*Ent*-kaur-2-one-16β,17-dihydroxy-acetone-ketal (DEK) (C23H36O3, MW: 360.54, Figure 2) was isolated from the leaves of *Rubus corchorifolius*, and exhibited cytotoxic activity (IC50 = 40.0 μM) in HCT-116 human colon cancer cells after 72 h treatment but was non-toxic up to 100 μM in human colonic myofibroblasts CCD-18Co cells [75]. Mechanistic studies found that DEK induced apoptosis by increasing the expression of cleaved caspase-3, -9, PARP, p53, BAX, and p21Cip1/Waf1, while decreasing the expression of cell cycle markers such as cyclin D1, CDK2, and CDK4. Moreover, the expression of two carcinogenic proteins including epidermal growth factor receptor (EGFR) and COX-2 were reduced and the activation of Akt was inhibited after DEK treatment.

#### *2.20. JDA-202*

JDA-202 (C21H30O5, MW: 362.47, Figure 2) was isolated from *Isodon rubescens*, and exhibited anticancer effects in esophageal cancer EC109 (IC50: 8.60 μM) and EC9706 (IC50: 9.40 μM) cells, yet was considerably non-toxic in normal cell lines KYSE-450 (IC50: 26.2 μM) and HET-1A (IC50: 36.1 μM) at 24 h drug incubation. Antiproliferative activity was related to direct binding of JDA-202 to the antioxidant protein Prx I, inhibition of its activity and expression, as well as induction of hydrogen peroxide (H2O2)-related cell death in esophageal cancer cell lines. Moreover, JDA-202 treatment also led to the increased phosphorylation of JNK, p38, and ERK, which were suppressed by ROS scavenger *N*-acetylcysteine (NAC) and H2O2 scavenger catalase. Intravenous administration of JDA-202 at 20 mg/kg/day in Balb/c nude mice significantly inhibited the tumor growth without loss of body weight and organ toxicities [26].

#### *2.21. Pterisolic Acid G*

Pterisolic acid G (PAG) (C20H28O6, MW: 364.44, Figure 2), isolated from *Pteris semipinnata*, doseand time-dependently inhibited the growth of human colorectal cancer cell line HCT-116 with IC50 values 20.4, 16.2, and 4.07 μM for 24, 48, and 72 h, respectively. Mechanistic studies found that PAG reduced the expression of disheveled segment polarity protein 2 (Dvl-2), GSK-3β, β-catenin, cyclin D1, and c-myc in HCT-116 cells. Moreover, it induced apoptosis by increasing the expression of p53, puma, cleaved PARP, and cleaved caspase-3, as well as decreasing the expression of p-p65, BCL-2, and BCL-xL [76].

#### *2.22. Rabdoternin B and Maoecrystal I*

Rabdoternin B (C21H30O7, MW: 394.46, Figure 2) and maoecrystal I (C22H30O8, MW: 422.47, Figure 2) were isolated from the aerial parts of *Isodon rosthornii* [77] and the leaves of *I. xerophilus* [78], respectively. After 48 h, rabdoternin B showed cytotoxicity in colon cancer SW480 (IC50: 23.2 μM), HT-29 (IC50: 36.3 μM), and HCT-116 (IC50: 20.7 μM) cells, but no toxicity in normal colon CCD-841-CoN (IC50: >40 μM) cells. The IC50 values of maoecrystal I were 16.2, 11.4, 26.2, and >40 μM in SW480, HT-29, HCT-116, and CCD-841-CoN cells, respectively. Both compounds induced G2/M phase arrest in SW480 cells. Further research with maoecrystal I found that its anticancer activity was related with downregulation of Wnt-signaling target genes such as c-myc, cyclin D1, surviving, and axin2 [79].

#### *2.23. CHKA*

Huang et al. (2016) isolated 3α-cinnamoyloxy-9β-hydroxy-*ent*-kaura-16-en-19-oic acid (CHKA) (C29H36O5, MW: 464.6, Figure 2) from the ethanol extract of *Wedelia chinensis*. CHKA from petroleum ether fraction showed most potent anti-angiogenic activity in zerbrafish model. Moreover, CHKA treatment also considerably blocked a series of VEGF-stimulated angiogenesis events such as proliferation, invasion, and tube formation of endothelial cells. Additionally, it also diminished the activity of VEGFR-2 tyrosine kinase and inhibited several downstream targets including p-VEGFR-2, p-mTOR, p-Akt, and p-ERK in HUVECs. In a mouse model, CHKA significantly blocked sprouts formation of the aortic ring, and inhibited subsequent formation of vessels [80].

#### *2.24. CrT1*

*Ent*-18-acetoxy-7β-hydroxy kaur-15-oxo-16-ene (CrT1) (C22H32O4, MW: 360.49, Figure 2) was isolated from the traditional Vietnamese medicinal plant *Croton tonkinensis*, and exhibited dose- and time-dependent antiproliferative activity in various cancer cell lines with IC50 values ranging between 8.40 and 31.2 μM but no toxicity in fibroblast NIH-3T3 cells (IC50 not reported) [81]. CrT1 induced apoptosis and G1 cell cycle arrest in human hepatocellular carcinoma SK-HEP1 cells by the activation of caspase-3, -7, -8, -9, and PARP. Moreover, the expression of p53 and BAX increased, while the expression of BCL-2 decreased after CrT1 treatment. CrT1 also increased the cytoplasmic translocation of cytochrome c, upregulated the expression of p-AMPK, while downregulating the expression of p-mTOR and p-p70S6K.

#### *2.25. Ent-16*β*-17*α*-dihydroxykaurane*

*Ent*-16β-17α-dihydroxykaurane (DHK) (C20H34O2, MW: 306.49, Figure 2) was isolated from the bark of *Croton malambo*, and exerted pro-apoptotic effects in human breast cancer cell line MCF-7 (IC50: 40.8 μM) after 72 h by downregulating BCL-2 expression at both the mRNA and protein levels, as well as decreasing human telomerase reverse transcriptase (hTERT) expression at the mRNA level [82]. The same research group later found dissociation of activator protein 2 alpha (Ap2α)–Rb activating complex in MCF-7 cells after DHK treatment, which affects the binding ability of the complex to BCL-2 gene promoter. This process leads to downregulation of BCL-2 as well as upregulation of E2F transcription factor 1 (E2F1) and its target pro-apoptotic gene puma [83].

**Figure 2.** Structures of plant-derived anticancer *ent*-kaurane diterpenoids.

#### *2.26. Ent-11*α*-hydroxy-16-kauren-15-one*

*Ent*-11α-hydroxy-16-kauren-15-one (KD) (C20H30O2, MW: 302.46, Figure 2) was isolated from the Japanese liverwort *Jungermannia truncata*, and induced apoptosis in human promyelocytic leukemia HL-60 (IC50: 0.56 μM) cells through activation of caspase-8. Treatment with KD also resulted in activation of caspase-9 but caspase-9-specific inhibitor could not diminish KD-induced apoptosis. Moreover, KD treatment resulted in time-dependent cleavage of caspase-8-substrate Bid as well as proteolytic processing of procaspase-8, suggesting association of caspase-8-dependent pathway in KD-induced apoptosis [84].

#### *2.27. Hydroxy-15-oxo-zoapatlin*

Hydroxy-15-oxo-zoapatlin (OZ) (C20H26O4, MW: 330.42, Figure 2) was isolated from plants in the genus *Parinari* and has been thoroughly investigated for its anticancer activity. Evaluation of this molecule for its proapoptotic activity in acute lymphoblastic leukemia Molt4 (IC50: 5.00 μM) cells showed that OZ induced the externalization of hypodiploidia and phosphatidylserine which are considered two hallmarks of apoptosis. Furthermore, OZ treatment also increased the expression of PARP and caspase-3 in Molt4 cells [85].

The most commonly reported anticancer pathways of the *ent*-kaurane diterpenoids with their biological targets from different pathways have been summarized in Table S1 (Supplementary Materials) and Figure 3.

**Figure 3.** The common biological targets and mechanistic pathways of anticancer *ent*-kaurane diterpenoids.

#### **3. Conclusions**

Nature has blessed humanity with a wide variety of drugs for the treatment of cancer, and over the last few decades a large number of natural products have been widely investigated for their anticancer potential. In recent years, *ent*-kaurane diterpenoids have drawn great attention from the scientific community to evaluate their cancer inhibiting effects. Despite the identification of mechanistic pathways of a large number of compounds in both in vitro and in vivo models, there still remains much to understand about their mechanism of action, direct drug binding activity, and most importantly, to design and/or conduct preclinical and clinical research. We hope this article will provide a new platform for encouraging thorough investigation of *ent*-kaurane diterpenoids for the development of new anticancer drug candidates to treat the growing number of cancer patients. Although we have discussed the anticancer activity and mechanistic pathways of several *ent*-kaurane diterpenes, there remain many compounds with similar structures that we have not included that are certainly worthy of further exploration as anticancer agents. Additional work on the structure–activity relationship (SAR) of the *ent*-kaurane diterpenoids with their various molecular targets could also shed light as a way towards bringing some of these potent anticancer molecules into the clinic.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2218-273X/10/1/144/s1, Table S1: Plant-derived anticancer *ent*-kaurane diterpenoids with their biological targets.

**Author Contributions:** Conceptualization, M.S.S. and H.-J.Z.; Writing—original draft preparation, M.S.S.; Review and editing, S.W.T.; Revising, M.S.S., Y.-X.X., and Z.-M.L.; Supervision, H.-J.Z. All authors read and approved the final version of the manuscript.

**Funding:** This work is a part of PhD study supported by Hong Kong PhD Fellowship Scheme (HKPFS). This work was funded by the Research Grants Council of the Hong Kong Special Administrative Region, China (Project no. HKBU 12102219), and Hong Kong Baptist University, Research Committee, Initiation Grant—Faculty Niche Research Areas (RC-IG-FNRA/17-18/12).

**Acknowledgments:** The authors offer heartfelt thanks to Stephen Deyrup, Associate Professor, Department of Chemistry and Biochemistry, Siena College, USA, for his generous effort to edit the manuscript. The authors are also grateful to Yu Zhu, School of Chinese Medicine, Hong Kong Baptist University, for checking and revising the chemical structures of the compounds.

**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* **Neuroprotective E**ff**ects of Quercetin in Alzheimer's Disease**

**Haroon Khan 1,\* , Hammad Ullah 1 , Michael Aschner 2 , Wai San Cheang 3 and Esra Küpeli Akkol 4**

Received: 14 November 2019; Accepted: 22 December 2019; Published: 30 December 2019 -

**Abstract:** Quercetin is a flavonoid with notable pharmacological effects and promising therapeutic potential. It is widely distributed among plants and found commonly in daily diets predominantly in fruits and vegetables. Neuroprotection by quercetin has been reported in several in vitro studies. It has been shown to protect neurons from oxidative damage while reducing lipid peroxidation. In addition to its antioxidant properties, it inhibits the fibril formation of amyloid-β proteins, counteracting cell lyses and inflammatory cascade pathways. In this review, we provide a synopsis of the recent literature exploring the relationship between quercetin and cognitive performance in Alzheimer's disease and its potential as a lead compound in clinical applications.

**Keywords:** quercetin; polyphenols; Alzheimer's disease; mechanistic insights; clinical directions

#### **1. Introduction**

Alzheimer's disease (AD) contributes to 60–80% of total dementia cases, and it mostly affects elder people (65 years of age or older) [1]. The pathogenesis of AD is typically associated with the accumulation of amyloid-β (Aβ) aggregates and the hyperphosphorylation of tau proteins, leading to neurofibrillary tangles (NFTs) and synaptic dysfunction [2–4]. Around 35.6 million people worldwide are estimated to be affected with AD, with a prevalence rate of 4.6 million new cases each year. The prevalence rate of AD increases with age: the rate doubles every 5 years from 60 years of age [5,6].

Early studies led to the cholinergic deficit hypothesis of AD, which states that deficiency in acetylcholine is the main cause of the disease. In the pursuit of drugs that are able to restore acetylcholine levels, the first acetyl cholinesterase inhibitors were developed, including tacrine. Since then, other drugs in the same class have been pursued, namely donepezil, rivastigmine, and galantamine. Current AD therapy consists of cholinesterase inhibitors and *N*-methyl-d-aspartate (NMDA) antagonists, including memantine. Acetyl cholinesterase inhibitors prevent the hydrolysis of acetylcholine, and memantine modulates NMDA receptor activity, causing a reduction in excitatory glutamate signals. However, these drugs offer little palliative effects, and they also have numerous undesirable safety profiles with a number of adverse side effects [7,8]. Acetyl cholinesterase inhibitors are associated with gastrointestinal side effects such as nausea, diarrhea, and abdominal pain, as well as urinary incontinence, insomnia, and nightmares. The use of tacrine has been limited because of its poor bioavailability and reported hepatotoxicity. Memantine is clinically less effective compared

to acetyl cholinesterase inhibitors. Additionally, these drugs are not targeting the root cause of the disorder [9].

Phytochemicals are best known to reduce the risk of chronic diseases, such as cardiovascular diseases, hypertension, diabetes, and cancers [10–12]. Flavonoids are the most diverse group of phytochemicals and are widely distributed in higher plants with outstanding therapeutic potential [13–16]. Flavonoids are further divided into six classes on the basis of their chemical skeleton: flavanols, flavanones, flavones, flavonols, isoflavonoids, and anthocyanidins [17]. While targeting multiple targets, they have been proven beneficial in the prevention of neurodegenerative disorders and may delay the process of neurodegeneration [18]. Flavonoids are extensively studied for their antioxidant and anti-inflammatory activities, both of which are important in triggering the pathogenesis of AD [19]. Studies suggested that flavonoids are capable of crossing the blood–brain barrier (BBB), which makes them potential agents in preventing neurodegenerative disorders; however, different flavonoid subclasses differ in their ability to cross the BBB [20,21]. In the case of AD, their efficacy is attributed to the reduction of Aβ toxicity and decreasing oxidative stress [22,23]. Nevertheless, anti-AD effects of certain flavonoids, such as myricetin, rutin, fisetin, catechins, quercetin, kaempferol, and apigenin have been reported [24–27].

