *triquetra* leaf extract. To achieve this purpose, 100 mL filtered RT leaves extract was steadily mixed with 1 gm NiNO3 salt followed by continuous heating (70 ◦C) and stirring at 500 rpm for 2 h to achieve homogeneous solution. Further, obtained solution was centrifuged at 4000 rpm/20 min. Supernatant was discarded and pellet containing NiONPs was carefully washed 3 times with distilled water to remove uncoordinated materials. The obtained powder assumed as NiONPs was incubated at ~100 ◦C until the water evaporated completely, followed by annealing. Further, NiONPs were stored in cool, dry, and dark place. Finally, NiONPs were thoroughly characterized. Figure 1 shows a schematic representation of the synthesis, characterization, and biological application of NiONPs.

**Figure 1.** General overview of the study plan.

#### *2.3. Physical Characterizations of NiONPs*

The physicochemical properties of RT-NiONPs were investigated using different analytical techniques. The optical properties and bioreduction of nickel ions to NiONPs was confirmed by measuring the absorption spectra of reaction solution using a UV-spectrophotometer, and the solution was scanned between 200 and 600 nm. DLS analyses has provided further insight into the average hydro-dynamic particles diameter (d. nm), ζ-potential and PDI of NiONPs using Malvern Zetasizer Nano (Malvern instrument). RT-NiONPs were analyzed by Fourier transform infrared (FT-IR) spectroscopy to detect different bioactive functional groups responsible for the synthesis and stabilizing NiONPs using various modes of vibrations. FT-IR measurement of the sample was scanned in the wavenumber region 500 cm−<sup>1</sup> to 4000 cm<sup>−</sup>1. The structural analysis and crystalline nature of biogenic NiONPs was carried out using XRD analysis (PANalytical XRD (Netherland). The nano-crystallite size was calculated from the width of the XRD peaks using Debye-Scherrer's equation. The vibrational characteristics of RT-NiONPs were studied using Raman spectroscopy. The elements of NiONPs were detected by EDX (energy dispersive X-ray). The morphological features (surface topology) of *R*. *triquetra*-NiONPs was studied using SEM (EM (NOVA FEISEM-450 applied with EDX detectors). In addition, the morphological structure and actual particle size was studied under TEM (transmission electron microscopy).

#### *2.4. Antileishmanial Potentials (ALP)*

The in vitro antileishmanial potential of NiONPs was investigated using MTT cytotoxicity assay [7]. To confirm the antileishmanicidal potential, *Leishmania tropica* "KWH23 strain" (promastigote and amastigotes parasites) was cultured in MI-99 media containing 10% FBS. The 200 μL reaction mixture is comprised of 100 μL of standardized culture, fresh media (50 μL) and colloidal nanoparticles (50 μL) suspension. Amphoterecin B served as positive while DMSO function as negative control. The leishmanial parasites *L*. *tropica* were kept in 96-well plate and were treated with different concentration of NiONPs (1100–8.595 μg/mL) to determine their antileishmanial potency. The test sample (NiONPs) was incubated in 5% CO2 incubator at 24 ◦C/72 h. After treatment and incubation with NiONPs, the reaction mixture was scanned at 540nm using micro-plate analyzer and readings were taken. Both parasites were counted and IC50 values were calculated to determine intensity/degree of antileishmanicidal potential using formula below:

$$\% \text{inhibtion} = \frac{1 - \text{sample absorbance}}{\text{absorbance of control}} \times 100 \tag{1}$$

#### *2.5. Anticancer Activity*

The in vitro anticancer potential of *R*. *triquetra* mediated NiONPs was investigated using HepG2 and HuH7 cancer cell lines using an MTT assay [27]. The cancer cells were cultured in flasks containing DMEM media supplemented with 10% FBS, Pen-Strep and kept in 5% CO2 incubator for 24 h/37 ◦C. The confluent HepG2 and HuH7 cancer cells (4000 cells/well) were carefully seeded in 96-well plate. Further, cells were treated with varying doses of RT-NiONPs (1100–8.595 μg/mL) for 48 h. DMEM media was removed and MTT solution (100 μL) was added in each well followed by further incubation (3 h in 5% CO2 incubator/37 ◦C). The DMEM media containing other components (FBS, Pen-Strep) was removed and DMSO (100 μL) was loaded in each well followed by incubation for ~20–30 min. The conversion of MTT solution to formazan by living cells was measured using micro plate analyzer 570 nm wavelength. The untreated cancer cells were considered as control and % inhibition of HepG2 and HuH7 cell lines exposed to different concentration of NiONPs was calculated:

$$\% \text{inhibition} = \frac{1 - \text{OD of sample}}{\text{OD of control}} \times 100 \tag{2}$$

#### *2.6. Biocompatibility with Human Erythrocytes (RBCs) and Macrophages*

To determine the non-toxic nature of NiONPs, hemolysis assay was done using erythrocytes cells as discussed in the previously published article [28]. To achieve this purpose, 1 mL freshly isolated human red cells was placed in an Ethylenediaminetetraacetic Acid (EDTA) tube to avoid blood coagulation. Further, erythrocytes were centrifuged at 12,000 rpm/10 min. After centrifugation, supernatant was discarded, and pellet was rinsed three times with PBS. Erythrocytes suspension was made adding 200 μL erythrocytes into 9.8 mL PBS. Further, erythrocytes suspension (100 μL) was treated with various concentration of NiONPs and reaction mixture was incubated at 36 ◦C for 1 h followed by centrifugation (12,000 rpm/15 min). Supernatant was transferred into 96-well-plate and release of hemoglobin was studied at wavelength (540 nm) using micro-plate analyzer. Triton X-100 was used as positive and DMSO as negative control respectively. The data obtained was calculated as % hemolysis caused by different doses of NiONPs using formula below:

$$\% \text{hemoglobin} = \frac{\text{Sample abs} - \text{Negative control abs}}{\text{Positive control abs} - \text{Negative control abs}} \times 100\tag{3}$$

The biosafe nature of RT-NiONPs was further confirmed using human macrophages (HM) following previously used protocol [14]. To confirm the biosafe nature, HM cells were culture in flasks containing RPMI media provided with 10% FBS, Hepes, Pen-Strep (antibiotic). For the proper growth and attachment of cells, flasks containing macrophages were transferred in 5% CO2 incubator for 24 h. After culturing, macrophages (4000 cells/well) were loaded into 96-well-plate. After incubation, macrophages were treated with varying doses of NiONPs (1100–8.595 μg/mL). Finally, the % inhibition of HM cells treated with different doses of RT-NiONPs was calculated using the formula below:

$$\% \text{ inhibition} = \frac{1 - \text{Absorbance of sample}}{\text{Absorbance of control}} \times 100\tag{4}$$

