FIGURE 7 | Scheme of the proposed mechanism by which SFI increases eNOS activity. Administration of SFI results in an increase in the gene and protein expression of eNOS and promotes the eNOS phosphorylation at Ser1177 but inhibits the eNOS phosphorylation at Thr 495 and decreases the Cav-1 content, ultimately resulting in enhanced NO production. This effect was antagonized by the PI3K/Akt signaling pathway inhibitor LY294002. The inhibitor used and its location of action is also indicated.

phosphorylation at the eNOS Ser 1177, which is involved in the basic activation of eNOS and agonist-induced activation (Fulton et al., 1999; Bauer et al., 2003). It is mainly present in the cytoplasm in an inactive form and must translocate to the cell membrane to activate itself and phosphorylate eNOS (Gonzalez et al., 2002). In addition, it is directly controlled by the PI3Kdependent phosphorylation pathway, and PI3K recruits Akt to the cell membrane, thereby phosphorylating it (Vanhaesebroeck et al., 1997). To investigate the mechanism by which SFI regulates eNOS activity, we used EA.hy926, a fusion of primary cultured human umbilical vein cells with thioguanine-resistant A549 clone under PEG stress, to detect the gene and protein expression of eNOS, the protein expression of Cav-1, total eNOS, and the phosphorylation of eNOS at Ser1177 and Thr495. The results showed that SFI significantly up-regulated the gene and protein expression of eNOS, decreased the Cav-1 content in the cell supernatant, and promoted the phosphorylation of eNOS at Ser 1177 but inhibited the phosphorylation of eNOS at Thr495, thereby enhancing eNOS activity. This effect was antagonized by the PI3K/Akt signaling pathway inhibitor LY294002, which suggested that SFI may promote the phosphorylation of eNOS through the PI3K/Akt signaling pathway (Figure 7).

Although this study preliminarily revealed that the mechanism behind SFI's endothelium-dependent vasodilatation is that it enhances eNOS activity through the PI3K/Akt signaling pathway in vitro, the mechanism of eNOS activation is extremely complex and can be divided into gene regulation (regulation of promoter and mRNA stability) and protein regulation (eNOS intracellular translocation, eNOS complex formation, and the phosphorylation of eNOS amino acid residue) (Dias et al., 2011; Zhu et al., 2015). Various regulatory mechanisms are interconnected to form a complex network structure. Therefore, more comprehensive and systematic research on other regulatory mechanisms is needed in the future to fully elucidate the mechanism by which SFI regulates eNOS activity.

It is well-known that vasomotor function is not only related to vascular endothelium but also requires co-regulation of vascular smooth muscle (Dias et al., 2011). Therefore, it is necessary to explore the effect of SFI on signal transduction between VECs and VSMCs in the future to clarify its mechanism of diastolic vasodilation further.

#### CONCLUSION

Our study revealed for the first time that endothelium-dependent vasodilatation effect of SFI occurs through the NO-cGMP

#### REFERENCES

pathway but not the PGI2 pathway. Moreover, we found that the vasorelaxation effect of SFI is mediated through the PI3K/ Akt/eNOS/NO signaling pathway in VECs. Overall, this study provides a scientific basis for the treatment of shock and HF with SFI and lays a foundation for the expansion of its clinical application and secondary development.

#### DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/ Supplementary Material.

#### ETHICS STATEMENT

This study was carried out in strict accordance with the recommendations in the Guidance Suggestions for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of China. The protocol was approved by the Laboratory Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine (Permit Number: LAEC2013002).

#### AUTHOR CONTRIBUTIONS

JZ and BW carried out the experiments. JZ, WS, SX, YM, and HW performed analysis and interpretation of the data. JZ and SH designed the experiments. JZ wrote and revised the manuscript. All of the authors gave final approval to the work.