Quercetin is one of the most potent antioxidants of plant origin and is one of the predominant flavonoids found more commonly in edible plants [28]. It belongs to the flavonols class of flavonoids, representing a major class of polyphenols. The dietary intake of total flavonoids is estimated to be 200–350 mg/day, and the intake of quercetin is 10–16 mg/day. The recommended dosage of quercetin aglycone as a dietary supplement is 1 g/day [29–31]. It exhibits numerous beneficial effects on human health, acting as anti-carcinogenic, anti-inflammatory, anti-infective, and psychostimulant agent. It also inhibits lipid peroxidation and platelet aggregation, and it stimulates mitochondrial biogenesis [32]. Several studies have reported on the neuroprotective effects of quercetin, both in vitro and in vivo models of neurodegenerative disorders, such as cognitive impairment [33], ischemia, traumatic injury [34], Parkinson's disease (PD) [35], and Huntington's disease (HD) [36]. The aim of the present review is to provide a summary of the recent literature exploring the relationship between quercetin and cognitive performance. Our primary focus is on the chemical basis and pharmacology of quercetin and its anti-AD mechanisms.

#### **2. Chemistry of Quercetin**

A flavonoid is structurally diphenyl propane containing 15 carbon atoms in its structure (Figure 1A). It contains a close heterocyclic pyran ring in addition to two benzene rings. The term 4-oxo-flavonoid (Figure 1B) is often used to describe flavonoid containing a carbonyl group on position C-4 of ring C. Substitution and oxidation in the heterocyclic pyran ring classifies flavonoids into subclasses, namely flavones, flavonols, flavanones, flavononol, isoflavones, and flavan-3-ols. Substitution in the benzene rings of flavonoid structures leads to differences in individual compounds within specific classes [37,38]. Quercetin belongs to the flavone class of flavonoids having a chemical formula of C15H10O7. Its IUPAC name is 3,3 ,4 ,5,7-pentahydroxyflavanone or 3,3 ,4 ,5,7-pentahydroxy-2-phenylchromen-4-one (Figure 2). Quercetin contains an OH group at positions 3, 5, 7, 3 , and 4 . Quercetin is an aglycone, (lacking an attached sugar molecule). Attaching a glycosyl group (glucose, rhamnose, or rutinose) most commonly at position 3 replacing the OH group leads to the formation of quercetin glycoside. Quercetin is insoluble or sparingly soluble in water, while it is quite soluble in alcohol and lipids. A glycosyl group increases its water solubility, and thus the quercetin glycoside is soluble in water [32,39]. In addition to antioxidant activities, multiple OH groups in the structure of quercetin may also lead to its photodegradation. Dall'Acqua et al. (2012) reported that OH groups at positions 3, 3 , and 4 are mainly involve in photolability, while OH groups at positions 5 and 7 do not play a crucial role in the photo-oxidative mechanism [40].

**Figure 1.** (**A**) Flavan nucleus, (**B**) 4-oxo-flavonoid nucleus.

**Figure 2.** Chemical skeleton of quercetin and derivatives. (**A**) Quercetin glucoside, (**B**) quercetin-3-*O*-sulfate, (**C**) quercetin-3-*O*-glucuronide, and (**D**) 3-*O*-methyl quercetin.

Several flavonoids have anti-inflammatory activity, among which flavanones such as naringenin have weak activity, while flavonols including quercetin, kaempferol, and myricetin have strong anti-inflammatory activity. Flavonols are more active against phospholipase A-2, given the presence of the C-ring-2,3 double bond. Furthermore, the glycosylation of quercetin, such as rutin, reduces the anti-inflammatory activity of quercetin [41]. Glycosylation at position 3 of quercetin leads to a decrease in the free radical neutralizing activity. Glycosylation also decreases the acetyl cholinesterase inhibition activity of quercetin [42]. The methylation of quercetin at positions 4 and 7 results in improved anticancer activities. The replacement of OH groups with O-methylated moieties also increases the metabolic stability of a compound [43]. The presence of a double bond between C2 and C3 as well as the OH group at ring B is essential for the thrombin inhibition activity of quercetin. Substituting the OH group with an OCH3 group in ring B and ring C reduces the thrombin inhibition activity, whereas replacement of the OH groups in ring A with an OCH3 group improves the thrombin inhibition activity of quercetin [44].

Quercetin may act as a pro-oxidant phytochemical by generating reactive oxygen species (ROS) and reactive electrophilic quinine type metabolites because of the presence of catechol moiety in the B ring, C-ring-2, 3 double bond, and free OH group at the C-3 position [45]. Furthermore, in vitro studies have confirmed that the esters-based precursors of quercetin increase the bioavailability of quercetin [46]. In considering the general structure of flavonoids, OH group substitution at positions C5, C7, and C3, the substitution of OCH3 or H at C3 , and OH or OCH3 substitution at position C4 improve their neuroprotective activities as confirmed by in vitro studies, utilizing neuronal cell cultures [47].

#### **3. Sources**

The name quercetin has been used since 1857. It is derived from Latin *Quercetum,* after *Quercus* (oak) [48]. It is a flavonoid that is most commonly occurring in higher plants and in the glycosidal form, such as rutin (quercetin-3-*O*-rutinoside), isoquercetin (quercetin-3-*O*-glucoside), and hyperin (quercetin-3-*O*-galactoside). It can also be isolated in free-form from leaf surfaces, fruits, or bud extracts. Plant families rich in quercetin are Compositae, Passiflorae, Rhamnaceae, and Solanaceae [49]. Onions, asparagus, red leaf lettuce, apples, capers, and berries contain relatively high concentrations of quercetin [29,50].The botanical sources of quercetin have been summarized in Table 1.

**Table 1.** Botanical sources of quercetin.

#### **4. Pharmacokinetic Parameters of Quercetin**

Studies have revealed that the low bioavailability of quercetin limits its use for therapeutic purposes, although it has a wide range of pharmacological properties. Low solubility, poor absorption, and rapid metabolism are major aspects of the low bioavailability of quercetin [80]. Ader et al. (2000) have explored the bioavailability and metabolism of quercetin in pigs [81]. Quercetin was administered to each animal in a single intravenous dose of 0.4 mg/kg body weight and a single oral dose of 50 mg/kg body weight one week later. Blood samples were collected to analyze the pharmacokinetic parameters of quercetin. The apparent bioavailability was 0.54 ± 0.19% when considering free quercetin alone, 8.6 ± 3.8% including conjugated quercetin, and 17.0 ± 7.1% including quercetin metabolites (kaempferol, tamarixetin, and isorhamnetin). The authors also reported that the conjugation of quercetin to glucuronic acid and sulfuric acid preferentially occurs in the intestinal wall.

Moon et al. (2008) have studied the pharmacokinetics of quercetin in human beings [82]. Quercetin was administered to subjects in doses of 500 mg three times daily, and the plasma and urine samples were collected from subjects to analyze the concentration of quercetin aglycone and metabolites. The average peak plasma concentration reported after the administration of quercetin at 500 mg three times daily was 463 ng/mL at 3.5 h. Re-entry peaks on plasma concentration versus time curves showed enterohepatic recirculation. The oral clearance of quercetin was reported to be high (3.5 × 104 L/h) with an average half life of 3.5 h. The urinary recovery of quercetin aglycone and conjugated metabolites were 0.05% to 3.6% and 0.08% to 2.6%, respectively. Previously, Day et al. (2001) while studying the identification of quercetin metabolites in plasma have reported that quercetin is found exclusively as a glucuronated or sulfate conjugate in plasma after oral administration [83].

Moreover, quercetin aglycone is less effective to cross the BBB, as it is a substrate for efflux transporter p-glycoprotein. On the other hand, quercetin conjugates are also predicted to less effectively cross the BBB because of high polarity [84,85]. However, the literature supported that some glucuronides are effectively crossing the BBB because of aided active transport [86]. Kawei et al. (2008) revealed that at sites of inflammation, the intracellular deconjugation of quercetin 3-*O*-glucuronide by β-glucuronidase increases, and this phenomena is more relevant inside the central nervous system (CNS) in the case of neurodegenerative disorders, rather than being of relevance in the penetration of the BBB [87].

#### **5. Pathophysiological Mechanisms of Alzheimer's Disease**

AD is mainly characterized by the accumulation of extracellular amyloid plaques, which are known as senile plaques. The amyloid plaques are associated with neuroinflammatory changes as well as neurofibrillary tangles, affecting processes that are essential in maintaining neuronal health: communication, metabolism, and repair. Aβ is produced by the sequential cleavage of amyloid β precursor protein by β and γ secretases [88,89]. The amyloid precursor protein (APP) exists in three different forms: the shortest isoform, APP695 mostly expressed in neurons, whereas two other isoforms APP751 and APP770 are expressed in glial cells, such as astrocytes. The physiological role of APP is to stimulate cell growth and proliferation. Aβ plaques lead to neuronal loss in several brain regions, including the entorhinal cortex, hippocampus, neocortex, amygdale, and subcortical areas [90,91]. Two different enzymatic processes of APP occur in the cells: the non-amyloidogenic and the amyloidogenic pathways. In the non-amyloidogenic processing pathway, the APP is cleaved by α and γ-secretases, which yields long secreted forms of APP (sAPPα) and C-terminal fragments. In the amyloidogenic processing pathway, APP is cleaved by β and γ-secretases, which yields long secreted forms of APP (sAPPβ), C-terminal fragments, and Aβs. The Aβ fragment is chemically sticky-forming plaques, and blocks cell-to-cell signaling via synapses, in turn leading to neuronal cell death [92,93].

Neurofibrillary tangles (NFTs) are produced by the hyperphosphorylation of microtubule-associated proteins, which are known as tau [94], representing another key feature of AD. The excessive phosphorylation of tau proteins decreases their binding ability to microtubules, resulting in the formation of NFTs [95]. Both the density and neuroanatomic localization of NFTs are important determinants in the pathogenesis of AD. NFTs in neocortical regions are commonly associated with severe cognition impairment [96]. Aβ induces the phosphorylation of tau proteins, thus triggering the formation of NFTs and causing neuronal cell death by increasing oxidative stress, altering calcium homeostasis and excitotoxicity [90]. Imbalance between Aβ production and clearance leads to the formation of Aβ aggregates, especially in late onset AD. The impairment of Aβ clearance most commonly occurs in late onset AD. Heparan sulfate proteoglycans (HSPGs) are most critical in the pathogenesis of AD by affecting Aβ metabolism and decreasing Aβ clearance. HSPGs bind to Aβ and accelerate its aggregation, mediating the neurotoxicity and neuroinflammation induced by Aβ. Under normal conditions, HSPGs perform various important physiological functions in development, growth factor signaling, cell proliferation, adhesion, migration, and homeostasis [97].

Oxidative stress commonly occurs in AD, contributing to neurodegeneration. ROS generation triggers Aβ aggregation. Oxidative stress is inherent to mild cognitive impairment (MCI) in the progression of AD. Patients with MCI usually develop cognitive impairment with a minimal impairment of instrumental activities of daily life, and it can be the first cognitive expression of AD. Alteration in the phosphorylation of proteins, such as heme oxygenase-1 and biliverdin reductase A, has been noted to occur, affecting the signaling of the most critical antioxidant pathways [98–100]. This leads to mitochondrial damage, which is concomitant with the decreased activity of mitochondrial energy-related proteins, including pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase. Defective mitochondria, in turn, trigger the generation of high levels of reactive oxygen species (ROS), for which antioxidant defenses may be deficient [101–103]. As such, the process proceeds unabated, as in a brush fire.

Neuroinflammation is a hallmark in the pathogenesis of AD. The innate immune cells of the brain are fast to respond to systemic events, mostly in aged and diseased brains. Misfolded and aggregated proteins binding to pattern recognition receptors on microglia and astroglia trigger an immune response, releasing a number of inflammatory cytokines and chemokines [104,105]. In contrast to oxidative stress, which is short lived, chronic inflammation is long lasting, resulting in the sustained release of inflammatory mediators.

Microglia surrounding senile plaques is commonly activated in AD, resulting in the upregulation of human leukocyte antigen-DR (HLA-DR). In addition, the generation of inflammatory mediators may lead to microglial activation and neurotoxicity secondary to the CD14-dependent process. Despite having phagocytotic activity, microglia fail to phagocytose Aβ due to the presence of inflammatory cytokines and various extracellular matrix proteins [106]. Receptor of advanced glycation end products (RAGE) activation leads to neurodegeneration as it triggers an increase in inflammatory mediators and oxidative stress. RAGE activation also leads to activation of downstream regulatory pathways such as the NF-kB, STAT, and JNK pathways [107].

Acetylcholinergic, glutamatergic, and serotonergic neurons are mostly affected in AD. Early histological changes involve the loss of cholinergic neurotransmission in the cerebral cortex. The latter, along with the hippocampus, receive the highest cholinergic input, and the loss of cholinergic neurons in these regions results in cognitive deficits and memory impairment. Choline acetyltransferase activity is greatly reduced in individuals with AD. It has also been shown that acetyl cholinesterase (AChE) accelerates the aggregation of Aβ [108]. The degeneration of serotonergic neurons in the raphe nucleus and noradrenergic neurons in the locus coeruleus mediates on cognitive symptoms associated with AD [109]. Figure 3 illustrates the pathogenesis of Alzheimer's disease.

**Figure 3.** Schematic presentation of the pathogenesis of Alzheimer's disease. [1] Amyloid precursor protein (APP) is hydrolyzed by β and γ secretases to form β-amyloid (Aβ), which aggregates to form fibril Aβs. Fibril Aβs upregulate oxidative stress, the inflammatory cascade, and caspase activation, which results into the hyperphosphorylation of the Tau protein to form neurofibrillary tangles(NFTs), and the ultimate result is neuronal cells loss. Extensive fibrils along with activated microglia accumulated to form senile plaques, which lead to neuronal and synaptic loss. [2] Upstream regulating acetyl cholinesterase (AChE) enzyme promotes acetylcholine (Ach) degradation, resulting in neurotransmitter deficit, which leads to cognitive impairment. Amyloid precursor protein (APP), amyloid beta proteins (Aβ), neurofibrillary tangles (NFTs), acetylcholine (Ach), acetyl cholinesterase (AChE).