#### *2.7. Antioxidant Activities*

Spectrophotometric procedures were used to confirm the antiradical potentials of RT-NiONPs using Total antioxidant capacity (TAC), CUPARAC, DPPH, and total reducing power (TRP). Total antioxidant capacity (TAC) was determined through the phosphomollybdenum method [29]. The incubation of RT-NiONPs with Molybdenum (VI) demonstrated the presence of antioxidants which were assessed by measuring absorbance at 695 nm. For this purpose, ascorbic acid was used as a positive control and DMSO was taken as negative control. Further, the cupric-ion assay (CUPRAC) was investigated for greenly orchestrated NiONPs [30] and the absorbance of solutions was taken at 515 nm using spectrophotometer. Moreover, the total reducing power (TRP) of greenly prepared NiONPs was studied using potassium ferricyanide method [31]. This method is based on the principle that reductones having reduction potential will reduce potassium ferricyanide (Fe3+) to form potassium ferrocyanide (Fe2+). When potassium ferrocyanide (Fe2+) reacts with FeCl3, it forms Fe+2-Fe+3 complex that has maximum absorption at 700 nm. The reducing power of RT-NiONPs was recorded as AAEq/mg. Further, the antioxidant scavenging potential of RT-NiONPs was evaluated. To investigate this potential, 2.4 mg DPPH was mixed with methanol (25 mL) to create a free radical environment. Further, various concentrations (1100–8.595 μg/mL) of NiONPs were prepared in DMSO and studied for their DPPH free-radical scavenging potential. The existence of reductones were determined by measuring maximum absorbance of reaction mixture at 517 nm using microplate analyzer.

$$1\,\% \text{DPPH savinging} = 1 - (\frac{\text{Absorbance of sample}}{\text{Absorbance of control}}) \times 100\tag{5}$$

#### *2.8. Enzymes Inhibition Potentials*

Proteins kinase (PK) inhibition potential of *R. triquetra* synthesized NiONPs was demonstrated using actinobacterium (*Streptomyces* 85E) [32]. To confirm the PK inhibition potential, SP4 minimal media was used to acquire uniform bacterial lawns. Briefly, 100 μL inoculum was taken from standard culture and was equally spread on petri plate to achieve uniform lawns. A sterilized filter disc (6 mm) loaded with various doses of RT-NiONPs were placed on *Streptomyces* 85E painted plate. The surfactin was used as positive and DMSO as negative control. Further, petri plates were incubated (30 ◦C/72 h) to determine the PK inhibition potential against *Streptomyces* 85E. After incubation, different clear and bald zones were appeared. These different zones determined inhibition potentials of spores/mycelia formation in *Streptomyces* 85E strain. Finally, ZIs were measured in millimeter to determine the PK inhibition potential of RT-NiONPs.

Further, antidiabetic potency of greenly orchestrated RT-NiONPs was studied using alpha amylase (AA) inhibition assay [33]. The reaction mixture was prepared by adding starch solution (45 μL), NiONPs (15 μL), AA enzyme (30 μL), and FBS (20 μL). Further, HCL (25 μL) and iodine solutions (95 μL) were added and incubated at 50 ◦C for 30 min. Acarbose was used as positive and distill water as negative control respectively. The micro-plate analyzer was used to calculate the optical density at 540 nm and IC50 value was recorded. The % inhibition was recorded utilizing below formula:

$$\% \text{ inhibition} = \frac{\text{S(ab)} - \text{NC(ab)}}{\text{Blark (ab)} - \text{NC(ab)}} \times 100 \tag{6}$$

#### *2.9. Antifungal Activity*

The disc-diffusion method (DDM) was used to study the antifungal potencies of RT-NiONPs using various fungal strains (*A. flavus*, *M. racemosus*, *C. albicans*, *A. niger*, *F. solani*). Fungal strains were cultured in flasks holding fungal growth media (sabouraud dextrose liquid media) (SDL) and kept in shaking incubator (37 ◦C/24 h). The SDL media were prepared, autoclaved, and poured in autoclaved petri plates to achieve antifungal activity. Further, 50 μL broth culture was spread on petri plate using autoclaved cotton swab to achieve uniform lawns. Filter discs (~6 mm) loaded with different concentrations (1100–34.38 μg/mL) of RT-NiONPs were kept on media plates. To compare the antifungal potential of RT-NiONPs, Amp B was taken as positive, and DMSO as negative control. Further, fungal plates were placed in an incubator for (24 h/37 ◦C) and zones of inhibition (ZIs) were observed with time intervals and MICs values were calculated.

#### *2.10. Antibacterial Activity*

To further evaluate the antimicrobial potential of RT-NiONPs, in vitro antibacterial potency was investigated using different bacterial strains, namely *P. aeruginosa*, *B. subtilis*, *S. aureus*, *K. pneumoniae*, and *E. coli*. Before antibacterial activity was investigated, bacterial cultures were refreshed in nutrient media and kept in shaking incubator at 37 ◦C (200 rpm /24 h). Further, bacterial strains were spread on media plates using sterilized cotton swabs. The DDM method was used to confirm the bactericidal potentials. For this purpose, 6 mm (filter disc) loaded with different concentration of RT-NiONPs (1100–34.38 μg/mL) were kept on bacterial lawns. Further, 10 μL of oxytetracycline loaded filter discs were used as positive control for five different bacterial strains. After loading test samples and positive control, petri plates were kept in incubator at 37 ◦C/24 h and observed after time intervals for ZIs. Finally, MICs were calculated to study the bactericidal potentials of RT-NiONPs.

#### **3. Results and Discussion**

The earlier research studies have confirmed the presence of different functional biomolecules such as emodin, Kaempferols-7-O-CH3 ether, Kaempferols-4-O-CH3 ether), gluside, quercetins, and physcion [24,25]. These biomolecules can act as a base source, bioreductant, stabilizers, and capping agents for the convenient synthesis of NiONPs. Previously, NiONPs have been fabricated using a variety of natural plants with potential medicinal values [26]. In the current study, NiONPs has been orchestrated using *R. triquetra* leaf extract via green method. The visual colour change from brown to greenish black revealed the formation of NiONPs. The photo-spectrometric analyses of NiONPs showed broad spectra at 333 nm (λmax), indicating the formation of NiONPs and validating optical observation. The UV visible spectra of green NiONPs are illustrated in Figure 2A. Elemental mapping and atomic content were confirmed using EDS analyses. Figure 2B shows EDS peak indicating strong signals at 0.94, 7.08, an 8.12 KeV for the presence of both nickel and oxygen. The presence of carbon in the spectra is ascribed to grid support. Moreover, no other peak for any elements apart from nickel 'Ni' and oxygen 'O' have been found, indicating the phase purity of greenly synthesized NiONPs.

**Figure 2.** UV visible spectra and EDS analysis for RT. NiONPs (**A**) UV visible spectra for NiONPs (**B**) Elemental composition of NiONPs using EDS analysis.