#### FUNDING

We are grateful for the financial support from the National Natural Science Foundation of China (No. 81973626, 81473381) and the Training Project of Tianjin Institution "Innovation Team" (TD13-5049).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2020. 00121/full#supplementary-material

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synthase activation and nitric oxide synthesis in cardiovascular system. Chin. J. Integr. Med, 1–9. doi: 10.1007/s11655-015-1964-1

Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Zhu, Song, Xu, Ma, Wei, Wang and Hua. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Phytochemical Profile, Pharmacological Attributes and Medicinal Properties of Convolvulus prostratus – A Cognitive Enhancer Herb for the Management of Neurodegenerative Etiologies

#### Edited by:

Valentina Echeverria Moran, Bay Pines VA Healthcare System, United States

#### Reviewed by:

Sefirin Djiogue, University of Yaounde I, Cameroon Vivekananda Mandal, Guru Ghasidas Vishwavidyalaya, India

> \*Correspondence: Anurag Varshney<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology

Received: 16 September 2019 Accepted: 07 February 2020 Published: 03 March 2020

#### Citation:

Balkrishna A, Thakur P and Varshney A (2020) Phytochemical Profile, Pharmacological Attributes and Medicinal Properties of Convolvulus prostratus – A Cognitive Enhancer Herb for the Management of Neurodegenerative Etiologies. Front. Pharmacol. 11:171. doi: 10.3389/fphar.2020.00171 Acharya Balkrishna1,2, Pallavi Thakur 1 and Anurag Varshney 1,2\*

1 Drug Discovery and Development Division, Patanjali Research Institute, Haridwar, India, 2 Department of Allied and Applied Sciences, University of Patanjali, Haridwar, India

Convolvulus prostratus Forssk., a nootropic herb used in traditional medicinal systems, is also frequently known by its taxonomic synonym Convolvulus pluricaulis. In Indian medicinal system – Ayurveda – it is named as Shankhpushpi. According to the ancient literature, this herb has been attributed with several therapeutic properties, such as anxiolytic, neuroprotective, antioxidant, analgesic, immunomodulatory, antimicrobial, antidiabetic and cardioprotective activities. This medicinal herb has been reported to contain many bioactive phytoconstituents, such as, alkaloid (convolamine), flavonoid (kaempferol) and phenolics (scopoletin, b-sitosterol and ceryl alcohol), that have been ascribed to the observed medicinal properties. Several research teams across the globe have highlighted the neuro-pharmacological profile of C. prostratus, wherein, the neuroprotective, nootropic and neuro-modulatory roles have been described. Besides, role of C. prostratus extracts in neurodegeneration has been well demonstrated. Despite of such elaborative preclinical pharmacological profile, detailed clinical investigations and mechanistic mode-of-action studies of this important herb are yet to be executed. The present review is attempted to showcase the phytochemical profile, pharmacological attributes and medicinal information of C. prostratus; with comprehensive research gap analysis. It is hoped that the scientific update on the ethnomedicinal aspects of this herb would thrive research propagation and development of the CNS phytopharmaceuticals, originated from C. prostratus.

Keywords: Convolvulus prostratus, Shankhpushpi, natural product, Ayurveda, neuroprotective, nootropic

## INTRODUCTION

In recent years, non-communicable diseases (NCDs) have become an emerging cause of morbidity and mortality (~ 12% global prevalence) (Islam et al., 2014). Neurological disorders constitute a significant proportion as the leading causes of death among all the other non-communicable diseases (Gourie-Devi, 2014). The most common disorders of the nervous system are schizophrenia (~ 40% prevalence in India) and epilepsy (~ 45% prevalence in India), which are clinically presented by dysfunction of the interneurons, misbalancing neuronal homeostasis, ultimately leading to neurophysiological disintegration (Gururaj et al., 2005). It is estimated that by next decade, mental and behavioural disorders will lead to a reduction in the average life expectancy, nearly by one-fifth proportion (Lopez and Murray, 1998). In India, an average of 65 people out of 1,000 inhabitants suffer from mental and behavioural disorders, wherein, maximum prevalence has been recorded for alcohol dependency afflicted mental disorders (~ 6% prevalence), child and adolescent behavioural disorders (~ 4.3% prevalence) and mood disorders (~ 1.6% prevalence). These statistics makes it obvious that the prevalence of neurodegenerative disorders is escalating, however, effective and safe treatment modalities are still under infancy (Gururaj et al., 2005).