#### **6. Neuroprotective E**ffi**cacy of Quercetin**

The neuroprotective effects of quercetin have been extensively studied. At low micromolar concentrations, it antagonizes cell toxicity by oxidative stress in neurons. It suppresses neuroinflammatory processes by downregulating pro-inflammatory cytokines, such as NF-kB and iNOS, while stimulating neuronal regeneration. After absorption, quercetin metabolites are glucuronated, methylated, or sulfated, and all have been shown to afford neuroprotection. The neuroprotective efficacy of quercetin has been studied in both in vitro and in vivo models [50,110,111]. A study using drug screening in *Caenorhabditis elegans* nematodes with neuronal expression of human exon-1 huntingtin (128Q) and mutant Htt striatal cells derived from knock-in HD mice, concluded that isoquercetin improved motor functions in acute spinal cord injury, reduced α-synuclein fibrillization, reduced hippocampal neuronal cell death, improved synaptic plasticity, and reversed histopathological hallmarks of AD [112]. Quercetin also protects against mitochondrial dysfunction and progressive dopaminergic neurodegeneration by activating PKD1-Akt cell survival signaling axis Cell Culture and MitoPark transgenic mouse models of Parkinson's disease [113].

Quercetin has shown therapeutic efficacy, improving learning, memory, and cognitive functions in AD [114]. Khan et al. (2009) and Shimmyo et al. (2008) concluded that quercetin administration resulted in the inhibition of AChE and secretase enzymes using in vitro models, thus preventing the degradation of acetylcholine, and decreasing Aβ production, respectively [115,116]. Sabogal-Guáqueta et al. (2015) have been reported that quercetin administration reverses extracellular β-amyloidosis and decreases tauopathies, astrogliosis, and microgliosis in the hippocampus and amygdale, thus protecting cognitive and emotional function in age triple transgenic Alzheimer's disease model mice [117]. Wand et al. (2014) studied the effects of the long-term administration of quercetin on cognition and mitochondrial dysfunction in a mouse model of Alzheimer's disease. They noted that quercetin ameliorates mitochondrial dysfunction by restoring mitochondrial membrane potential, decreases ROS production, and restores ATP synthesis. It also increased the expression of AMP-activated protein kinase (AMPK), which is a key cell regulator of energy metabolism. Activated AMPK can decrease ROS generation by inhibiting NADPH oxidase activity or by increasing the antioxidant activity of enzymes such as superoxide dismutase-2 and uncoupling protein-2. The activation of AMPK also decreased Aβ deposition, regulating APP processing and promoting Aβ clearance. These mechanisms likely account for some of the therapeutic efficacy of quercetin on cognition and the attenuation of Aβ-induced neurotoxicity [118]. Quercetin and rutin have also been reported to function as memory enhancers in scopolamine-induced memory impairment in zebrafish, thus possibly enhancing cholinergic neurotransmission [119].

#### **7. Anti-Alzheimer's Disease Mechanisms of Quercetin**

#### *7.1. Inhibition of A*β*Aggregation and Tau Phosphorylation*

The aggregation of Aβ is a key hallmark of AD [120]. Quercetin interferes with the formation of neurotoxic oligomeric Aβ species and displays fibril destabilizing effects on preformed fibrillar Aβ, reversing Aβ-induced neurotoxicity [110]. The structure of efficient polyphenolic inhibitors of Aβ contains two aromatic rings with two to six atom linkers. The aromatic rings contain a minimum number of three hydroxyl groups, which play an important role in fibril inhibition through hydrophobic interaction between the aromatic rings with β-sheet structures, forming hydrogen bonds. The phenolic hydroxyls increase the electron density in the aromatic rings, which may increase the binding of quercetin with the aromatic amino acids of the peptide beta-sheet structures. Quercetin possesses these structural requirements containing hydrophobic moieties and thus arrests fibril formation. The more hydroxyl groups present in the structure of the molecule, the higher its anti-amyloidogenic activity [121,122]. It is also suggested that the catechol structure may be auto-oxidized to form o-quinone on ring B, which then forms an O-quinone-Aβ42 adduct by targeting Lys residues at positions 16 and 28 of Aβ42. This phenomenon explains why quercetin has higher Aβ aggregation inhibitory activities compared to kaempferol, morin, and datiscetin [123].

Quercetin is also reported from in vitro and in silico studies to inhibit beta-secretase-1 (BACE-1) enzyme activity through the formation of hydrogen bonds. The OH group at position C-3 has a significant role in BACE-1 inhibition [116]. It has been documented from in vitro and molecular docking studies performed by Paris et al. (2011) that NF-kB regulates the production of Aβ by regulation of the β cleavage of APP, and that the quercetin-induced inhibition of NF-kB affects the regulation of BACE-1 expression [124]. Tauopathy commonly begins in the hippocampus, affecting hippocampal-dependent cognitive tasks followed by progression to other brain areas. Quercetin has been shown to decrease the phosphorylation of tau proteins and to inhibit the formation of NFTs in age triple transgenic Alzheimer's disease model mice [117]. Kinases and protein phosphatases such as protein phosphatase 2A (PP2A) play a role in regulating the hyperphosphorylation of tau proteins. The hyperphosphorylation of tau proteins is mostly due to the imbalance between phosphorylation and dephosphorylation mechanisms. Thus, the inhibition of PP2A may lead to the hyperphosphorylation of tau proteins. Quercetin reverses

the hyperphosphorylation of tau proteins via MAPKs and PI3K/Akt/GSK3β signaling pathways in HT22 cells (a cell line from mouse hippocampal neurons) [125].

#### *7.2. Acetylcholinesterase Inhibition*

The inhibition of AChE is one of the therapies most commonly pursued in the treatment of mild to moderate AD. AChE is an enzyme that is responsible for the degradation of acetylcholine, and its inhibition results in increased acetylcholine levels, thus improving the cognitive symptoms of AD. In vitro studies have shown that quercetin is a competitive inhibitor of AChE and butyrylcholinesterase (BChE). It inhibits both enzymes in a concentration-dependent manner [126]. Quercetin inhibits AChE secondary to hydrophobic interactions and strong hydrogen bonding with the enzyme, reducing the hydrolysis of ACh, thus increasing ACh levels in the synaptic cleft, as reported by Abdalla et al. (2013) using cadmium-exposed rats as a model of study [127]. Studies have linked the presence/absence of OH groups on the phenyl rings of the test compound to the inhibition of AChE and BChE, given that the OH groups form hydrogen bonds with amino acid residues at the active site of the enzyme. This phenomenon may explain the inhibitory efficacy of quercetin for both of these enzymes. Moreover, quercetin exhibits greater potency for the inhibition of AChE and BChE than its glycosidal form, rutin. Previous studies have posited the presence of a sugar moiety in the molecule is essential for its enzymatic inhibition [115,128,129]. Jung et al. (2007) studied the acetylcholinesterase inhibiting the potential of flavonoids, including quercetin isolated from *Agrimonia pilosa* and they reported the IC50 value of quercetin to be 19.8 [130].

#### *7.3. Attenuation of Oxidative Stress*

Oxidative stress is caused by the accumulation of ROS in cells, and is an important factor in various neurodegenerative disorders. It is the most common mechanism of age-related degenerative processes. Mitochondria are the primary site for ROS production, and mitochondrial dysfunction leads to the overproduction of ROS followed by ATP depletion and ultimately cell death [50,131]. The major source of ROS is the superoxide anion radicals generated by the electron transport chain during oxidative phosphorylation. Superoxide dismutase (SOD) converts superoxide ions to hydrogen peroxide, which can be detoxified by catalase and glutathione peroxidases [132]. It has been reported that Aβ triggers oxidative stress, leading to lipid peroxidation and protein oxidation, which results in damaged mitochondria and the dysfunction of key enzymes associated with various pathways, including glucose metabolism [133].

Among the phytochemicals, quercetin is a potent antioxidant. It has been shown to effectively reduce the concentrations of superoxide anion free radicals, and its antioxidant potential makes it a versatile choice in the management of various disorders, including AD [49]. Previous studies have shown that quercetin has direct radical scavenging action. The presence of two pharmacophores in its structure is responsible for its antioxidant activities: one is a catechol group in the B ring, and the other is the OH group at position C-3. Quercetin also modulates the cell's own antioxidant pathways, by inducing Nrf-2-ARE and paraoxonase 2 (PON2), which is an antioxidant enzyme. Nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) is an important regulator of cellular defenses against oxidative stress. Heme oxygenase-1, glutamate cysteine ligase, glutathione S-transferase, glutathione peroxidase, SOD, catalase, sulfiredoxin, and thioredoxin are enzymes downstream of Nrf-2-ARE. Thus, the activation of the Nrf-2-ARE pathway likely modulates the formation and degradation of misfolded protein aggregates in AD [50,64,134].

#### *7.4. Attenuation of Neuroinflammation by Quercetin*

Reducing the neuroinflammatory events in microglia might afford a beneficial strategy for the prevention of the progression of inflammatory-mediated neurodegenerative disorders. Quercetin has been reported to have anti-inflammatory actions, and is a suitable candidate among phytochemicals for future studies on its efficacy to reverse neuroinflammation [135,136]. Quercetin has already been shown to inhibit neuroinflammation by reducing nitric oxide production, iNOS gene expression in microglia, the production of inflammatory cytokines such as tumor necrosis factor-α (TNF-α), IFN-γ, interleukin-1β (IL-1β), IL-6, IL-12, and COX-2 in activated macrophages, as well as a reduction in cytokine expression as reported from in vivo studies. Quercetin also downregulates JNK/Jun phosphorylation and inhibits TNF-α production in mice, thus protecting neurons against LPS-induced inflammation [137]. Figures 4 and 5 have been summarizing anti-Alzheimer's targets and mechanistic insights of quercetin, respectively.

**Figure 4.** Anti-Alzheimer's disease targets of quercetin. Quercetin may utilize several mechanistic targets for neuroprotection in Alzheimer's disease such as the downstream regulation of oxidative stress and neuroinflammation, which leads to the direct protection of neurons, inhibiting AChE enzymes and resulting in increasing acetylcholine levels and reducing Tau phosphorylation and Aβ aggregation. Tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), interleukin-6 (IL-6), reactive oxygen species (ROS), nitric oxide (NO), acetyl cholinesterase (AChE), and amyloid beta protein (Aβ).

**Figure 5.** Mechanistic insights of quercetin in Alzheimer's disease. Quercetin has inhibitory effects on JNK, PI3K/Akt pathways, acetyl cholinesterase (AChE), nuclear factor (erythroid-derived 2)-like 2 (Nrf-2), beta-secretase-1 (BACE-1) enzyme activity, and the hyperphosphorylation of tau proteins. On the other hand, it stimulates the expression of AMP-activated protein kinase (AMPK), which thereby decreases reactive oxygen species (ROS) generation by inhibiting NADPH oxidase activity or by increasing the antioxidant activity of enzymes such as superoxide dismutase-2 (SOD2).

#### **8. Anti-Alzheimer's Disease Potential: In Vitro Studies on the E**ffi**cacy of Quercetin**

Quercetin protects neurons from oxidative damage, while reducing lipid peroxidation. Furthermore, given its antioxidant properties, quercetin inhibits the fibril formation of Aβ proteins, counteracting cell lyses and inflammatory cascade pathways [7,138,139]. In vitro studies on quercetin have shown that its antioxidant activities are concentration-dependent. It acts as an antioxidant at lower concentrations (neuronal cells treated with 5 μM and 10 μM), but it possesses toxic effects at higher doses (neuronal cells treated with 20 μM and 40 μM) [140]. It also inhibits iNOS and regulates the expression of COX-2 in various models, reflecting upon its anti-inflammatory activities. Most of the absorbed quercetin is present as metabolites, including glucuronidated, methylated, and sulfated metabolites. All exert neuroprotective effects, but limited testing of these metabolites has been carried out to date [117]. It has also been reported that quercetin decreases APP maturation, thus altering Aβ synthesis and aggregation [141]. The neuroprotective effects of quercetin glycosides have also been reported, which are characterized by antagonizing changes in gene expression, such as Park2, Park5, Park7, Casp3, and Casp7 genes [142].

#### **9. Anti-Alzheimer's Disease Potential: In Vivo Studies on the E**ffi**cacy of Quercetin**

The protective effects of a diet rich in polyphenols have been reported in several pathologic conditions, such as cardiovascular diseases, metabolic disorders, infections, cancers, and neurodegenerative disorders. It has been reported in several studies that quercetin exerts

neuroprotective effects when administered in vivo. It protects neuronal cells from oxidative stress induced by various chemicals and prevents hippocampal apoptosis [50,143]. Quercetin improves memory, learning, and cognitive functions, and all these effects have been shown to be associated with its antioxidant properties [144]. In vivo studies using mice as an animal model have supported that quercetin increases spatial memory tasks, and decreases β-amyloidosis, tauopathies, astrogliosis, and microgliosis by increasing AMPK activity and decreasing mitochondrial dysfunction [117,118].

Keddy et al. (2012) investigated the neuroprotective and anti-inflammatory effects of the flavonoids-enriched fraction containing quercetin and its glucosides in a mouse model of hypoxic-ischemic brain injury. The study had concluded that the repeated administration of the flavonoid-enriched fraction prior to an experimental stroke produced by hypoxic-ischemia prevents the neuronal loss in the striatum and dorsal hippocampus. Due to the low bioavailability of quercetin and its glucosides, it required injection through the intraperitonial or intravenous route to achieve neuroprotective effects [145]. Tota (2010) et al. studied the effects of quercetin on cerebral blood flow and memory impairment in mice and linked the ability of quercetin to increase cerebral blood flow and energy metabolism to its memory-enhancing effects [146]. After oral administration in humans, quercetin is extensively metabolized during its absorption from the gut, affecting its bioavailability. Clinical efficacy trials/studies of quercetin have yet to be carried out, which is likely given its low BBB penetrability [147]. Furthermore, the metabolites of quercetin have long half-lives in vivo, and repeated dosing may lead to plasma accumulation [145]. These are important factors, which will need to be considered in the design of quercetin analogs for clinical studies.

#### **10. Conclusions**

Quercetin is a flavonoid with notable pharmacological effects and promising therapeutic potential. It is widely distributed among plants and found commonly in our daily diet, such as in fruits and vegetables. It has beneficial properties against general mechanisms of AD etiology in a variety of in vitro and in vivo models. It protects neuronal cells by attenuating oxidative stress and neuroinflammation. The anti-Alzheimer's disease properties of quercetin include the inhibition of Aβ aggregation and tau phosphorylation. It restores acetylcholine levels through the inhibition of hydrolysis of acetylcholine by AChE enzyme. Although showing neuroprotective efficacy in several in vitro and animal models, in vivo studies have reported that it is extensively metabolized upon absorption from the gut, affecting its bioavailability. It also has low BBB penetrability, thus limiting its efficacy in combating neurodegenerative disorders. Therefore, future clinical trials of quercetin and its analogs as neuroprotective agents must improve its bioavailability, developing related molecules with greater gut and brain penetrability, which will likely improve clinical efficacy.