The hydrodynamic size and stability of RT-NiONPs were demonstrated by DLS and Zeta potential analysis. Zeta-potential or electro-kinetic potential refers to the measure of an effective surface functionality and surface charge on nanoparticle. The magnitude of zeta potential confers to particle stability. Nanoparticles with a high zeta potential exhibit increased stability, i.e., the dispersion or solution will resist the aggregation and agglomeration of nanoparticles. In our research study, data revealed a particle size of 65 nm, zeta-potential of −11 mV, and PDI of 1.000 (Figure 3A,B). Our DLS results are in agreement with previous studies using *Rhamnus virgata* mediated NiONPs [26]. DLS is mostly investigated to confirm the size of nanoscale particles in different suspensions. The mean hydro-dynamic particles diameter (d. nm) in aqueous medium show particles aggregation [11,20,34].

Raman spectral study was further used to analyze the vibrational modes of *R*. *triquetra* mediated NiONPs. Raman spectra in Figure 4A revealed the positioning of major modes at 358.38 (1 Phonon), 559.57 (1 Phonon), 697.34 (2 Phonon), 1106.18 (2 Phonon), and 1646.51 cm−<sup>1</sup> (2 Magnon). The intense peaks concluded that the greenly orchestrated nanoparticles are defect rich. The broad peak attributes to the antiferromagnetic behaviour and is related to the spin of individual Ni++. This antiparallel spin behaviour of 'Ni++' also signifies that NiONPs are nano-size in nature (crystal size: ~25 nm). Raman shifts revealed purity and correspond to earlier research studies using *S. thea* and *G. wallichianum* orchestrated NiONPs [7,35]. The difference in Raman scattering peaks might be due to the relative positions, size, intensity and effects of stress and strain [36]. Further, FT-IR analysis was performed to determine the qualitative distribution of functional groups adsorbed on the surface of NiONPs. The infrared absorption bands in Figure 4B revealed significant vibrations at 545.17 cm−<sup>1</sup> and 657.43 cm−<sup>1</sup> (Ni-O vibrations in stretching mode), 1025.26 cm−<sup>1</sup> (Ethers = C-O-C symmetric stretching), 1729.59 cm−<sup>1</sup> (Aldehyde group of carbonyl '-CHO'), and 3579.43 cm−<sup>1</sup> (alcohols and phenols of OH stretching). According to previously reported research work, IR bands between 470 and 800 cm−<sup>1</sup> indicate Ni-O vibrations in the stretching mode [37,38].

**Figure 3.** (**A**) Zeta potential of NiONPs (**B**) Size distribution of RT-NiONPs.

**Figure 4.** (**A**) Raman spectra of NiONPs (**B**) FT-IR spectra of biogenic NiONPs.

In addition, SEM analysis was performed to know the shape and surface morphology of greenly fabricated NiONPs. Figure 5A,B depicts SEM images of NiONPs confirming the spherical/agglomerated shape of NiONPs. The crystallographic structure and accurate particle size of the biogenic NiONPs were studied by TEM analysis (Figure 5C).

**Figure 5.** (**A**,**B**) Various SEM images of RT-NiONPs using nickel nitrate salt as a precursor (**C**) TEM images of RT- NiONPs.

Moreover, phase structure of NiONPs was assessed by XRD analyses. The XRD pattern of the synthesized NiONPs and miller indexation have been illustrated in Figure 6A,B, which indicates the diffraction bands at 36.52 (101), 43.44 (012), 63.11 (110), 76.28 (113), and 79.2 (202), corresponding to fcc symmetry in NiONPs crystalline lattice. Previously, Khalil et al. [35] and Iqbal et al. [26] have synthesized biogenic NiONPs from *Sageretia thea* and *Rhamnus virgata* leaf extracts and reported similar results.

**Figure 6.** (**A**) XRD Spectra of the RT-NiONPs (**B**) Size calculation and Miller indexation.

#### *3.1. Antimicrobial Potentials*

The antibacterial activities of NiONPs were demonstrated against different bacterial strains (*E. coli*, *P. aeruginosa*, *B. subtilis*, *K. pneumoniae*, *S. aureus*) in concentrations ranging from 34.38 to 1100 μg/mL. Most of the BSs were found susceptible using NiONPs and have shown significant antibacterial activities. Different MIC values were calculated for different bacterial strains *P*. *aeruginosa* (275 μg/mL), *K. pneumoniae* (137.5 μg/mL), *E. coli* (68.75 μg/mL), and *S. aureus* and *B. subtilis* (34.38 μg/mL). *S. aureus* and *B. subtilis* were found to be the most susceptible strains with MIC: 34.38 μg/mL while *P*. *aeruginosa* was found to be the least susceptible strain (MIC: 275 μg/mL), as shown in Figure 7A. Oxytetracyclines was taken as positive control and no single concentration of NiONPs determined a stronger potential than Oxytetracyclines. Overall, NiONPs have determined significant antibacterial activities which are in agreement with previous studies of greenly orchestrated NiONPs using *G*. *wallichianum* and *R*. *virgata* [7,26]. The strong bactericidal potency of NiONPs might be due to biomolecules adsorbed on NPs surface. In conclusion, RT-NiONPs have shown dose-dependent results. Some studies have explained that the bactericidal potential of NPs is due to ROS generation. Further, NPs damage membrane (membrane proteins) and result in bacterial cell death. Similarly, surface defects in the symmetry of NPs is responsible for the inhibition of bacteria and result in cell damage [39].

**Figure 7.** (**A**) Various antimicrobial activities of RT mediated NiONPs. Data represents the mean of three replicates and each alphabet indicates significance at *p* < 0.05 (**A**) MICs values of RT-NiONPs against various pathogenic bacterial strains (**B**) Antifungal potencies of RT-NiONPs against different fungal strains.

Numerous research studies have been conducted on the bactericidal potential of NiONPs while only limited research work has been published on fungicidal activities of NPs. In the present study, the fungicidal potency of RT-NiONPs was investigated using different fungal strains (FS). The different FS such as *F. solani*, *M. racemosus*, *A. niger*, *A. flavus* and *C. albicans* were exposed to different concentration of RT-NiONPs (34.38–1100 μg/mL) (Figure 7B). Amp-B was taken as positive control to confirm the inhibition potential of RT-NiONPs. According to our literature review, the current study, for the first time, reported the antifungal potential of RT-NiONPs. Our *R*. *triquetra*-NiONPs revealed a concentration-dependent inhibition response against different fungal strains where *A. flavus* was the least susceptible fungal strain (MIC: 275 μg/mL while *A*. *niger* was the most susceptible strain (MIC: 34.38 μg/mL). Previously, concentration mediated fungicidal activities were reported using different fungal strains [7] and are in line with our presently synthesized RT-NiONPs. The MIC values for different pathogenic bacterial and fungal strains are provided in Figure 7.