The chemotherapeutic moieties used currently for the treatment of neurological disorders face a daunting challenge of systemic delivery of the drugs to the central nervous system. Moreover, crossing the blood brain barrier after strenuous systemic administration is another challenge. Ultimately, the bioavailability of these drugs becomes low and therefore, lead to below- optimal efficacies (Tonda-Turo et al., 2018). Several drug delivery vehicles and nano-formulations have been devised so as to facilitate the targeted delivery of the drug to the site of action, i.e., the central nervous system, thereby bypassing the blood brain barrier (Saraiva et al., 2016; Tonda-Turo et al., 2018). However, these strategies have also shown little success. Most of the neurodegenerative disorders are progressive in nature, and the cycle of etiological events usually have an early-onset which might get unnoticed or under-detected. In such a case, the therapies initiated after the onset of neuropathological asymptomatic etiologies will only have limited value for the patients (Solanki et al., 2016). Moreover, the foresaid neuropathological conditions are manifested with several additional etiologies, wherein the chemotherapeutic modalities act typically on a single target, thereby providing only palliative care (Chen and Pan, 2014). Hence, natural compounds serve as the holistic option which act on multiple neural targets and are enriched with free radical scavenging polyphenolic compounds (Park et al., 2018). These phytoconstituents have well-described antioxidant and anti-inflammatory properties which aids in protecting the neuronal cells against oxidative stress. Additionally, the natural compounds, such as flavonoids also aid in modulating the neuronal cell signalling pathways (Solanki et al., 2016).

There are various herbs which are used in traditional medicine for the management of neurodegenerative diseases, for instance: Centella asiatica (L.) Urb., Glycyrrhiza glabra L., Tinospora cordifolia (Willd.) Hook. f. & Thomson, Bacopa monnieri (L.) Wettst. and Nardostachys jatamansi (D. Don) DC. (Kulkarni et al., 2012). One such example is Convolvulus prostratus Forssk. (Syn. Convolvulus pluricaulis Choisy), which is commonly known as Shankhpushpi in Ayurveda and is widely recognized for its anxiolytic, antidepressant and nootropic activities (Kulkarni et al., 2012). As per an ancient Indian medicinal scripture—Charaka Samhita—this plant is superior to other nootropic drugs (Medhya rasayana) of Ayurveda, however, a detailed ethnomedicinal update yet needs to be presented (Dev, 2006). The ensuing sections will provide an ethnomedicinal, phytochemical and pharmacological update of this cognitive booster herb, C. prostratus (CP).

### ETHNOMEDICINAL UPDATE ON C. prostratus

C. prostratus belongs to Convolvulaceae family and is ubiquitous in the north-western regions of India (Nisar et al., 2012). In Ayurveda, this herb is classically described as a memory and intellect booster. Moreover, it is employed in a variety of formulations used for the treatment of nervous disorders, such as insanity, epilepsy, hysteria, insomnia, and psycho-neurosis (Khare, 2004). Mechanistically, it reduces the spontaneous motor activity, thereby controlling the refluxes and frightening responses. It ultimately acts as a sedative moiety which initiates a persistent fall in blood pressure and cardiac contraction, thereby managing neurological pathologies, such as anxiety, insanity and epilepsy (Chaudhary, 1996). The neuromechanistic aspects of this herb has been elucidated in Figure 1A. Besides, this plant has manifold therapeutic utilities, wherein, a decoction of its shoots is used as a remedy for anaemia and weakness (Singh et al., 2003). More specifically, in ancient texts, this plant has been mentioned as sara, medhya, vrsya and rasayana, which refers to the laxative, nootropic, aphrodisiac and rejuvenator properties of this herb, respectively. Additionally, one of the revered ancient Indian medical practitioner, Acharya Charaka had used whiteflowered variety of C. prostratus (Shankhpushpi) along with the juice of Bacopa monnieri (Brahmi), Acorus calamus (Vacha) and Saussurea lappa (Kushtha) for alleviating insanity and epilepsy. Similar views had been presented in Chikitsasangraha written by Chakradatta; Kaideva Nighantu; and Ayurveda Saar Sangraha (Khare, 2004).