**Funding:** MA was supported in part by grants from the National Institutes of Environmental Health Sciences R01 ES10563, R01 ES07331 and R01 ES020852 (MA).

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

#### **References**

© 2019 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*

### **Anticancer Plants: A Review of the Active Phytochemicals, Applications in Animal Models, and Regulatory Aspects**

**Tariq Khan 1,\* , Muhammad Ali 2,\* , Ajmal Khan 3, Parveen Nisar 2, Sohail Ahmad Jan 4 , Shakeeb Afridi 2 and Zabta Khan Shinwari 2,5**

Received: 15 November 2019; Accepted: 25 December 2019; Published: 27 December 2019 -

**Abstract:** The rising burden of cancer worldwide calls for an alternative treatment solution. Herbal medicine provides a very feasible alternative to western medicine against cancer. This article reviews the selected plant species with active phytochemicals, the animal models used for these studies, and their regulatory aspects. This study is based on a meticulous literature review conducted through the search of relevant keywords in databases, Web of Science, Scopus, PubMed, and Google Scholar. Twenty plants were selected based on defined selection criteria for their potent anticancer compounds. The detailed analysis of the research studies revealed that plants play an indispensable role in fighting different cancers such as breast, stomach, oral, colon, lung, hepatic, cervical, and blood cancer cell lines. The in vitro studies showed cancer cell inhibition through DNA damage and activation of apoptosis-inducing enzymes by the secondary metabolites in the plant extracts. Studies that reported in vivo activities of these plants showed remarkable results in the inhibition of cancer in animal models. Further studies should be performed on exploring more plants, their active compounds, and the mechanism of anticancer actions for use as standard herbal medicine.

**Keywords:** cancer; apoptosis; herbs; cell lines; in vivo

#### **1. Introduction**

The burden of cancer rose to 18.1 million new cases and 9.6 million deaths in 2018. With 36 different types, cancer mainly affects men in the form of colorectal, liver, lung, prostate, and stomach cancer and women in the form of breast, cervix, colorectal, lung, and thyroid cancer [1]. Treating cancer has become a whole new area of research. There are conventional as well as very modern techniques applied against cancers. A variety of techniques i.e., chemotherapy, radiation therapy, or surgery are used for treating cancer. However, all of them have some disadvantages [2]. The use of conventional chemicals bears side effects and toxicities [3]. But as the problem persists, new approaches are needed for the control of diseases, especially, because of the failure of conventional chemotherapeutic approaches. Therefore, there is a need for new strategies for the prevention and cure of cancer to control the death rate because of this disease.

Herbal medicine has become a very safe, non-toxic, and easily available source of cancer-treating compounds. Herbs are believed to neutralize the effects of diseases in a body because of various characteristics they possess [4]. For instance, among the many anticancer medicinal plants, *Phaleria macrocarpa* (local name: *Mahkota dewa*) and *Fagonia indica* (local name: *Dhamasa*) have been used traditionally for the anticancer properties of their active ingredients [5,6]. Metabolites extracted from the plant material are used to induce apoptosis in cancer cells. Gallic acid as the active component was purified from the fruit extract of *P. macrocarpa* and has demonstrated a role in the induction of apoptosis in lung cancer, leukemia, and colon adenocarcinoma cell lines [7,8]. It is a polyhydroxy phenolic compound and a natural antioxidant that can be obtained from a variety of natural products i.e., grapes, strawberries, bananas, green tea, and vegetables [9]. It also plays a critical role in preventing malignancy transformation and the development of cancer [10]. Similarly, other compounds such as vinca alkaloids, podophyllotoxin, and camptothecin obtained from various plants are used for the treatment of cancer.

With the advancement in the industrial sector and industrial medicine, the use of herbs was forgotten for a long period of time [11]. Hurdles regarding natural compounds are reduced because of the advent of new techniques and interest has been developed in the use of such natural ingredients in the pharmaceutical industry [12,13]. It has been estimated by the world health organization that 80% of the world is using traditional treatment methods [14]. Understanding of the effects or actions of herbs on various targets comes with the help of modern biomolecular science which recognizes some important properties i.e., anticancer, anti-inflammatory, and anti-virus. With the increasing understanding of the effects of such herbal medicine, their effects against different types of cancers have also been identified. For instance, hepatocellular carcinomas (HCC) are considered as the fifth most common malignancy in the world with increasing incidence [15,16]. Many studies have been performed on the treatment and prevention of using herbal medicine against HCC in which it is shown that all phases of HCC such as initiation, promotion, and progression could be affected by components of herbs [17,18].

However, as far as herbal compounds are considered as drugs, it is erroneously believed that they have no issues in terms of safety and side effects. There are hundreds of species of plants that are toxic to health. In the same way, there are many compounds in otherwise friendly plants that cause cytotoxicity. Based upon testing it has been proved that even anticancer plants result in cytotoxic effects [19].

Herbs are regulated under the "dietary supplement health and education act" as a dietary supplement in the United States of America. This review highlights the mechanism of some very important anticancer plants, the research related to their mechanism of action, their active ingredients, and the guidelines in place for their regulations.

#### **2. Sources and Methodology**

The most relevant literature was retrieved through a meticulous search on the electronic databases, Web of Science, Scopus, PubMed, and Google Scholar. The keywords and phrases used during the search were "Medicinal plants," "Anticancer activity," "Anticancer herbs," "Anticancer plants," "Mechanism of action," "Animal models," "in vitro activity," and "in vivo activity." The number of relevant articles finalized after extraction and analysis through the combination of the above keywords/phrases and the inclusion criteria was 200. The inclusion was based on two sets of criteria. According to the first set, i.e., "general criteria," articles selected for this manuscript had (i) reported the traditional anticancer activity of plants and their parts, (ii) reported the anticancer role of extract or pure compounds from plants.

The second set of criteria was used for selecting specific anticancer plants whose phytochemicals are discussed in detail. For this purpose, twenty plants were selected for which recent articles were available that (a) studied in vitro and in vivo anticancer activities of herbal products, (b) reported the anticancer/antitumor activity of active compounds from the plants, and (c) assessed the in vivo anticancer activity of the herbal anticancer products.

All the data were extracted in a table and the mechanisms of action were explained in respective subheadings and demonstrated through different figures.

#### **3. Selected Plants and Their Anticancer Activity**

Research so far has tested the anticancer activity of a plethora of plants and plant-based compounds. Some of these plants and their compounds prove to be very effective against one or more types of cancers. Based on their activities, the following plants are selected for the in vitro and in vivo anticancer activities of their compounds. The rest of the important plants shortlisted for their activities are presented in Table 1 along with their activities.

#### *3.1. Artemisia annua*

The genus Artemisia, widespread in Europe, Asia, North America, and South Africa has approximately 400 species worldwide [20]. Plants of the genus were used for centuries in classical medicine [21]. *Artemisia annua* is an annual short-day plant that belongs to family Asteraceae, having a brownish rigid stem. *A. annua* is known as sweet wormwood (Chinese: q¯ınghao) and "dona" in ¯ the Urdu language in India and Pakistan [8]. *A. annua* was used by old Chinese for the preparation of anti-malarial drugs known as artemisinin (Figure 1). Having a unique ability of environmental adaptation it consistently resists insects and pathogens [22].

**Figure 1.** Structural representation of important anticancer secondary metabolites from plants. The structures are adapted from NCBI cited as National Center for Biotechnology Information. PubChem Database. (**a**) 2-Methylanthraquinone, Compound identification number (CID) = 6773; (**b**) albanol A, CID = 44567218; (**c**) artemisinin, CID = 68827; (**d**) baicalein, CID = 5281605; (**e**) berberine, CID = 2353; (**f**) curcumin, CID = 969516; (**g**) D-amygdalin, CID = 656516; (**h**) garcinol, CID = 5281560; (**i**) oblongifolin A CID = 53364454; (**j**) oridonin, CID = 5321010; (**k**) platycodin D, CID = 162859; (**l**) polyphyllin C, CID = 44429637; (**m**) scutellarein, CID = 5281697, and (**n**) triptolide, CID = 107985. (**o**) isoegomaketone, CID = 5318556; https://pubchem.ncbi.nlm.nih.gov/compound/ (accessed on 18 July 2019).

*A. annua* also synthesize scopoletin and 1,8-cineole compounds. Similarly, semi-synthetic derivatives of artemisinin are also generated such as arteether, artemether, and artesunate. Artesunate has been studied to be a very effective anticancer compound.  They suggested that artesunate was most effective against leukemia and colon cancers. Furthermore, it was observed through these studies that the artesunate was more active than the drugs used for such cancers.

The stem and leaves *A. annua* were subject to extraction with the help of 80% ethanol and water. Several quantitative phenolic compounds from *A. annua* were identified using high-performance liquid chromatography (HPLC). The extracts were tested against HeLa and AGS cell lines. The cell growth inhibition activity of stem extracts was lower compared to leaf extracts. The ethanolic extracts of leaves lead to growth inhibitions (57.24% and 67.07%) in HeLa and AGS cells, respectively at a concentration of 500 mg/mL. HPLC analysis showed that the amount of phenolic acids was lower in stem extract than in leaves extract of *A. annua*. It was concluded from the data that the antioxidant and anticancer capacity was the result of phenolic compounds as well as unidentified compounds within *A. annua* [24].

#### *3.2. Coptis chinensis*

*Coptis chinensis*, the Chinese goldthread, is a herb used as a traditional medicine in China thus officially enlisted in the Chinese pharmacopeia [25]. It is widely known for its traditional use against various diseases like diarrhea, dysentery, acute febrile, and supportive infections. The organic extract of *C. chinensis* possesses anti-inflammatory and anti-oxidant properties [26,27]. *C. chinensis* extract has wide use in the treatment of cholera, dysentery, diabetes, blood and lung cancer because of its strong antibacterial activity [28]. *Coptis* genus contains the most important and active components, such as an alkaloid i.e., berberine (Figure 1). Berberines alkaloids are used frequently as criteria in the quality control of *Rhizoma coptidis* (Huang Lian) products and lead to the apoptosis of human leukemia HL-60 cells by down regulating nucleophosmin/B23 and telomerase activity.

#### *3.3. Curcuma longa*

*Curcuma longa* (Turmeric) belongs to the ginger family Zingiberaceae. It is a rhizomatous herbaceous perennial plant [29]. It is naturally found in Southeast Asia and the Indian subcontinent. These plants are annually collected for their rhizomes and are then propagated from some of those rhizomes [30]. *C. longa* possesses a broad range of pharmacological activities including anti- HIC (human immunodeficiency virus), anti-inflammatory, antioxidant effects, nematocidal and anti-bacterial activities.

Curcumin, the main component of *C. longa*, plays an important role in the therapeutic activities of *C. longa* [31]. Curcumin shows anticancer and anti-inflammatory activities as reported by many different studies. Cyclooxygenase (COX)-2 plays a vital role in the formation of colon cancer. In a study conducted by Goel et al. [32], the HT-29 colon cancer cells of humans were treated with different concentrations of curcumin to study the effect of curcumin on the expression of COX-2. The cell growth of HT-29 cells was inhibited by curcumin in a concentration- and time-dependent manner. Curcumin affected COX-2 by inhibiting its mRNA and protein expression, but no such inhibitory effect was found against COX-1. From this data, it can be suggested that the in vitro growth of HT-29 cells is significantly affected by a non-toxic concentration of curcumin. Curcumin may thus play an important role in the prevention of colon cancer. Furthermore, the anticancer effects of curcumin on human breast cancer cell lines (MCF-7) were assessed through lactate dehydrogenase and 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide assays to assess cytotoxicity and cell viability, respectively. The results showed that curcumin induced cytotoxicity and inhibited cells in a time- and concentration-dependent manner. This was observed through increased caspase 3/9 activity and induction of apoptosis. The results also indicated that curcumin downregulated miR-21 the expression of miR-21 in MCF-7 cells by upregulating the PTEN/Akt signaling pathway [33].

#### *3.4. Fagonia indica*

*Fagonia indica*, locally known as "dhamasa" is a flowering plant and belongs to the family of caltrop, Zygophyllaceae [34]. Members of Fagonia genus are known for their use as traditional medicine and are found effective in the treatment of many skin problems [35]. Traditionally, it was also used as a medicine for curing cancer as well as ailments resulting from poisons [5]. Amino acids and proteins [36], flavonoids [37], alkaloids [38], saponins [39], and terpenoid [40] are the phytochemicals found in the Fagonia species. *F. indica* is found to have liver protective [41] and antioxidant properties as well [42].

The aqueous extracts of *F. indica* have been found very effective against different types of cancer specifically breast cancers. For instance, Waheed et al. [43] performed bioactivity-guided fractionation to isolate the active and potent fraction of the *F. indica* extract.  The results through different pieces of evidence such as the activity of pan-caspase inhibitor Z-VAD-fmk, caspase-3 cleavage, and DNA ladder assays suggested that apoptosis was stimulated in MDA-MB-468 and Caco-2 cells. Furthermore, a new steroidal saponin glycoside caused necrosis through cell lysis in MCF-7 cells. Similarly, Lam et al. [44] also demonstrated significant activity against breast cancer cells line MCF-7 through an aqueous extract of *F. indica*.

**Figure 2.** Illustration of activity of plants against several types of cancers. The icons were taken from Biorender illustrator and constructed through ChemBiodraw v14.0.

#### *3.5. Garcinia oblongifolia*

*Garcinia oblongifolia* (Lingnan Garcinia) belongs to the family of Clusiaceae and has a wide range of pharmaceutical activities. The important metabolites of the *G. oblongifolia* species; polyisoprenylated benzophenones and xanthones have anticancer, antioxidant, antifungal, apoptotic, and anti-pathogenic properties [45,46]. In vitro study showed that the bark of *G. oblongifolia* contains important secondary metabolites including oblongifolin A–G, oblongixanthones A–C along with other important compounds. These metabolites showed maximum apoptotic activities in HeLa-C3 cell lines and cytotoxic properties in the cervical cancer cells [47,48]. Li et al. [49] isolated about 40 different compounds from fruit, leaves, branches, and other parts of *G. oblongifolia*. They noted very high cytotoxic activities of these metabolites in the tested MCF-7 breast cancer cell line. However, they found the higher anti-cytotoxic activity of branch as compared to other plant parts. A small vacuole body formation was found at a low bark concentration of 0.250 g/mL. The vacuole size was increased at high concentrations of 500 g/mL and 1000 g/mL. The leaf part showed mild vacuole formation at a high concentration of 500 g/mL. Similarly, Feng, Huang, Gao, Xu and Luo [48] tested the pro-apoptotic activities of twenty different isolated compounds from *G. oblongifolia* in cervical cancer HeLa cells. Among all tested compounds the oblongifolins F and G, xanthone, nigrolineaxanthone T, and garcicowin B gave high pro-apoptotic properties at 10 μM concentration.