#### *3.2. Antileishmanial Potentials*

Leishmaniasis is a widespread tropical disease caused by leishmanial parasites [27]. The drug antimonial was developed as a potential candidate to cure leishmaniasis but has lost its therapeutic potential as they have developed resistance. Thus, scientists are involved in designing alternative routes to fight and manage this global disease. Therefore, extensive research studies are needed to design some novel and effective nanomaterials. Various nanomaterials are being used to study their antileishmanial potential [11,14]. However, greenly orchestrated NiONPs are rarely studied to investigate their cytotoxic potential. In current study, antileishmanial potentials of *R. triquetra* orchestrated NiONPs was investigated against *L*. *tropica*. The parasites were treated with different doses of RT-NiONPs (8.595−1100 μg/mL) (Figure 8A). The antileishmanial potential increased with RT-NiONPs, thus indicating a dose-dependent response. The RT-NiONPs displayed significant potential against *L*. *tropica* promastigote (IC50: 27.32 μg/mL). Similarly, the antileishmanial potential of RT-NiONPs was reported against *L*. *tropica* amastigotes (IC50: 37.4 μg/mL). Our results of RT-NiONPs are in agreement with the previous reports of *Sageretia thea* mediated NiONPs [35].

**Figure 8.** Biocompatibility, cytotoxic and antioxidant properties of NiONPs. Data represents the mean of three replicates and each alphabet indicates significance at *p* < 0.05 (**A**) Antileishmanial activities against Leishmanial parasites (**B**) Anticancer potentials of RT-NiONPs against HUH-7 and HepG2 cell lines (**C**) Biocompatibility of NiONPs against RBCs and Macrophages (**D**) Antioxidant activities of RT-NiONPs.

#### *3.3. Anticancer Potential of NiONPs*

Among the different types of cancer, liver cancer is the second deadliest cancer in males and sixth deadliest in females [15,40,41]. Different risk factors are involved in regulating the rate of cancer such as viral infections, heavy consumption of alcohol and toxin exposures (aflatoxin). In the current study, the anticancer potential of greenly orchestrated RT-NiONPs was investigated against HUH-7 and HepG2 liver cancer cell lines using an MTT assay [28]. To achieve this purpose, HUH-7 and HepG2 cancer cells were exposed to different concentrations of RT-NiONPs (1100–8.595 μg/mL) as summarized in Figure 8B. The NiONPs have determined strong reduction in metabolic activities of both HUH-7 and HepG2 cancer cells using different doses of NiONPs. The metabolic activities were decreasing while increasing RT-NiONPs concentration. The highest anticancer activity recorded was 81.41% for HuH-7 and 84.41% for HepG2 at 1100 μg/mL and anticancer activity decreased with NiONPs concentration. Further, IC50 values were recorded for RT-NiONPs which are 11.3 μg/mL for (HuH-7) and 20.73 μg/mL (HepG2) cell lines respectively. The anticancer potential induced by RT-NiONPs even at low concentration (8.595 μg/mL) could be due to different functional molecules adsorbed from leaves broth on the surface of NiONPs. The reduction in the metabolic activities have determined that RT-NiONPs have strong anticancer activities. The results of RT-NiONPs using HUH-7 and HepG2 are in correspondence with the previously published reports using *G*. *wallichianum* and *Euphorbia heterophylla* [7,42].

#### *3.4. Biocompatibility Assays with Human Red Cells and Macrophages*

Considering the interest in biomedical applications, the biosafety and biocompatible nature of RT-NiONPs were investigated using previously optimized protocol [7]. According to biosafety principle guidelines, biological and chemical substances having hemolysis >5% are hemolytic, 2−5% are slightly hemolytic, while <2% is non-hemolytic [43]. If the tested NPs are hemolytic, it will damage erythrocytes and result in hemoglobin release from RBCs. To confirm the hemolytic potential, red cells were treated with different doses of RT-NiONPs (1100−8.595 μg/mL) and revealed dose dependent response. The hemoglobin release was 24.23% at highest concentrations of 1100 μg/mL (Figure 8C). Research studies concluded that RT-NiONPs are non-hemolytic at 17.19 μg/mL, slightly hemolytic at 68.75 μg/mL, and hemolytic at >68.75 μg/mL. On the whole, RT-NiONPs are non-toxic and biocompatible at low concentration against red cells. Our biocompatibility results of RT-NiONPs are in line with the previously synthesized *S. thea* and *G*. *wallichianum* mediated NiONPs [7,35].

The biosafe and biocompatible nature of RT-NiONPs was further determine using normal human macrophages (HM). For this purpose, confluent HM cells were seeded in sterilized 96-well plate containing RPMI media and were cultured for 24 h. Further, the seeded cells were exposed to different doses of RT-NiONPs (1100−8.595 μg/mL). Further, an MTT cell viability assay was performed to confirm the biosafe nature of RT-NiONPs. The results shown in Figure 8C indicate that biosynthesized RT-NiONPs at 1100 μg/mL inhibited the growth of HM cells by ~30.89% confirming its biosafe nature, thus indicating a dose-dependent response. Normally, HM cells have developed a natural strategy to neutralize ROS produced from external sources. Previous research studies reported that ROS are non-toxic to both red cells and HM cells at low concentrations unless concentrations increase beyond the limit, which will result in toxicity to both erythrocytes and macrophages [44]. Previously, Iqbal et al. [26] reported the biocompatibility potential of greenly fabricated NiONPs against HM cells using *Rhamnus virgata*.

#### *3.5. Antioxidant Activities*

The antioxidant potential of phytogenic NiONPs was evaluated (Figure 8D). The maximum value for TAC of *R. triquetra* mediated NiONPs in terms of AA Emg−<sup>1</sup> was reported as 71.93% at 1100 μg/mL. Generally, a TAC assay is performed to evaluate the scavenging potential of reductones/antioxidants present in the test sample towards ROS species. Our TAC results are in correspondence to

Abbasi et al. [7] using *Geranium wallichianum* mediated NiONPs. Further, a cupric-ion assay was investigated to assess the scavenging potentials of antioxidants species adsorbed on the surface of RT-NiONPs. The maximum score for cupric ion assay of green RT-NiONPs was obtained as 81.83%. According to our literature review, a cupric-ion assay was demonstrated for the first time on RT-NiONPs. Moreover, greenly orchestrated NiONPs were further explored to determine the surface adsorbed antioxidant molecules. To achieve this goal, TRP assay was performed. The maximum value for TRP was recorded as 83.41% at 1100 μg/mL which is corroborated with the earlier research report using *Rhamnus virgata* mediated NiONPs [26]. Further, DPPH free radical scavenging assay was demonstrated to assess the presence of radical scavengers (antioxidants) adsorbed on the surface of greenly fabricated NiONPs. The highest DPPH value reported is 77.91% and our DPPH data are in agreement with the previous studies using *Sageretia thea* mediated NiONPs [35]. Together, the antioxidant potential confirmed the presence of radical scavengers on RT-NiONPs which play a potential role in the stabilization, reduction, and capping of nano nickel oxide particles [7].