#### *3.6. Garcinia indica*

*Garcinia indica*, commonly known as kokum, is also an important medicinal plant that belongs to the Garcinia genus. The garcinol of *G. indica* shows positive activities in the experimental HT-29 and HCT-116 colon cancer cells along with normal immortalized intestinal cells (IEC-6 and INT-407). In another study, the fruit extract of *G. indica* was used for the isolation of garcinol. The garcinol at IC50 values (3.2–21.4 μM) for 72 h treatment shows strong inhibitory properties in all intestinal cells. The anticancer properties were higher in the cancer cells as compared to normal immortalized cells [50].

Similarly, Liao et al. [51] also observed a high tumor-inhibiting activity of *G. indica* in a human colorectal cancer cell line (HT-29). The garcinol at 10 μM concentration retarded the cell invasion activities several folds. The fruit extracts of *G. indica* has been shown very effective in the activation of caspase-3/CPP32 and the breakdown poly (ADP-ribose) polymerase (PARP) protein to inhibit leukemia in humans in the HL-60 cells [52]. These results indicated that garcinol (IC50 = 9.42 μM) shows strong growth inhibitory effects against human leukemia HL-60 cells.

#### *3.7. Hedyotis di*ff*usa*

*Hedyotis di*ff*usa* (Chinese: sheshecao) is a member of the family Rubiaceae. It is spread over the northeast regions of Asia. *H. di*ff*usa* has been commonly used to cure inflammatory diseases i.e., urethritis, bronchitis, and appendicitis [53,54].

Because of the recent advances in pharmacological practices, this herb received importance for having antitumor properties and showed effective results in treating cancers of the liver, colon, lungs, brain, and pancreas [55]. *H. di*ff*usa* contains important bioactive derivatives of polysaccharides, triterpenes, and anthraquinones [56,57].

Methyl anthraquinonesare, one of the bioactive compounds in *H. di*ff*usa*, is responsible for apoptosis of many cancers. It shows apoptosis and inhibitory effect on the MCF-7 cell line of breast cancer via activation of the caspase-4/Ca2+/calpain pathway when applied in a concentration of 18.62 μM for 24 h. It was observed that the S phase of the cell cycle and the percentage of the apoptotic cells were markedly increased when methyl anthraquinone was applied to MCF-7 cells [58]. Similarly, a concentrated extract of *H. di*ff*usa* cause an inhibitory effect on the cervical cancer proliferation and induces apoptosis of Hela cells. Studies on the effect of *H. di*ff*usa* ethanolic extracts on anti-colorectal cancer showed that these extracts cause an inhibitory effect on the Ct-26 cells by applying different concentrations (0.06 mg/mL, 0.08 mg/mL, 0.10 mg/mL and 0.12 mg/mL) with the rate of 35.46% to 71.84% [59].

#### *3.8. Loranthus parasiticus and Scurrulus parasitica*

*Loranthus parasiticus*, also known as Sang Ji Sheng (in Chinese), is a member of the Loranthaceae family and is widely distributed in the Southwestern regions of China. *L. parasiticus* is a semiparasitic plant, historically used as traditional folk medicine in China and Japan [60]. Since they are parasitic in nature, their biological activities including phytoconstituents are highly dependent on the host trees [61,62]. In fact, certain studies indicated the reverse effects of *L. parasiticus* on a host tree in decreasing sugar and chlorophyll content [63]. In vitro and in vivo studies have been conducted to evaluate the possible anticancer and antitumor potential of *L. parasiticus*. Cytotoxicity analysis of aqueous extracts of *L. parasiticus* has shown positive activity against ovarian cancer cell lines; SKOV3, CAOV3, and OVCAR-3 [64].

A comparative study conducted by Xiao et al. [65] on flavonoids extracted in 80% ethanol from *Scurrulus parasitica* harvested from different hosts showed a good anticancer potential on acute myeloid leukemia cell line HL-60. Flavonoids from *S. parasitica* on *N. indicum* induced apoptosis and inhibited cell proliferation with an IC50 value of 0.60 mg/L on HL-60 cells and arrested cell cycle at G0–G1 phase. *S. parasitica* parasitizing on *Morus alba* also showed an IC50 value of 2.49 mg/L. Similarly, Xiao et al. [66] isolated a polysaccharide in aqueous and ethanol extract from the leaves of *S. parasitica* and conducted proliferation inhibition assay on S180, K562, HL-60 cell lines. They observed inhibition in sarcoma S180 growth in mice with a 54% tumor inhibition rate Loranthus parasiticus on the optimal dose of 100 mg kg−1 d−1. *S. parasitica* downregulate expression of CyclinD1, Bcl-2, and Ki-67 protein, and upregulate the expression of Bax protein which helps in the inhibition of cancer cell line and apoptosis of cancer cell in vivo.

#### *3.9. Morus alba*

*M. alba*, commonly called white mulberry, is native to China, Japan, India and is cultivated throughout the world where silkworm is raised. Their leaves are the main source of food for silkworms. Extracts from *M. alba* are traditionally used to cure cough, edema, insomnia, bronchitis, asthma, nose bleeding, wound healing, eye infections, and diabetes [67]. *M. alba* contains many pharmaceutically important compounds like kuwanol, hydroxymoricin, moranoline, morusin, calystegin, albafuran, and albanol. The leaves of *M. alba* contain some active compounds such as quercetin, rutin, apigenin, 1-deoxynojirimycin [67].

A study by Chon et al. [68] on methanolic extract of *M. alba* leaves showed anti-proliferative effects on different human cell lines like pulmonary carcinoma (Calu-6), colon carcinoma (HCT-116) and breast adenocarcinoma (MCF-7). These results showed a strong link to the concentrations of the investigated extracts. Anti-proliferative activity in the methanolic extract of *M. alba* leaves was observed on the human cell line of gastric carcinoma (SNU-601) in a concentration of 1000 mg/mL.

In another study on albanol A, isolated from *M. alba* root extract, Kikuchi et al. [69] showed apoptosis-inducing, cytotoxic activity (IC50 = 1.7 μM) in HL-60 cell line. It induced topoisomerase II (IC50 = 22.8 μM), clearly reduced the levels of pro-caspases 3, 8, and 9. Furthermore, the Bax/Bcl-2 ratio also increased and induced HL-60 apoptotic cell death through stimulation of the death receptor. The results of a study conducted on *M. alba* leaves in different extracts on human cell line hepatoma (HepG2) showed that methanolic leaf extract also showed inhibition (IC50 = 33.1 μg/mL) of HepG2 cell. It was concluded that *M. alba* leaves extract contains different phenolic compounds in different solvents which showed an anti-proliferative effect on the HepG2 cell line through the arrest of the cell cycle in G2/M phase. This was achieved with p27Kip1 protein expression, activated caspases to induced cell apoptosis and inhibited topoisomerase IIα activity [70].

Furthermore, lectin was isolated from the leaves of *M. alba* which showed anti-proliferative activity on the human breast cell line (MCF-7) at a concentration of 8.5 μg/mL. This compound also showed cell cycle arrest and cytotoxicity in a human colorectal cell line (HCT-15) with inhibiting concentration of 16 μg/mL. The mechanism of inhibition of cancer cell lines was linked to the induction of apoptosis through activation and release of caspase-3 [71].

Methanolic extracts of *M. alba* showed significant anti-proliferative activity on the HepG2 cell line. These extracts showed significant inhibition of cells reducing cleared viable cell count. The cell growth was inhibited by suppressing the activity of NF-kB gene expression and biochemical marker modulation [72].

Further, Qin et al. [73] isolated 15 compounds from the root bark of *M. alba* which contain six diels-alder adducts and nine prenylated flavanones. The study observed that two new compounds, soroceal B and sanggenol Q showed cytotoxic activity. One of the isolated compounds showed selective cytotoxic activity in cells (HL-60 and AGS) at inhibiting concentrations of 3.4 μM.

#### *3.10. Paris polyphylla*

*Paris polyphylla* (called "Love Apple") belongs to family Liliaceae and contains 24 species throughout the world [74]. *P. polyphylla* is mostly used by Indian and Chinese traditional medicine system for having potential anticancer properties. *P. polyphylla* consists of important secondary metabolites such as polyphyllin D, formosanin C, β-ecdysterone, dioscin, daucosterol heptasaccharide, oligosaccharides, octasaccharide, protogracillin, trigofoenoside A, yunnanosides G-J, padelaoside B, pinnatasterone, and other saponins [75]. Steroidal saponins are the main active components because of its structural diversity and bio-activities such as antitumor, immune-stimulator, analgesic, and hemostatic properties [76–81].

Aqueous and ethanol extracts of *P. polyphylla* showed potential antitumor activity against human liver carcinoma (HepG2 and SMMC-7721) cell line, human gastric (BGC-823) cell line, human colon adenocarcinoma (LoVo and SW-116) cell line, and human esophagus adenocarcinoma (CaEs-17) cell lines. Ethanolic extract showed a strong inhibitory effect with IC50 values ranging from 10 μg/mL to 30 μg/mL [82]. Extract of *P. polyphylla* also showed an antitumor effect in esophageal cancer ECA109 cells by increasing the connexin26 mRNA and protein expression. Studies reported that *P. polyphylla* extracts increased the *Bad* genes expression and decreased the expression of *Bcl-2* genes, inhibiting the growth of ECA109 cells by proliferation and inducing cell apoptosis [83].

#### *3.11. Perilla frutescens*

*Perilla frutescens*, commonly called perilla or Korean perilla or Beefsteak plant, is widely distributed in Vietnam, China, Japan, and most Asian regions belong to the Labiatae family [84,85]. Economically, one of the most significant crops, cultivation of *P. frutescens* in China and some other Asian countries is more than 2000 years old [86,87]. Stem, seed, and leaf parts of *P. frutescens* have been used to treat poisoning, cold, bloating, and headache [74,88]). Multiple in vivo and in vitro studies have been conducted to evaluate the anticancer and antitumor potential of *P. frutescens*. Leaf extract of *P. frutescens* showed the highest anticancer activity in HepG2 cells through cell proliferation inhibition and upregulation of apoptosis-related gene expression [89]. Other studies revealed that ethanolic leaf extract of *P. frutescens* promoted apoptotic induction and tumorigenesis through death-mediated receptors and scavenging the reactive oxygen species (ROS) [90,91]

Similarly, Lin, Kuo, Wang, Cheng, Huang and Chen [89] used leaf extract of *P. frutescens* to evaluate the proliferation and apoptosis in HepG2 cells. Anti-proliferation activity was observed in HepG2 cells treated with *P. frutescens* leaf extract at a concentration of 105 μg/mL. The study reported that significant apoptosis was observed through flow cytometry. Furthermore, microarray results showed apoptosis-related gene expression in a time-dependent manner. The activity of *P. frutescens* leaf extract was compared to the activity of rosmarinic acid which showed less effective results in apoptosis-related gene expression and apoptosis induction in HepG2 cells.

The essential oil component "isoegomaketone" isolated from *P. frutescens* also induced apoptosis in human colon cancer (DLD1) cells. A study by Cho et al. [92] reported the inhibition of cell growth by isoegomaketone when treated for over 24 h, cleaved caspase-3, 8, and 9 in a time-dependent and dose-dependent manner. Isoegomaketone treatment triggered PARP cleavage, translocation of the protein Bax, cleaved Bid protein and the release of cytochrome *c* to the cytoplasm from mitochondria, induced apoptosis and translocation of apoptosis-inducing factor from mitochondria into the nucleus. It was suggested that isoegomaketone from *P. frutescens* induced apoptosis in DLD1 cells via both caspase-dependent and caspase-independent pathways.

#### *3.12. Platycodon grandiflorus*

*Platycodon grandifloras*, commonly known as balloon flower, or Chinese bellflower, belongs to the family Campanulaceae, which is distributed through Northeast Asia. The rhizomes of *P. grandiflorus* are very effective and are used as a traditional medicine in China, North Korea, and Japan for treatment of different diseases like cough, sore throat, phlegm, and other ailments [93]. *P. grandiflorus* contains many biologically active compounds which include saponins, flavonoids, anthocyanins, phenolics, and polysaccharide. These compounds have significant immune-stimulatory [94], anti-inflammatory [95], hepatoprotective [96], and antitumor activities. The antitumor activity of *P. grandifloras* was shown in a dose-dependent manner by reducing PKC enhancement of matrix metallopeptidases (MmP-9 and MmP-2), which caused the death of HT-80 cells [97]. Yu and Kim [98] isolated platycodin D from the root of *P. grandiflorus* and treated MCF-7 cells with a concentration of 5–100 μM which reduce cell viability and proliferation in a dose-dependent and time-dependent manner as compared with controlled cells. Induction of anticancer activity was observed because of caspase 8 and 9 activation and PARP cleavage. In addition, platycodin D upregulate cellular levels of protein Bax and Bcl-2 and downregulate the activation of caspase-9. Further, it also induces proteolytic activation of Bid (a protein of the proapoptotic Bcl-2 family).

Platycodin D is a triterpene saponin isolated from the roots of *P. grandiflorus* shows cytotoxic effects on the human leukemia cells. It inhibited telomerase activity and showed a cytotoxic effect in a dose-dependent manner with a concentration of 10–20 μM. This was shown to be achieved through downregulating the expression of human telomerase reverse transcriptase (hTERT). The results of the study by Kim et al. [99] showed that suppression of telomerase activity and cytotoxic effect on leukemia cells by platycodin D was through post-translational and transcriptional inhibition of hTERT. Platycodin D induced apoptosis and cell death of human leukemia (U937) cell by inducing the production of ROS through *Egr-1* gene activation and as a result, decreased in mitochondrial membrane potential, activating caspase-3 and PARP cleavage [100].

The extract of *P. grandiflorus* also showed antitumor effects on ovarian (SKOV3) cancer cells. Results showed that induced apoptosis occurs through the downregulation of Bcl-2 expression, upregulation of Bax expression, activation of caspase (3, 8, 9), and mitochondrial cytochrome *c* released to the cytosol [101].

#### *3.13. Prunus armeniaca*

*Prunus armeniaca* (Armenian plum) belongs to an important plant family Rosacea. Various parts of the plant are used as the major source of some important antioxidant substances and are commonly used against cancer and some other cardiovascular diseases [102]. The fruit part of *P. armeniaca* contains various important secondary metabolites like β-carotene, flavonoids, organic acids, thiamine, minerals, and oils [103]. The seeds of *P. armeniaca* contains plenty of cyanogenic glycosides, used against different types of cancers [104]. Amygdalin is one of the important glycosides of *P. armeniaca*, used for the treatment of prostate cancer [105]. Gomaa [106] reported the antioxidant and anticancer activities of *P. armeniaca* at different combinations of methanolic and ethanolic extracts with water. The study concluded that kernels of *P. armeniaca* have better antioxidant capacity compared to almonds that belong to the same genus. The study showed the highest antioxidant activity (67%), total lycopene content (4.70 mg/mL), and total phenolic content (3.290 mg/g of dry extract) in *P. armeniaca* kernels. Furthermore, *P. armeniaca* kernels were also found active in the inhibition of certain cancers when tested on the colon (HCT-116), human breast (MCF-7), and HepG2 cell lines in different concentrations and combinations. The highest cytotoxicity was observed from methanolic extract (IC50 = 10.1 μg) against HepG2 cells. According to Madrau et al. [107], the fruit part contains a large amount of phenolic

compound that shows strong antioxidant and immune-stimulant properties. Similarly, the study conducted by Liu et al. [108] found low anti-scavenging activity against DPPH of dry seed extract of apricot. Vardi et al. [109] reported the strong protective activity of apricot against intestinal oxidative damage in the rat model experiment.

#### *3.14. Rabdosiae rubescens*

*Rabdosiae rubescens* (Chinese: Dong Ling Cao) is a Chinese medicinal herb that belongs to the family Lamiaceae. It possesses multiple biological activities like antibacterial, anti-inflammatory, anti-parasitic, and anticancer [110]. *R. rubescens* contain important chemical compounds including monoterpenes, sesquiterpene, diterpene, and terpenoids. Oridonin, a tetracyclic terpenoid, is the main active compound in *R. rubescens* [111]. Oridonin gained its attention because of the remarkable properties of growth inhibition and the induction of apoptosis in cancer cells. In vitro and in vivo studies showed the induction of apoptosis in a variety of cancer cells by oridonin as in hepatocellular carcinoma, breast, gastric, skin, colorectal, gallbladder, and pancreatic cancers [112].

Bao et al. [113] isolated oridonin from *R. rubescens* which showed a potent anticancer potential in gallbladder both in vitro and in vivo. SGC996 and NOZ cells treated with oridonin results in inhibition of colony formation and growth of tumor cells in S-phase and induced apoptosis. Wang, et al. [114] showed the molecular mechanism of oridonin as an anticancer compound in HepG2 cancer cells. Oridonin in a concentration of 41.77 μM inhibited the growth of HepG2 cells while in a concentration of 44 μM, it induced G2/cell cycle arrest and apoptosis when applied for 24 h. The expression of nine different proteins was observed through proteomic analysis. Eight out of the nine proteins are already reported to be involved in anticancer activity. Oridonin up-regulate STRAP, Hsp70.1, Sti1, TCTP, and PPase, downregulate hnRNP-E1 which are significantly involved in the G2/M cell cycle arrest and apoptosis. The upregulation of HP1 beta and GlyRS by oridonin are responsible for the inhibitory effects on tyrosine kinase and telomerase.

Wang et al. [115] also reported that oridonin from *R. rubescens* is a potent cytotoxic agent that inhibited the growth and migration of highly metastatic human breast cancer cell lines (MCF-7 and MDA-MB-231). Oridonin significantly inhibited the growth of human breast cancer cells in a dose- and time-dependent manner, by arresting the cell cycle in the G2/M phase and accumulated cells to SUB-G1 phase. Results from the study also showed that oridonin triggered apoptosis by reducing Bcl-2/Bax ratio, NF-κB (p65), caspase-8, phospho-mTOR, IKKβ, IKKα and increasing the expression level of PPARγ, PARP, and Fas cleaving in a time-dependent manner. Oridonin significantly suppressed MDA-MB-231 cell invasion and migration through decreasing MmPs expression and regulation of integrin β1/FAK pathway and inhibited the growth and apoptosis via DNA damage and activated the extrinsic and intrinsic apoptotic pathway in breast cancer cells.

#### *3.15. Scutellaria baicalensis*

*Scutellaria baicalensis* is one of the important medicinal plants species of family Lamiaceae. It is commonly known as Baikal skullcap or Chinese skullcap and is found in different regions of the world including East Asia, Europe, and the Russian Federation. Its root part is known as *Scutellariae radix* and used as traditional Chinese medicine for the treatment of hepatitis, respiratory, and gastrointestinal diseases [116]. The root parts have maximum flavonoid content having multiple pharmacological properties [117].

About 60 different flavonoids have been identified in *S. baicalensis* which showed maximum antioxidant activities [116,118]. The four flavones metabolites also showed antimutagenic properties [119]. Wo´zniak et al. [120] studied the antioxidant potentials of four flavones: baicalein, baicalin, wogonin, and their glucuronides compounds and wogonoside. These flavones have different antioxidant capacity depending on the chemical structure and mechanisms of activity. However, among these, baicalein showed maximum antioxidant activities while the wogonin provided high protection to the linoleic acid from oxidation but did not show any antioxidant activity [121].

 The aqueous extracts of roots led to programmed cell death, and thus inhibit the growth and development of apoptosis. They suppressed the growth of lymphoma and myeloma cell lines via disturbing normal expression level of *Bcl* and *c-myc* genes, while increased the expression level of cyclin-dependent kinase inhibitor p27 (KIP1) [121]. Hongwei et al. [125] used three different concentrations (80, 120, and 160 μmol/L) of baicalin for 2 days against BGC-823 and MGC-803 gastric cancer cells. The results of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay showed a lower rate at different doses. Flow cytometric analysis showed that baicalin induces apoptosis in a dose-dependent way. Also, it was found that baicalin increase the gene expression of caspase-3, caspase-9, and other B cell lymphoma (Bcl-2)-associated X protein but lowers the expression of the *Bcl-2* gene.

A recent study reported the angiogenic properties of baicalin by using chick embryo chorioallantoic membrane [126]. The aqueous extract of baicalin showed both inhibitory and angiogenic activities while the baicalein showed only anti-proliferative property. Quantitative PCR results showed the expression of 84 different angiogenesis-related genes at different doses of baicalin and baicalein. The low dose significantly increases angiogenic genes expression while the high concentration showed the opposite activity by decreasing angiogenesis and increasing the lethality. The low to high levels of baicalein decrease the level of gene expression of multiple angiogenic genes and decreased cell proliferation.

Sato et al. [127] studied the anticancer potential of the root extract of *S. baicalensis* by using human oral squamous cell carcinoma (OSCC) cell line. The findings of the study showed 100 μg/mL root extract retard the monolayer and anchorage-independent growth rate many times but do not affect the cell adhering ability. Downregulated expression of cyclin-dependent kinase 4 cyclin D1, G1 phase arrest, and PARP cleavage was observed with the application of the root extract of *S. baicalensis*.

#### *3.16. Scutellaria barbata*

*Scutellaria barbata*, the barbed skullcap is a key medicinal plant species of family Lamiaceae, used to treat inflammatory and cancer diseases [128]. It is rich in important secondary metabolites like alkaloids, flavones, steroids, and polysaccharides [129,130]. In vitro studies showed positive activities against a vast range of cancers i.e., colon cancer, lung cancer, hepatoma, and skin cancer [128].

The apigenin and luteolin isolated from *S. barbata* gave cytotoxic activity against both human breast cancer cell line MDA-MB-231 and non-transformed breast cell line (MCF10A) [131]. Similarly, scutellarein was found to possess strong anti-breast cancer activity demonstrated in MDA-MB-468 cell lines [132]. Scutellarein increased the concentration of mitochondrial superoxide and peroxide while decreasing the level of glycolysis, retarding the growth of cancer cells by lowering ATP synthesis. Other secondary metabolites of *S. barbata* showed cytotoxic properties by leading ROX and DNA damage [131]. The other important alkaloid of *S. barbata*, scutebarbatine A (SBT-A) also resulted in high antitumor and apoptosis activities in experimental A549 cells.

#### *3.17. Tripterygium wilfordii*

*Tripterygium wilfordii* of the family Celastraceae is also known as "Thunder God Vine," and is native to Korea, China, and Japan. It is commonly used for the treatment of multiple diseases such as rheumatoid arthritis, systemic lupus erythematosus, nephritis, asthma, and cancers [133–135]. *T. wilfordii* produces important bioactive compound triptolide which is used as an immunosuppressive and anti-proliferative agent [134]. It has a five-membered unsaturated lactone ring and is used against different breast cancer cells by activation of pro-apoptotic compounds by modulating several signaling pathways [136].

In vitro studies showed anti-proliferative and pro-apoptotic activities against tumor cell lines [137–139]. He et al. [140] studied the in vitro properties of triptolide of *T. wilfordii* activity by using human umbilical vein endothelial cells (HUVECs). The IC50 value for HUVECs proliferation

was 45 nM. The results of semi-quantitative RT-PCR and Western blot analysis in human umbilical vein endothelial cells showed that triptolide cause downregulation of proangiogenic Tie2 and VEGFR-2 expression after 24 h at 50 nM concentration. At high concentration (100 nM), the VEGFR-2 mRNA expression was completely blocked. But on the other hand, the knockdown of Tie2 decreases the inhibitory activities of triptolide on endothelial network formation. The overall results showed that anticancer properties of triptolide are directly correlated with the blockage of two endothelial receptor-mediated signaling pathways. Triptolide showed more antagonistic activities against the proliferation of HUVECs as compared to normal cells like skin keratinocytes HaCaT cells and other liver cells L-02 [141].

Celastrol is another important compound of *T. wilfordii*, having cytotoxic activity against a broad range of cancer cells [142]. It demonstrates its anticancer activities by blocking the NF-κB via targeting IκB kinase and TAK1-induced NF-κB activation [143,144]. Other studies showed that triptolide of *T. wilfordii* has anticancer properties in many model systems such as experiments have also demonstrated triptolide's therapeutic efficacy in several model systems including neuroblastoma in nude mice model [145], other xenografts of human melanoma, breast cancer, bladder cancer, gastric carcinoma [146].

#### *3.18. Tussilago farfara*

*Tussilago farfara* (commonly called coltsfoot) is one of the important medicinal plants, grown in Europe and various regions of western and central Asia, commonly used against cancer. It possesses a high quantity of flavonoids and other phenolic compounds and some trace elements (Zn, Mg, and Se). The presence of these substances plays a key role in the anticancer activities of this plant. Maximum scavenging activity was recorded in water extract as compared to ethanol extract. It shows a 20.9% antioxidant activity. Further, this plant showed maximum antioxidant activity both using DPPH and yeast model [147]. The quercetin-glycosides isolated from the flower bud of *T. farfara* shows the highest antioxidant activity [148]. Lee et al. [149] reported the (TF)-induced cytotoxic and apoptotic activities of the flower part of *T. farfara* in human colon cancer cell line (HT-29) by using a methanolic extract. Fatykhova et al. [150] showed the genotoxic activity of *T. farfara* herb juice against known genotoxic compounds like nalidixic acid in SOS chromotest and furacilin in Rec assay. Their findings showed that dilution of the herb juice gave maximum antimutagenic properties in SOS chromotest as compared to furacilin in Rec assay.

Lee et al. [151] reported the activity of *T. farfara* as the TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis via MKK7/JNK activation by inhibition of mitogen protein kinase-TOR signaling pathway regulator-like protein (MKK7-TIPRL) in human hepatocellular carcinoma cells. The *T. farfara* extract decreases 50% interaction between MKK7-TIPRL. HPLC data further verified the presence of many important phenolic compounds that decrease MKK7-TIPRL interaction and also examined the activation of MKK7/JNK.

#### *3.19. Wedelia chinensis*

*Wedelia chinensis* (Chinese: Peng qi ju), indigenous to India, South-East Asia, and China, is one of the important anticancer plants belonging to family Asteraceae which is rich in many important secondary metabolites like phenol, flavonoids, and tannin [152].

The essential oils of *W. chinensis* give a positive effect on lung cancer during the in vitro study. The GC-MS analysis recorded the presence of two important compounds carvacrol and trans-caryophyllene. High anti-scavenging activities were found at different levels of dose. The study of B16F-10 melanoma metastatic cell line showed that the concentrations of some important antioxidant enzymes (including catalase, superoxide dismutase, and glutathione peroxidase) increased many folds in the treatment groups. Similarly, the amount of glutathione also increased while the concentrations of other compounds such as lipid peroxidation and nitric oxide were decreased. The histopathology studies further verified that these essential oils show negative effects on cancer development [153].

#### **4. In Vivo Studies of Anticancer Herbal Medicine: An Overview**

The herbal medicines are tested both in vitro and in vivo. The anticancer activities of the various medicinal plants have been tested in vivo using different animal models (Figure 3). There are many studies available on in vivo experiments of the many different anticancer plants in mice models. For instance, dihydroartemisinin was reported to inhibit tumor tissue, increase the level of interferon-gamma (IFN-γ), and decrease interleukin 4 (IL-4) in tumor-bearing mice [154]. Similarly, artesunate, a derivative of artemisinin is also reported to be a promising drug against angiogenic Kaposi s sarcoma [155], growth inhibition of A549 and H1299 lung tumors by 100 mg/kg dose [156], the suppression of human prostate cancer xenograft [157] and the inhibition of leukemia growth in mice [158].

**Figure 3.** A depiction of general strategies applied for assaying extracts/phytochemicals from important medicinal plants for their anticancer activity both in vitro and in vivo.