#### *3.6. Enzymes Inhibition Potentials*

Greenly orchestrated RT-NiONPs were examined for their protein kinase (PKs) inhibition activity. Figure 9A shows the significant PK inhibition potential of *R. triquetra* mediated NiONPs using different doses of NiONPs (34.38–1100 μg/mL). Moderate PK inhibition potential was revealed for RT-NiONPs. The maximum value for ZIs was 15.5 mm with IC50 > 1000 μg/mL. On the whole, ZIs obtained for greenly orchestrated NiONPs were smaller as obtained from surfactin (positive control). These results suggested cell viability at lower concentrations of NiONPs, consistent with a previous research report using *G. wallichianum*−NiONPs [7]. Further, A-amylase assay was demonstrated to evaluate the inhibition potentials of *R. triquetra*-NiONPs using different concentrations (1100−34.38 μg/mL). The biogenic NiONPs were observed to cause increased % inhibition (39.31%) at 1100 μg/mL (Figure 9B). However, the percent inhibition significantly decreased with a decrease in concentration. Overall, a moderate inhibition potential is reported. The results of our current report are in agreement with a previous research study using *S*. *thea* mediated NiONPs [35].

**Figure 9.** (**A**) Inhibition potential against protein kinase of RT orchestrated NiONPs (**B**) Inhibition potential against alpha amylase.

#### **4. Conclusions and Future Directions**

This study has established a simple, eco-friendly, and economically viable method to synthesize NiONPs simply by mixing NiNO3 with aqueous broth of *R*. *triquetra* leaves which are free of toxicants and rich in functional biomolecules. The actions of different functional biomolecules in the leaves extract may result in the reduction, stabilization, and capping of NiONPs. The microscopic analyses from SEM and TEM confirmed the predominant spherical shape and small size of NiONPs (~25 nm). Further, spectroscopic studies from UV–vis, Raman, FT-IR, EDX, zeta potential, and DLS supported the fabrication and stability of NiONPs. Significant anticancer potentials were revealed against different cancer cell lines (HepG2: IC50: 20.73 and HuH-7: IC50: 11.3 μg/mL). Further, antileishmanial potential was investigated against leishmanial parasites (promastigotes; IC50: 27.32, amastigotes: IC50: 37.4 μg/mL). The outcomes of a biocompatibility assay revealed that NiONPs are non-toxic and biocompatible. NiONPs determined significant free radical scavenging and moderate enzyme inhibition activities. Further, NiONPs determined significant antimicrobial studies against different bacterial and fungal strains. In conclusion, our results unequivocally indicate that RT-NiONPs may be used as a safer alternative in biotechnological, biomedical, and pharmaceutical industries. Further, more in vitro and in vivo studies are recommended in different animal models before bringing NiONPs into clinical trials.

**Author Contributions:** J.I., B.A.A., designed and performed the experiments at Quaid-i-Azam University Islamabad, Pakistan. R.A., and M.M., analyzed the data. A.M., S.A.Z., A.S., M.S., S.K., S.U. draft, revised and improved the manuscript. T.M., and R.C. supervised the study. All authors have read and agreed to the published version of the manuscript.

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

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

#### **References**

© 2020 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 (http://creativecommons.org/licenses/by/4.0/).

## *Review Trametes versicolor* **(Synn.** *Coriolus versicolor***) Polysaccharides in Cancer Therapy: Targets and E**ffi**cacy**

#### **Solomon Habtemariam**

Pharmacognosy Research Laboratories & Herbal Analysis Services UK, University of Greenwich, Chatham-Maritime, Kent ME4 4TB, UK<EMAIL_ADDRESS>Tel.: +44-208-331-8302

Received: 5 May 2020; Accepted: 21 May 2020; Published: 25 May 2020

**Abstract:** *Coriolus versicolor* (L.) Quél. is a higher fungi or mushroom which is now known by its accepted scientific name as *Trametes versicolor* (L.) Lloyd (family Polyporaceae). The polysaccharides, primarily two commercial products from China and Japan as PSP and PSK, respectively, have been claimed to serve as adjuvant therapy for cancer. In this paper, research advances in this field, including direct cytotoxicity in cancer cells and immunostimulatory effects, are scrutinised at three levels: in vitro, in vivo and clinical outcomes. The level of activity in the various cancers, key targets (both in cancer and immune cells) and pharmacological efficacies are discussed.

**Keywords:** *Coriolus versicolor*; *Trametes*; cancer; polysaccharides; PSP; PSK; immunostimulation; adjuvant therapy

#### **1. Introduction**

According to the recent WHO figure [1], cancer is the second most leading cause of mortality in the world and accounts for an estimated 9.6 million deaths in 2018. Most of the cancer death (~70%) occur in developing or the so-called low- and middle-income countries where access to modern medicines are not widely available. The common cancer deaths are from the lung, colorectal, stomach, liver and breast cancers, respectively, while other common cancers include prostate and skin cancers [1]. The major control measure for cancer is chemotherapy by using a variety of small molecular weight compounds and biological agents. As always, nature has its fair share of abundance as a source of these agents and drugs like paclitaxel (Taxol®), podophyllotoxin derivatives and vinca alkaloids (vinblastine and vincristine) are our excellent examples for potential exploration of more novel anti-cancer agents from higher plants. On the other hand, doxorubicin, daunomycin, mitomycin C, and bleomycin are good representative examples of anti-cancer agents explored from fungal sources, particularly *Streptomyces.*

In addition to their nutritional value, medicinal mushrooms have emerged in recent years not only as a source of drugs but also as adjuvants to conventional chemo- or radiation-therapy to either enhance their potency or reduce their side effects ( see [2] and references therein). In this regard, one of by far the best investigated medicinal mushroom in recent years is *Coriolus versicolor* (L.) Quél. *(Syn. Polyporus versicolor)* which is now known by its accepted scientific name as *Trametes versicolor* (L.) Lloyd (family Polyporaceae). Its most common name in the Western world is Turkey Tail, and its distinct morphological features include the concentric multicoloured zones on the upper side of the cap (no stalk) and spore-bearing polypores underside (Figure 1). The fungus is common in temperate Asia, North America and Europe, including the UK, where it has been recorded in all regions [3]. Its medicinal value as part of the Chinese traditional medicine dates back for at least 2000 years and includes general health-promoting effects [4], including endurance and longevity. Both in China and Japan, preparations such as dried powdered tea of the fungus are employed in traditional medicine practices. In this communication, the main components of the fungi, polysaccharides, that have given

the fungi its medicinal value in cancer therapy are assessed by reviewing the chemistry, pharmacology and therapeutic potential at three levels: in vitro, in vivo and clinical studies. Readers should note that nearly all the published literature in this field is available under the name *Coriolus versicolor (Trametes versicolor*).

**Figure 1.** Morphological features of *Coriolus versicolor*. The various morphological features of the fungus grown in the UK are shown. While the upper surface shows concentric zones of colours (red, yellow, green, blue, brown, black, and white), the picture in the lower-right shows the polyporous nature of the underside portion of the fungus. Pictures are a kind gift of *first-nature.com* (https: //www.first-nature.com/fungi/trametes-versicolor.php#distribution).