Irradiation of C57BL/6 mice combined with a dose of 2 mg/kg twice a week was proved effective against lung carcinoma [159]. The effectiveness of berberine was enhanced when it was used in combination with other agents. Coptisine, another alkaloid of *Coptidis rhizoma* is proved to have anticancer effects when used in concentrations of 150 mg/kg against BALB/c nude mice by suppressing tumor growth and reducing cancer metastasis. The inhibition of the RAS-ERK pathway was suggested as the mechanism for this activity [160]. Another study was also performed on the nude mice on the HepG2 cells by applying the aqueous extract of *H. di*ff*usa* which inhibits proliferation of cells in a dose-dependent manner, also delay S phase and arrest cells in G0/G1 phase [161].

Similarly, a high anticancer activity of SBT-A was found in transplanted tumor nude mice. Yang et al. [162] reported the anticancer activity of the polysaccharides isolated from *S. barbata* by 95-D Xenograft model. The results showed that polysaccharides give strong anti-proliferative activities against a 95-D cell line. It also lowered the expression of phospho-c-Met and other signaling elements like phospho-Erk and phospho-Akt. In vivo study also gave maximum antitumor activity by using a 95-D subcutaneous xenograft model. After one daily intraperitoneal injection for 3 weeks, the tumor growth was significantly decreased (47.72 % and 13.6%) at 100 and 200 mg/kg treatments. The ex vivo studies also showed that polysaccharides of *S. barbata* inhibit the phosphorylation of c-Met signaling pathway.

Furthermore, Li et al. [163] isolated a steroidal saponin from *P. polyphylla* which inhibited tumor growth in Lewis bearing-C57BL/6 mice and induced apoptosis in A549 cells. Results showed that steroidal saponin in concentration of 2.5, 5.0, and 7.5 mg/kg showed significant inhibition rate of 26.49 ± 17.30%, 40.32 ± 18.91%, and 54.94 ± 16.48%, remarkably increased thymus and sleep indices, decreased inflammatory cytokines (TNF-α, IL-8, and IL-10). This in turn inhibited the tumor growth in C57BL/6 mice by reduced volume and weight of tumor. Nuclear changes, DNA condensation, chromatin fragmentation, and apoptosis are induced in A549 cells with a concentration of 0.25, 0.50, and 0.75 mg/mL steroidal saponin. Tumor growth inhibited by steroidal saponin was associated with decreased ROS, inflammatory response, and induction of apoptosis.

Furthermore, Wanga et al. [164] reported the effect of isoegomaketone from *P. frutescens* on Huh-7 hepatoma cell carcinoma and tumor-xenograft nude mice. Results showed that isoegomaketone inhibited cells and decreased tumor weight and volume. Isoegomaketone in the concentration of 10 nM/L decreased pAkt without affecting Akt. Hepatoma cell carcinoma tumor growth was suppressed by isoegomaketone from *P. frutescens* through PI3K/Akt signaling pathway blocking. *R. coptidis* is also showed anticancer activity in rats as suggested by the inhibition of cyclooxygenase 2 activity. The number of aberrant crypt foci in the rat colon was decreased by 54% after the administration of *R. coptidis* extracts [165].

Manjamalai and Grace [166] reported the apoptosis along with lowering angiogenesis and lung metastasis activities of the essential oils of *W. chinensis* by using B16F-10 melanoma cell line in C57BL/6 mice. The mice were injected with B16F-10 melanoma cells through the tail vein and treated with different doses of essential oil. A 50-μg essential oil concentration showed maximum cytotoxic activities with 65.17% lethality within 24 h. The numbers of apoptotic cells increased many times in experimental samples as compared to the control group. They also recorded high levels of important proteins like p53 and caspase-3 in essential oil-treated samples compared to other non-treated samples. They recommended this plant for the treatment and control of cancer.

In vivo activities of oridonin from *R. rubescens* showed a potent anticancer potential in the gallbladder [113]. When injected intra-peritoneally with a concentration of 5, 10, 15 mg/kg for 3 weeks to athymic nude mice, oridonin significantly inhibited NOZ xenografts growth. Oridonin also inhibited NF-κB nuclear translocation, increased Bax/Bcl-2 ratio, activated caspase-3, caspase-9, and PARP-1 which showed that the mitochondrial pathway is concerned with apoptosis mediated by oridonin.

Studies have reported anticancer activities of two artemisinin dimer, dimer-hydrazone (dimer-Sal) and dimer-alcohol (dimer-OH) and one monomer dihydroartemisinin (DHA) compared to the control against MTLn3 breast tumors in rats. Results of the study reported that dimer-Sal, dimer-OH, and DHA significantly suppressed tumors in rats compared to the control group. It was also observed that the dimers were more potent as compared to the monomers [167].

It is also reported that artemisinin is responsible for preventing breast cancer in rats treated with a single oral dose (50 mg/kg) of 7,12-dimethalbenz anthracene (DMBA) which is known for rapidly inhibiting the multiple breast tumors. After the feeding DMBA with 0–2% artemisinin to the target group and plain food in powdered form to the control group, both groups of experimental rats were monitored for breast tumors for 40 weeks. Oral artemisinin significantly reduced the development of breast tumors (57%) as compared to control fed (96%). The research indicates that artemisinin might be a potent cancer chemoprevention agent, having lesser side effects [168]. Similarly, oral administration of curcumin to rats reduced the level of Gp A72 (glycoprotein) by 73% hence lowering paw inflammation [169].

Tanaka et al. [170] observed activities of fruit extracts of *G. indica* in the azoxymethane (AOM)-induced colonic aberrant crypt foci in male model rats (F344). They found lower proliferating cell nuclear antigen index and high concentrations of glutathione S-transferase and quinone reductase. They also observed the maximum chemo-preventive activities of garcinol.

Besides mice and rats, there are many other animal models employed for studying anticancer activities. Zebrafish models are also employed for technical advantages including the ease of

advanced genetic studies, expression of tumor in any organ and the striking resemblance to human malignancies [171]. Zhu et al. [172] used furanodiene which is a terpenoid isolated from *Rhizoma curcumae*, for their anticancer effects in zebrafish models. They observed that furanodiene showed anticancer effects in a pancreatic cell line (JF 305) and human breast cancer cells (MCF-7) transplanted into zebrafish. Furanodiene showed effective results through ROS production, anti-angiogenesis, apoptosis induction, and DNA strand breaks.

Similarly, the artemisinin type compound can have anticancer activities against different types of tumors including leukemia, carcinomas of breast, kidneys, lungs, and ovaries, lymphoma, melanoma, and brain tumors [23,173,174]. Currently, reports of in vivo activities of *A. annua* are accumulating.  The animals were treated with various doses of 150 mg/day (3 capsules), 450 mg/day (2 capsules), 450 mg/day (3 capsules), and 450 mg/day (2 capsules). All the animals showed complete reduction with no tumor relapse [175].

#### **5. Regulatory Aspects of Herbal Anticancer Drugs**

It is generally established that the drugs including the anticancer compounds require phase III clinical research trials for marketing permissions. The Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines require at least one controlled trial in Phase III with statistically significant results for the green signal to market them [176]. Except for exceptional circumstances, all the drugs need to go through all the phases of trials according to the guidelines of international agencies such as the FDA and EMA. However, it has been observed that pharmaceutical companies deviate from the standard protocol and start testing new compounds on human subjects earlier than the defined timeline. The reason for such practices is to accelerate the approval of these compounds under the pressure of investors [176]. This means that the drug is presented for approval with insufficient data on its quality, safety, and efficacy.

Although plant-based compounds have shown be less toxic compared to conventional synthetic compounds, there is growing evidence on the side effects of the unregulated use of these plants against different diseases. The problem is that there is insufficient data available regarding the quality, safety, and efficacy of herbal drugs. *F. indica*, for instance, has shown potent activity against breast cancer when tested in the MDA-MB-231 cell line [44]. *F. indica* is used traditionally to treat many disorders and people have even started the use of its herbal tea against breast cancer. However, the question remains that there are only a few reports available on the anticancer activity of the plant. Globally, the process of oncology drug development and marketing is regulated through the involvement of experts and an advisory process mediated by regulatory authorities [177].

There are several regulatory framework models available for prescribing such drugs but there is a need for harmony among regulating agencies and improvement in the regulation process. For instance, the FDA has recently adopted the questions and answers guidelines of the International Council for Harmonization on the nonclinical evaluation of drugs intended to treat cancer. These guidelines include 41 questions and answers which provide additional information about anticancer drug development and are aimed at bringing harmonization in the process of anticancer drug development [178]. It is, however, suggested that regulatory authorities, while bringing harmony with other agencies working for regulating anticancer herbal compounds, should increase the focus on combining information from traditional knowledge about that drug and the scientific studies on it [179].

Moreover, it is evident that plants of the same species grown in different areas vary in their profile of medicinal compounds [180]. This calls for the need to focus on the production of uniform and high-quality plants with a uniform metabolite profile that once tested is declared safe or unsafe once and for all. This might be achieved through the help of in vitro growth and biotechnological and genetic studies on these anticancer plants [181,182].

#### **6. Modern Trends in Traditional Medicine Informatics and Opportunities for Anticancer Plant Products**

With the advancement of information technology and bioinformatics, there is an increasing trend to build resources and databases that report herbal formulations, active components of the herb, and related information. There are several efforts like Chinese Medicine Integrated Database (TCMID) [183], Collective Molecular Activities of Useful Plants (CMAUP) [184], SymMap [185], encyclopedia of traditional Chinese medicine (ETCM) [186] etc. In addition, several researchers have developed strategies for in silico pharmacokinetic properties of molecules/drugs [187–191]. Such approaches are also applicable to phytochemicals and plant-based active drug components for their virtual screening, possible mode of action, and advanced drug discovery [192–195]. Several plant-based anticancer compounds have been evaluated using in silico and systems pharmacology tools [196–201]. The current study encourages further studies on anticancer active ingredients (of plant origin) for their in silico screening and pharmacokinetic activities. Considering the fact that plant-based drug formulations usually consists of several phytochemicals or even more than one plants. The major challenge on this direction would be to predict the role of phytochemicals other than active compounds and are present in the traditional medicine.

#### **7. Conclusions**

This detailed analysis of different plants showed that medicinal herbs promise a huge anticancer potential. This article comprehensively highlights the mechanism of antitumor action of some of the important plants. This is generally done through regulating signaling pathways. Many studies have reported inhibition of enzymes that stops tumor growth. These studies are mainly performed in human cell lines. It is highlighted that these plants play an important anticancer role through their different classes of secondary metabolites (Table 1). However, the study of these plants should not limit the study of a plethora of anticancer plants some of which are still unexplored. Studies are needed to highlight the mechanism of anticancer action of many already explored and many unexplored plants.

**Table1.**Someoftheimportantanticancermedicinalplants,theiractivecomponents,andinvitroandinvivo

*Biomolecules* **2020**, *10*, 47

**Table 1.***Cont.*

*Biomolecules* **2020**, *10*,

**Author Contributions:** Conceptualization, T.K., and M.A.; methodology, T.K. and M.A.; software, T.K.; investigation, T.K.; resources, S.A.J. and A.K.; data curation, P.N.; writing—original draft preparation, T.K., P.N., S.A. and A.K.; writing—review and editing, T.K. and M.A.; visualization, T.K.; supervision, Z.K.S. and M.A.; project administration, M.A. All authors have read and agreed to the published version of the manuscript.

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

**Acknowledgments:** The authors acknowledge the efforts of all researchers working in the area of traditional medicine against cancer. The authors apologize to all scientists whose work has not been cited because of space limitation or unknowingly due to the great amount of literature in the field of anticancer phytochemicals.

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

#### **References**

© 2019 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/).

### **Bioactivities of** *Geranium wallichianum* **Leaf Extracts Conjugated with Zinc Oxide Nanoparticles**

**Banzeer Ahsan Abbasi 1,**†**, Javed Iqbal 1,\*,**† **, Riaz Ahmad 2, Layiq Zia 3, Sobia Kanwal 4, Tariq Mahmood 1, Canran Wang 5 and Jen-Tsung Chen 6,\***

Received: 26 October 2019; Accepted: 17 December 2019; Published: 26 December 2019

**Abstract:** This study attempts to obtain and test the bioactivities of leaf extracts from a medicinal plant, *Geranium wallichianum* (GW), when conjugated with zinc oxide nanoparticles (ZnONPs). The integrity of leaf extract-conjugated ZnONPs (GW-ZnONPs) was confirmed using various techniques, including Ultraviolet–visible spectroscopy, X-Ray Diffraction, Fourier Transform Infrared Spectroscopy, energy-dispersive spectra (EDS), scanning electron microscopy, transmission electron microscopy, and Raman spectroscopy. The size of ZnONPs was approximately 18 nm, which was determined by TEM analysis. Additionally, the energy-dispersive spectra (EDS) revealed that NPs have zinc in its pure form. Bioactivities of GW-ZnONPs including antimicrobial potentials, cytotoxicity, antioxidative capacities, inhibition potentials against α-amylase, and protein kinases, as well as biocompatibility were intensively tested and confirmed. Altogether, the results revealed that GW-ZnONPs are non-toxic, biocompatible, and have considerable potential in biological applications.

**Keywords:** Zinc oxide nanoparticles; anticancer; antileishmanial; antimicrobial; biocompatibility

#### **1. Introduction**

Nanotechnology is one of the important thriving and rapidly developing interdisciplinary sciences involving a combination of knowledge from materials science, physics, chemistry, and biology, etc. The term "nano" is a Greek word which means "extremely small or dwarf" in size range of about one to one-hundred nanometers. NPs possess unique and fascinating magnetic, electrical, optical, and chemical properties with small size, different shapes, and surface effects compared to bulk materials [1–3]. NPs possess breakthrough applications in different fields such as food, agriculture, medicine, cosmetics, energy, environment, and many more [4,5]. Among the different metal nanoparticles (MNPs), zinc oxide nanoparticles (ZnONPs) has received due attention due to their multifunctional and tunable nature. ZnONPs possess 3.37 eV direct band gap with high excitation energy (60 meV), which make it perfect to be utilized in UV photodetector, transistors, and semiconductor diodes [6–8]. In addition, ZnONPs are used in the field of bio-imaging, drug delivery,

mineral based sunscreens, lotions, and ointments; biomedicine, especially in the fields of anticancer and antibacterial fields, which are involved with their potent ability to trigger excess reactive oxygen species (ROS) production, release zinc ions, and induce cell apoptosis [9].

Numerous physical and chemical routes have been developed to synthesize ZnONPs with disparate morphologies and sizes [10–13]. These physical synthesis routes face numerous problems; they need high energy, costly instruments, and require high temperature and pressure [14]. Meanwhile, chemical approach involves synthesis of ZnONPs using several reducing agents, costly metal salts, toxic solvents, and reductants. These physical and chemical methods are not only expensive at industrial level production, but also possess potential environmental and biological hazards [15–18]. In contrast, biological/biogenic synthesis of NPs is an emerging area and economically feasible option in the field of "green chemistry" [19–22]. It is considered to be simple, safer, greener, easily scaled up, non-toxic, eco-friendly, energy-efficient, cost-effective, and performed at room temperature and pressure in the absence of non-hazardous solvents and reductants [19–21]. Biological fabrication of NPs can be accomplished using different bacteria, algae, diatoms, and medicinal plants [22,23]. The major disadvantage associated with microbial sources is maintaining contamination free environment, high isolation cost and their maintenance in culture media. Therefore, plants (phytofabrication) promise to be an excellent route in the formation of NPs due to simplicity, replacing chemicals to lessen or even remove materials that are harmful to human health and the ecosystem [24]. The use of a plant-extract in the synthesis of NPs has recently gained significant popularity [20,25]. The phytochemicals available in plants act as a reducing agents, leading to a synthesis of capped NPs, and thus reducing costs and chemical reagents [25,26]. Further, phytochemicals and aqueous environment replace many harmful organic/inorganic solvents and chemical compounds [26]. Currently, plant extracts of various plant parts have been used in the biofabrication of NPs [25]. Plant extracts possess potential biological activities in biomedical applications due to the presence of different phytomolecules: alkaloid, flavonoid, phenolic, terpenes, amino acids, and vitamins may function as strong reducing and stabilizing agents, reducing cost and eliminating the use of toxic chemicals agents [27–30].

Numerous investigations have focused on the green synthesis of ZnONPs and have reported different biological activities [31–34]. Considering the importance of green synthesis, in the present study, ZnONPs have been synthesized using GW leaves extract. The plant possesses many therapeutic application in the treatment of rheumatism, leucorrhoeas, arthritis, gonorrhea, heart, and liver related problems. Among the various plants used in the formation of green NPs, GW was selected due to large amount of bioactive compounds; ursolic acid, herniarin, stigmasterol, β-sitosterol, herniarin, etc. which can help in the reduction, stabilization, and capping of metal ions [35].

The aim of the current research study was to biosynthesize ZnONPs (Zinc nitrate hexahydrate) using natural GW leaves extract without utilizing any surfactants and chemical solvents. The reaction condition and synthesis protocol have been discussed comprehensively. To the best of our knowledge, this is the first research study reporting green fabrication of ZnONPs using GW leaves extract. Furthermore, ZnONPs were extensively studied using various characterizations techniques, followed by investigations of its diverse bio-potentials using number of activities and assays.

#### **2. Experimental**

#### *2.1. Plant Sampling and Extract Preparation*

*Geranium wallichianum* (Geraniaceae) was collected in its flowering stage from Nathiagali Mountains (34◦04 N 73◦23 E), Khyber Pakhtunkhwa, Pakistan. The soil is typically moist, loamy, and very shallow. The weather remains cool, pleasant and foggy in summers, cold and chilly in winters with heavy snow [35]. The leaves of GW were thoroughly washed with deionized water, shade dried for 10–15 days so that water content gets removed entirely. The leaves were crushed to powder and preserved in dry and airtight container. The plant extract was prepared by mixing 10 g of GW fresh leaves powder with 250 mL distilled water. The mixture was heated for 2 h at 80 ◦C under continuous stirring. The resulting solution was cool down at room temperature and filtered three times utilizing Whatman filter papers. The resulting filtered extract was preserved at 4 ◦C for future use in the biofabrication of ZnONPs.

#### *2.2. Synthesis of ZnONPs*

Synthesis of ZnONPs was successfully performed by reducing Zn(NO3)2·6H2O using GW leaf extract. In detail, 50 mL filtered GW leaves extract was taken and mixed with 3 gm Zn(NO3)2·6H2O salt, heated at 60 ◦C, and continuously stirred at 500 rpm for 2 h. The obtained solution was centrifuged at 12,000 rpm/30 min. The pellet containing ZnONPs was carefully washed 3–4 times with double distilled water. The obtained powder assumed as ZnONPs was placed in an oven at ~100 ◦C for 3 h. Further, ZnONPs were calcined in a furnace to obtain crystalline ZnONPs. The calcined ZnONPs were kept in a cool, dry, and dark place, and their characterizations were performed. Figure 1 shows a study layout of ZnONPs from synthesis to characterization and biological applications.

**Figure 1.** The process of green synthesis of leaf extract-conjugated zinc oxide nanoparticles.

#### *2.3. Characterization of ZnONPs*

The biogenic ZnONPs were characterized morphologically, physically, and chemically using various analytical techniques. The bio reduction of zinc ions to ZnONPs was determined by measuring the absorption spectra of reaction solutions via UV-4000 UV-Vis spectrophotometer (Germany) between 200–700 nm. The morphology of the ZnONPs was analyzed through SEM (EM (NOVA FEISEM-450 applied with EDX detectors). TEM analyses was performed to study size and shape of ZnONPs. The average particle size and particle size distribution of ZnONPs were studied via DLS system, using Malvern Zetasizer Nano (Malvern instrument). FTIR (Alpha, Bruker, Germany) analysis was performed between 500–4500 cm−1 to study diverse types of functional groups which are responsible in reduction and effective stabilizations for ZnONPs using various modes of vibrations. The crystalline structure of biogenic ZnONPs was determined by PANalytical XRD (Netherland) and crystal size was calculated. The Raman spectroscopy analyses was performed for ZnONPs to study their vibrational properties. The EDS analyses were done to detect the elemental composition of ZnONPs.

#### *2.4. Bio-Potentials of Green GW-ZnONPs*

#### 2.4.1. Metabolic Activity of GW-ZnONPs against HepG2 Cells

The MTT cytotoxicity assay was performed according to the previously described method to assess the survival (viability) of the HepG2 (liver cancer cells) by assessing the mitochondrial activity

inside the cells after treatment with ZnONPs. For this purpose, HepG2 cells were cultured in DMEM media provided with 10% FBS, 1% Pen-Strep. Cells were seeded in 96-well plates and placed in 5% CO2 incubator at 37 ◦C for cell attachment. To assess the cytotoxicity potentials of green ZnONPs, HepG2 cells were treated with different doses of zinc oxide nanoparticles (7.8125–1000 μg/mL) for 48 h and MTT assay was performed. The ZnONPs treated cells were placed in DMEM medium and placed in an incubator for 24 h. After incubation, 100 μL MTT solutions was loaded and set aside for ~2 h. The MTT dye reacted with the oxidoreductase enzyme present in the mitochondria and was then converted into crystals of formazan. This process occurred only in viable cells. Further, the formazan crystals were dissolved with dimethyl sulfoxide (DMSO) and its absorbance was measured using a plate reader at 570 nm. Untreated cells were considered as control, while % inhibition of HepG2 cell lines treated with the synthesized ZnONPs nanoparticles was calculated using the formula below.

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

#### 2.4.2. Antileishmanial Potential of ZnONPs

The cytotoxicity potentials of ZnONPs were further evaluated using *Leishmania tropica* "KWH23 strain" (both amastigotes and promastigotes culture) utilizing cytotoxicity assay [30,36]. The MI99 media was used added with 10% FBS to culture leishmanial parasites. The leishmanial parasites were treated with various concentrations of ZnONPs (1–200 μg/mL) to evaluate their antileishmanial potentials. During the experiment, Amphotericin-B was considered positive and DMSO as negative control. The Leishmanial parasites in 96 well plates were treated with various concentrations of ZnONPs and kept in 5% CO2 incubator for 72 h at 24 ◦C, and absorbance was measured at 540 nm. After treatment, all living leishmanial parasites were counted under a microscope and IC50 values were recorded. Median lethal concentration (IC50) was calculated using GraphPad software, while percent inhibition was calculated using the following formula:

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

#### 2.4.3. Alpha Amylase (AA) Inhibition Potential

The AA inhibition potential of biogenic ZnONPs was determined using a previously described method [36]. The reaction mixture for the activity was made by mixing 25 μL of AA enzyme, 10 μL ZnONPs, 40 μL starch solutions, and 15 μL FBS. The reaction solution with all component was incubated at 50 ◦C for 30 min by adding 1 M HCL (20 μL) and iodine solutions (90 μL). The acarbose was utilized as positive and distilled water was utilized as negative control during experiment. Median lethal concentration (IC50) was calculated using GraphPad software while percent inhibition was calculated using the following formula:

$$\% \text{inhibtion} = \frac{\text{sample absorbance} - \text{absolute of negative control}}{\text{absolute of blank} - \text{absolute of negative control}} \times 100\tag{3}$$

#### 2.4.4. Protein Kinase (PK) Inhibition Potential

The ZnONPs were also evaluated for its PK inhibition potential using *Streptomyces* 85E strain according to a previously published method [37]. The PK inhibition activity was conducted in a sterile environment. The SP4 minimal media was utilized to prepare even lawns of *Streptomyces*. 10 μL of ZnONPs loaded on filter discs were placed on the petri plates to determine their PK inhibition potential. The surfactin was taken as positive and DMSO as negative control. To target the growth of *Streptomyces* 85E strain, incubation was performed at 30 ◦C for 72 h. After 24 h, clear and bald zones appeared around the discs, which showed spores inhibition and mycelia development. Finally, zone of inhibition (ZOI) were measured.

#### 2.4.5. Antifungal Assays of ZnONPs

Fungicidal potentials of biogenic ZnONPs were investigated against different fungal strains via disc diffusion method. Before fungicidal assay was performed, fungal spores were sub-cultured in Sabouraud Dextrose liquid media and kept in an incubator for 24 h at 37 ◦C. The liquid cultures of different fungal strains were adjusted to OD of 0.5. Further, SDA solid media was made for culturing fungal strains and poured into petri plates. Filter discs laden with different concentrations of ZnONPs were kept on media plates. The Amphotericin-B was taken as positive and DMSO as negative controls. After loading test samples and both positive and negative controls, fungal plates were kept in an incubator for ~48 h at 28 ◦C to observe the ZOI. The fungal strains were treated with various concentrations of ZnONPs ranging from 31.25–1000 μg/mL, and their MIC value was recorded to determine their antifungal potential.

#### 2.4.6. Antibacterial Activity of ZnONPs

The bacterial inhibition potential of GW-ZnONPs were evaluated using various bacterial strains through discs-diffusion method. Before the activity was conducted, bacterial strains were sub-cultured overnight in nutrients broth media and incubated at 37 ◦C for 24 h. To determine the antibacterial potency of ZnONPs, an overnight culture of bacterial strains was spread on pre-prepared agar media and allowed to dry for 5 min. Subsequently, filter discs laden with different concentration of ZnONPs (31.25–1000 μg/mL) were dried and kept on surface of plates. The plates were kept in incubator at 37 ◦C for 24 h and observed for ZOI. The standard antibiotic oxytetracycline was taken as positive and 5% DMSO as negative control. Further, MIC values of GW-ZnONPs were determined by calculating ZOI.

#### 2.4.7. Antioxidant Capacities

The radical scavenging potential of ZnONPs was determined using spectrophotometric procedure. The working solution was prepared by mixing DPPH (2.4 mg) into 25 mL of methanol as free radicals. Before the activity was started, various concentrations (1–200 μg/mL) of ZnONPs were prepared and evaluated for their antioxidant potential. The ascorbic acid (AA) was taken as positive and DMSO as negative control. The 200 μL of reaction mixtures was comprised of 180 μL of reagent solution and 20 μL ZnONPs sample. The reaction mixture was then kept for 2 h under dark conditions and absorbance of reaction mixture was measured at 517 nm. The scavenging potential of GW-ZnONPs on free radicals are presented as follows:

$$\% \text{DPH savenging} = 1 - \left(\frac{\text{Absorbance of sample}}{\text{Absorbance of control}}\right) \times 100\tag{4}$$

The antioxidant potential of ZnONPs was further studied by total antioxidant capacity (TAC) using previously described phosphomollybdenum method [38]. The absorbance was recorded at 695 nm and results were indicated as microgram equivalent of AA per/mg of test samples. AA was used as positive control and DMSO as negative control. Furthermore, total reducing power (TRP) of the asynthesized ZnONPs were studied using Potassium-ferricyanide procedure [39]. AA was taken as positive and DMSO was taken as negative controls. The absorbance of mixture solutions was recorded at 630 nm. The reducing power of asynthesized ZnONPs was measured as AA equivalents per milligrams (AAE/mg).

#### 2.4.8. Biocompatibility of ZnONPs with Human Macrophages

The biocompatible nature of green ZnONPs were investigated using human macrophages via previously used method [40]. The macrophages were cultured in RPMI media supplemented with FBS (10%), Herpes (25 mM), antibiotics (Pen-Strep. Further, macrophages were seeded and cultured in 96-well plates, kept in 5% CO2 incubator for 24 h for cell attachment. The macrophages were exposed to various concentrations of biogenic ZnONPs (1–200 μg/mL) for 24 h. The absorbance was measured and % inhibition was calculated using the equation below.

$$1\% \text{ inhibition} = \frac{1 - \text{Absorbance of sample}}{\text{Absorbance of control}} \times 100\tag{5}$$

#### 2.4.9. Biocompatibility of ZnONPs with Human RBCs

The biocompatible nature of asynthesized ZnONPs was further confirmed using human RBCs through previously described method [41]. For hemolytic assay, 1 mL of fresh human RBCs was taken and stored in EDTA falcon to avoid blood coagulation. Further, centrifugation was performed for human RBCs at 12,000 rpm for 10 min. The erythrocytes suspension was made by adding 200 μL erythrocytes into 9.8 mL of PBS (pH 7.2). The 100 μL erythrocytes suspensions was treated with various doses of ZnONPs and incubated at 35 ◦C for 1 h. Further, centrifugation was performed at 12,000 rpm and supernatants was removed. Further, the supernatant was shifted into 96-well-plate and hemoglobin release was studied at 540 nm. During experiment, Triton X-100 was utilized as positive and DMSO as negative control. The results are calculated as % hemolysis produced by different concentration of ZnONPs and can be calculated employing the formula below:

$$\% \text{ hemoglobin} = \frac{\text{Sample abs} - \text{Negative control abs}}{\text{Positive control abs} - \text{Negative control abs}} \times 100\tag{6}$$

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

#### *3.1. Biosynthesis of ZnONPs*