#### **2. Overview of Chemistry**

#### *2.1. Small Molecular Weight Compounds*

Like all other mushrooms, the fruiting body of *C. versicolor* is harvested for its nutritional and medicinal values. The bracket or shelf mushroom body in the wild or the mycelial biomass collected from the submerged fermentation could all be used for this purpose. In addition to the major macromolecules (proteins, carbohydrates, and lipids) and minerals, the fungus is known to contain potential pharmacologically active secondary metabolites belonging to small molecular weight compounds. The study by Wang et al. [5] reported the isolation of four new spiroaxane sesquiterpenes (Figure 2), tramspiroins A–D (1–4), one new rosenonolactone 15,16-acetonide (5), and the known drimane sesquiterpenes isodrimenediol (6) and funatrol D (7) from the cultures. Readers should bear in mind that these compounds isolated from the ethyl acetate fraction are non-polar and are not expected to be available in the polysaccharide fractions of the fungus (see below). Janjuševi´c et al. [6] studied the phenolic composition of the fruiting body of *C. versicolor* of European origin. In their HPLC–MS/MS-based study, they identified 38 phenolic compounds belonging to the flavonoid

(flavones, flavonols, flavanone, flavanols, biflavonoids, isoflavonoids) and hydroxy cinnamic acids. Although the ethanol and methanol extracts are generally the richest sources of these phenolic compounds, the water extracts were also shown to contain (μg/g dry weight) considerable amount of baicalein (21.60), baicalin (10.7), quercetin (31.20), isorhamnetin (14.60), catechin (17.20), amentoflavone (17.20), *p*-hydroxybenzoic acid (141.00) and cyclohexanecarboxylic acid (80.40). The biological activities of the water extracts of *C. versicolor*, especially in the antioxidant area, must, therefore, account for the cumulative effects of the phenolic compounds. These compounds are, however, not established as the main components of the fungus, and further research is required to establish their potential contribution to the known biological activities of *C. versicolor.*

**Figure 2.** Terpenoids from *C. versicolor.*

#### *2.2. Polysaccharides*

Like other edible mushrooms, the fruiting body of *C. versicolor* is composed of carbohydrates, proteins, amino acids, and minerals. The main bioactive components of *C. versicolor* are the polysaccharopeptides (PSPs), which are isolated from the mycelium as well as fermentation broth. As a commercial product, the main sources of these PSPs are China and Japan that produce them from the strains of "COV-1" (PSP in China) and "CM-101" (polysaccharide K (PSP Krestin or PSK, in Japan), respectively. Both products have been approved as medicines primarily as adjuvants in cancer therapy. Given that over 100 strains of the fungi are known to occur, one must recognise the diversity of these products coming from different genetic and environmental sources, including the in vitro culture conditions of their mycelial production. They are made from polysaccharides covalently bonded to peptides through *O*- or *N*-glycosidic bonds. Numerous studies have established that D-glucose is the principal monosaccharide of PSP and PSK, although other sugars such as arabinose and rhamnose are also found in small amounts (e.g., [6–8]). One noticeable difference in these products could be the composition of polysaccharide:peptide ratio, and their relative molecular weight. The (PSK and PSP) are both proteoglucans of about 100 kDa with variations in the individual sugar compositions such as glucose, fucose, galactose, mannose, and xylose.

The distinction between the extracellular (EPS) and intracellular polysaccharides have also been made with respect to their backbone structure [7,8]. The EPS contains small amounts of galactose (Gal), mannose (Man), arabinose (Ara), xylose (Xyl) and predominantly glucose (Glc) and are composed of β-(1→3) and β-(1→6)-linked D-glucose molecules. On the other hand, PSP and PSK contain α-(1→4) and β-(1→3) glucosidic linkages in their polysaccharide moieties. D-glucose is the major

monosaccharide present while fucose (Fru), Gal, Man, and Xyl are the other principal monosaccharides in PSK. Earlier studies [9] established the distinctive features of these two polysaccharides with the presence of fucose in the PSK and rhamnose and arabinose in PSP. Analysis of the polysaccharide moiety of PSP, showed the predominance of 1→4, 1→2 and 1→3 glucose linkages (molar ratio 3:1:2), together with small amounts of 1→3, 1→4 and 1→6 Gal, 1→3 and 1→6 Man, and 1→3 and 1→4 Ara linkages [9]. On the other hand, the peptide moiety of PSP contains 18 different amino acids, with aspartic and glutamic acid residues being most predominant [8]. More importantly, PSK and PSP polymers are soluble in water.

The complexity of *C. versicolor* can be seen from the detailed structural analysis, as shown for PSP-1b1 backbone by Wang et al. [10] as follows: "→4)-α-Gal*p*-(1→4)-α-Gal*p*-(1→2)-α-Man*p*- (1→4)-α-Gal*p*-(1→2)-α-Man*p*-(1→4)-α-Gal*p*-(1→4)-α-Gal*p*-(1→2)-α-Man*p*-(1→4)-α-Gal*p*-(1→2)-α-Man*p*-(1→4)→, with branches of α-1,6-Man*p*, β-1,6-Glc*p*, β-1,3,6-Glc*p*, α-1,3-Man*p*, α-1,6-Gal*p*, α-1,3-Fuc*p*, T-α-Glc*p* and T-α-Gal*p* on the *O*-6 position of α-Man*p* of the main chain, and secondary branches linked to the *O*-6 position of β-Glc*p* (β-glucose-pyranose(*p*)) of the major branch." Awadasseid et al. [11] also isolated a water-soluble glucan extracted from *C. versicolor* called CVG with the general backbone structure of [→6)−α−D−Glc*p*−(1→] n. In comparison to the PSK and PSP, CVG was small with a molecular weight of 8.8 Kda and carbohydrate composition of D-Fuc, D-Ara, D-Man, D-Gal and D-Glc, in a molar ratio of 1.0/1.1/3.0/3.9/350.7, respectively. The polysaccharide isolated by Zhang et al. [12] called β-1→3 was with the main chain consisting of β -D-1,4-Glc and β-D-1,3-Glc, and branch chains situated at β-D-1,3,6-Glc and β-D-1,4,6-Glc. More research is, however, required to identify the structures of all polysaccharides from this fungus.

#### **3. Anticancer E**ff**ect through Direct Toxicity to Cancer Cells**

Studies during the 1990s established that *C. versicolor* polysaccharides such as PSK could inhibit hepatic carcinogenesis in rats induced by 3 -methyl-4-dimethylaminoazobenzene [13]. The direct effect of PSK on gene expression profile in cancer cells was also established back in the 1980s [14]. Studies on combination therapy with radiation further showed the increased survival rate of mice bearing MM46 tumours [15]. Corriolan as a β-(1→3) polysaccharide with some (1→6) and no (1→4) branched glucan from *C. versicolor* was shown to be effective (100 mg/kg for 30 days) in suppressing sarcoma 180 tumours in mice [16]. Since then, the direct anticancer effect of *C. versicolor* polysaccharides has been demonstrated in the various experimental models in vitro, in vivo and clinical trials (see below).

#### *3.1. Evidence of E*ffi*cacy through In Vitro Studies*

The direct toxicity of *C. versicolor* polysaccharide preparations to cancer/tumour cells has been demonstrated in the various in vitro models [17–49] (Table 1). The number of cancer types that could be targeted by the polysaccharides is incredibly large and include breast (e.g., MCF-7, HBL-100, T-47D, ZR-75-30, MDA-MB-231 and Walker 256) [18,20,32,37,44,46], lung (e.g., A-549, and SWi573) [20,21], melanoma (e.g., SKMel-188 and B16) [17,31], colon (e.g., LoVo, HT-29, SW480, WiDr, LS174 and LS174-T) [19–26,48], leukaemia (e.g., Jukart, K562, THP-1, OCI-AML3, HL-60 and U937) [20,22,24–26,28, 30,35,36,38–40,48,49], cervix (e.g., HeLa [20,21], gastric cancer (e.g., AGS, KATOIII, SCG-7901 [24–26,48]), prostate (e.g., PC-3, JCA-1, LNCaP, DU-145 [29,43], glioma (e.g., C6) [42], hepatoma (e.g., HepG2) [45,48], and ovarian (e.g., H4-II-E) [47] cancers. The vast majority of studies are on the two known commercially available *C. versicolor* polysaccharides, PSP and PSK (Table 1), while others include small polypeptide of about 10 Kd [48], refined polysaccharide peptide fractions [23,45], and aqueous or alcoholic extract [19–21,31,35,37,41,43]. Given the high molecular weight nature of the polysaccharides and even most are crude products, their observed anticancer activity mostly demonstrated in less than 1 mg/mL concentrations should be considered good but many experiments even showed activity at concentrations less or equal to 100 μg/mL [17,19,24–26,28,32,36,38,41,46,48]. The inhibition of cell proliferation by *C. versicolor* is associated with cell cycle arrest which could vary depending on the concentration and cell type. For example, disruption of cell cycle progression and arrest at G0 phase [22], G0/G1 phase [31,33] or G1/S and G2/M phases [36] have been reported. As a mode of cell death, induction of apoptosis has been shown for many cell types which was associated with caspase-3 activation via the mitochondrial pathway [24–26,28,36,39].

**Table 1.** Direct cytotoxic effects in vitro.

**Table 1.** *Cont*.

**Table 1.** *Cont*.

**Table 1.** *Cont*.

**Table 1.** *Cont*.

Abbreviations: CAT, catalase; COX-2, cyclooxygenase 2; GPx, glutathione peroxidase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated kinase; MMP, matrix metalloproteinase; NBT, nitroblue tetrazolium; NF-κB, nuclear factor κB; PARP, poly(ADP-ribose) polymerase; ROS, reactive oxygen species; SOD, superoxide dismutase; STAT, signal transducer and activator family of transcription.

As expected for apoptosis-inducing agents, genes and proteins that are associated with cancer cell survival (anti-apoptotic BCL-2*,* Bcl-xL, survivin) are shown to be suppressed while those markers of apoptosis (proapoptotic Bax) induction are upregulated by the *C. versicolor* polysaccharide preparations [18,32,35,36]. Induction of the intracellular level of reactive oxygen species (ROS) in cancer cells is a well-established mechanism of cell death by chemotherapeutic agents and this appears to be the case for *C. versicolor* in several cell lines [17,46]. The critical cell growth and death regulator mitogen-activated protein kinase (MAPK) is involved in the induction of cell death by *C. versicolor* polysaccharides, as shown by the enhancement of p38 MAPK phosphorylation [24–26,28]. Accordingly, the cytotoxicity of these polysaccharides in melanoma cells could be abolished in the presence of c-Jun N-terminal kinase (JNK) inhibitors [17] or the p38 MAPK inhibitor [24–26]. Key transcription factors that are involved in cancer development and metastasis could be inhibited by *C. versicolor* polysaccharides. This includes the well-defined cancer modulator, NF-κB, or its induced protein product, cyclooxygenase-2 (COX-2) [36,40]. The potential combination of *C. versicolor* polysaccharides with conventional chemotherapeutic agents has been demonstrated in vitro, as shown for camptothecin [30], doxorubicin and etoposide [32,38], and cisplatin [46]. Even at concentrations where direct toxicity was not observed, inhibition of cell migration and invasion was evident along with inhibition of key angiogenic enzymes such as matrix metalloprotease (MMP)-9 [23] or MMP-2 [19].

#### *3.2. Evidence of E*ffi*cacy through Animal Models*

Many in vitro experiments that showed promising effects in direct cytotoxicity study were also extended to animal models of tumour-bearing mice. This was based on the injection of the cancer cells into mice and assess the size and spread (metastasis) of the tumour in the presence or absence of *C. versicolor* polysaccharides. Table 2 has a good summary of these data with the description of the polysaccharides, their route of administration and main outcomes [14,23,29,31,37,42,45,50–55]. It appears that *C. versicolor* polysaccharides in the form of PSP, PSK, refined fractions, water or aqueous

extracts exhibit anticancer effects in vivo when administered by either oral (p.o.), intraperitoneal (i.p.) or intravenous (i.v.) routs. In a combination approach, favourable responses were obtained with metronomic zoledronic acid [50] and docetaxel–taxane [51]. In addition to a reduction in the size and volume of the implanted tumours, the incidence of tumours [29,45] and angiogenesis via vascular endothelial cell growth factor (VEGF) expression have been shown to be inhibited.

**Table 2.** *Cont*.

Abbreviations: VEGF, vascular endothelial cell growth factor.

#### *3.3. Evidence of E*ffi*cacy through Clinical Trials*

Chay et al. [56] employed a human study on *C. versicolor* extract by recruiting fifteen eligible cases of hepatocellular carcinoma patients in Singapore who failed or were unfit for standard therapy. The randomised placebo-controlled trial using 2.4 g as a daily treatment for ~5.9 weeks) showed a better quality of life without a significant difference in primary endpoint measure of the median time to progression. On the other hand, a pilot study of randomised, double-blind, and multidose study on dogs (not humans) revealed that treatment with PSP (e.g., 100 mg/kg capsules daily) could delay the progression of metastases of canine hemangiosarcoma [57]. The systematic review and meta-analysis studies by Eliza et al. [58] assessed the survival outcome in cancer patients from 13 clinical trials on *C. versicolor*. They reported an impressive result showing a significant survival advantage when compared with standard conventional anti-cancer agents alone. For example, a 9% absolute reduction in 5-year mortality was recorded with one additional patient alive for every 11 patients treated. They also reported a better 5-year survival rate in patients receiving combination treatment in

cases of breast cancer, gastric cancer, or colorectal cancer. Database on ClinicalTrial.gov shows one terminated clinical trial on the potential benefit of *C. versicolor* for hepatocellular carcinoma and one currently recruiting for a vaginal gel based on *C. versicolor* medical device (PAPILOCARE) as a phase III trial.

#### **4. Anti-Cancer E**ff**ect Via Immunostimulation**

Studies on the immunotherapeutic potential of the *C. versicolor* polysaccharides in cancer started in the late 1970s and accelerated in the 1980s and 1990s. In 1977, Kataoka et al. [59] reported that immuno-resistance in mice could be induced when protein-bound polysaccharides are administered together with L1210 murine leukemic cells. The suppression of TNF-α production in mice by cytotoxic antitumour agents (5-fluorouracil, cyclophosphamide and bleomycin) was shown to be ameliorated by PSK with an implication of immunotherapy potential [60]. The immunosuppressive effect of cyclophosphamide in rats could also be abolished by PSP [61]. Myelosuppressed mice due to chemotherapy could also be reversed by PSK, particularly when used in combination with granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF) or IL-3 [62]. These general immunostimulations or ameliorations of immunosuppression under cancer and depressed immune systems, either by cancer, splenectomy or other experimental agents, have been observed for *C. versicolor* polysaccharides [63–69].

Further studies in vitro showed the direct lymphocyte proliferative effect of PSP, while in mice, it reversed the inhibition of IL-2 production induced by cyclophosphamide along with restoration of the T-cell-mediated response [70]. The study by Kanoh et al. [71] also demonstrated that PSK could enhance the anti-tumour effects of IgG2a monoclonal antibody in the human colon cancer cell line, colo 205, both in vitro and in vivo via antibody-dependent macrophage-mediated cytotoxicity. Studies on PSK using mice bearing syngeneic plasmacytoma X5563 also showed that it enhances anti-tumour immunity by ameliorating the immunosuppressive activity of serum from tumour-bearing mice [72,73]. The tumour-induced immunosuppression could also be abolished by PSK in various cancer models in vivo [74]. Earlier in vitro studies further confirmed the direct effect of the polysaccharides on peritoneal macrophages [75], namely, interleukin-1 production by human peripheral blood mononuclear cells [76]. Further insight into the immunotherapeutic potential of *C. versicolor* polysaccharides is outlined below under the headings of in vitro, in vivo and human studies.

#### *4.1. Evidence of Immunotherapy Potential through In Vitro Studies*

Perhaps the best characterised pharmacological activity of *C. versicolor* relates to its immunostimulatory effects. Some of the key outcomes from in vitro studies with implications to cancer are shown in Table 3 [8,49,77–94]. The proliferative effect of the polysaccharides on mononuclear cells, such as lymphocytes [78,79,86,92], monocytes [85] or macrophages [77] and others, including splenocytes [81], has been shown for the polysaccharides. The immunostimulatory effect also includes activation of immune cells, as shown in the LPS-induced cytokine (interleukin (IL)-1β and IL-6) expression by peripheral blood mononuclear cells (PBMCs) [77] or by blood lymphocytes) [78]. Enhancement of antibody production such as IgM and IgG1 by splenocytes was reported [82], while activation of dendritic cells was evident from the expression level of surface markers in mature cells [84]. Increased level of IgM production in B cells by PSK has also been reported. Similarly, cytokines expression, including upregulation of TNF-α expression, leads to enhanced breast cancer cell killing) [79]. The production of IL-10 in mouse B cells could be enhanced by up to 60-fold for some preparations [86], while the antibody-mediated cytotoxicity of natural killer (NK) cells against cancer cells could be enhanced through IL-12-dependent and independent mechanisms [87]. Selective induction of cytokines expression that promotes Th1 and Th2 lymphocytes have been shown [92]. Also, increased nitric oxide (NO) production in polymorphonuclear cells (PMNs) or mononuclear cells such as U937 and THP-1 have been reported for PSK and its fractions [49,94].

Considerable levels of research have been devoted to understanding how *C. versicolor* polysaccharides interact with the immune cells. One of the established recognition sites for the polysaccharides is the toll-like receptors (TLRs), of which effects via TLR4 are well-documented. In mouse peritoneal macrophages, the expression of cytokines and NF-κB activation by PSP was shown to be coupled with TLR4 activation [81]. Furthermore, the induction of TNF-α and IL-6 secretion in J774A.1 cells and primary splenocytes by PSP via TLR4 has also been well established and correlated with its effect on NF-κB p65 transcription and phosphorylation of c-Jun [88]. The expression of both TLR4 and TLR5 by PSP was shown in PBMCs of human origin, while TLR9 and TLR10 appear to be downregulated [89]. Fractionation of PSK further led to the identification of two motifs: a β-glucan recognised by the Dectin-1 receptor and lipid fraction with agonistic activity towards TLR2.

**Table 3.** Immunomodulatory effects related to cancer: in vitro studies.

**Table 3.** *Cont*.

**Table 3.** *Cont*.

Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; CTL, cytotoxic T lymphocytes; DC, dendritic cells; FITC, fluorescein isothiocyanate; HLA, human leukocyte antigen; IFN-γ, interferon-γ; LPS, lipopolysaccharides; MHC, major histocompatibility complex; PMBCs, peripheral blood mononuclear cells; PMN, polymorphonuclear cells; SARM, sterile-alpha and Armadillo motif-containing protein; TOLLIP, Toll interacting protein; TRIF, TIR domain-containing adaptor protein-inducing interferon β; TRAM, (TRIF)–related adaptor molecule; TRAF, tumor necrosis factor receptor (TNF-R)-associated factor.

#### *4.2. Evidence of Immunotherapy Potential through In Vivo Studies*

The animal studies on *C. versicolor* polysaccharides also support the general immunostimulatory effect (Table 4) [23,31,48,78,81,84,87,95–104]. Increased cytokine and ROS production and NF-κB activation have been reported in rats [95]. Through IL-10-dependent mechanism, an enhancement of cytokine production that was associated with T helper (Th2 and Th17 cells) (e.g., IL-2, -4, -6, -10, -17A and IFN-α and -γ) were observed for a glucan product of *C. versicolor* in cancer-bearing mice [96]. By increasing the level of IL-6, PSP could also increase the duration of endotoxin fever in rats [98]. The proinflammatory effect of *C. versicolor*is also evident from in vivo effect of PSP in inducing a writhing response in animals, which was associated with induction of the release of prostaglandin-E2 (PGE2), TNF-α, IL-1β, and histamine from macrophages and mast cells [101]. In agreement with the in vitro experiments, combination with acacia gum resulted in a selective increase in IgG level in mice treated by PSP while the IgA or IgE levels were not affected [97]. Small peptide fractions of the polysaccharides have also shown to increase IgG level in vivo as well as white blood cell (WBC) count in tumour-bearing nude mice [48]. The animal studies in rats also suggest that high temperature exposure (hyperthermia) could suppress the cytokine production by *C. versicolor* polysaccharides [78]. Furthermore, PSP has been shown to rapidly lower temperature in rats by elevating the level of TNFα [99].

The correlation between TLR4 activation by PSP and anti-tumour potential was studied in mice. This was substantiated from the fact that its anti-tumour effect and increased thymus index and spleen index were evident in tumour-bearing C57BL/10J (TLR4+/+) mice but not in C57BL/10ScCr (TLR4−) mice [81]. PSK could also enlarge lymph nodes, activate dendritic cells, and stimulate T-cells to produce