Besides Ayurveda, C. prostratus (CP) has also been used in Siddha system of medicine, wherein an oil obtained from this plant is used as a keratogenic agent for promoting hair growth (Gogte, 2012). It is also believed that a paste prepared from its roots and flowers act as anti-aging agents, thereby indicating its apparent anti-oxidant activity (Adams et al., 2007). Furthermore, in Unani medicinal system, a syrup prepared with C. prostratus and Piper nigrum is prescribed in bleeding piles and venereal diseases (Khare, 2004). All the above mentioned ethnomedicinal uses of C. prostratus have been tabulated in Table 1.

FIGURE 1 | (A) Neuro-mechanistic aspects of Convolvulus prostratus (CP). Schematic representations of C. prostratus (CP) target and mode of action in neural system. CP reduces the lesions formed in the hippocampus (A); and amyloid plaque accumulation in hippocampus (A), cortex (P), basal ganglia (M) and cerebellum (E). CP also acts as GABA-A agonists and binds to lateral prefrontal cortex (O) and anterior cingulate cortex (N). CP deactivates the wake promoting areas, namely, tuberomamillary nucleus (J) located in the hypothalamus (K), locus coeruleus (I) and raphe nucleus (H). It also activates the sleep inducing areas of the brain, namely, preoptic nucleus (L). Moreover, CP activates the hypoactive regions situated in the prefrontal cortex (shaded pink area; O), and deactivates hyperactive regions situated in the anterior cingulate cortex (shaded green area; N). CP blocks the excessive production of dopamine as produced via substantia nigra (D). CP also removes the excessive calcification formed at the cranio-cervical junction (F). Additionally, CP removes the formation of thrombus in the carotid artery (G). (B) Main mechanisms and phytoconstituents responsible for neuro-pharmacological activities of C. prostratus (CP). This herb exhibits anti-convulsant, anti-depressant, anxiolytic, sedative, anti-inflammatory, anti-oxidant, analgesic, nootropic, spasmolytic and neuroprotective activities. Phyto-constituents belonging to diverse chemical families, namely, coumarins, alkaloids and polyphenols (see Table 2) are responsible for such evident neuro-pharmacological profile of the CP plant (Nolte, 1999; Carter, 2014). Reported mechanisms of CNS action of CP phyto-constituents has been depicted in these schematics, with putative site of action has been marked with fluorescent green blobs.

## INDUSTRIAL SIGNIFICANCE OF C. prostratus

C. prostratus is extensively used in pharmaceutical, cosmeceutical and nutraceutical industries (Jalwal et al., 2016). In the pharmaceutical industry, various extracts, syrups and tablets are produced, speci fi cally for targeting neurodegenerative diseases, hypertension, hypercholesterolemia and gastric ulcers. A few examples of such marketed herbal pharmaceutical formulations include Patanjali Divya Shankhpushpi Churna™, Divya Pharmacy Shankhpushpi Sharbat™, Baidyanath Shankhpushpi Sharbat™, Dabur Shankhpushpi Syrup™, Biotrex Shankhpushpi™, Maxmind capsule™, Herbal Hills Shankhpushpi Tablets™ and many more (Bhowmik et al., 2012). Similarly, in the cosmeceutical industry, the CP herb is used as a general tonic for rejuvenating

#### TABLE 1 | Ethnomedicinal uses of Convolvulus prostratus (CP) with predominant plant parts and mode of herbal preparation.

† Shaded rows indicate specific neurological diseases which can be managed by the administration of C. prostratus.

the skin and hairs, thereby treating skin related ailments as well as keratogenic disorders. A few examples for elucidating the use of CP (Shankhpushpi) as cosmeceutical ingredients include Econature Shankhpushpi Hair oil™, Khadi Natural Shankhpushpi oil™ and Alps Shankhpushpi hair Mask Powder™. Moreover, the CP powder and juice, such as, Jain Shankhpushpi Powder™ and Axiom Jeevan Ras Shankhpushpi Juice™, are also being used as skin mask for rejuvenating the skin and managing skin problems such as acne, blemishes and sun spots (Hindu, 2012). Additionally, food grade CP powder and syrups are also available in the market for being used as a nutraceutical nootropic supplement, for example, Divya Pharmacy Shankhpushpi Sharbat™, Baidyanath Shankhpushpi Sharbat™, Shivalik Herbals Shankhpushpi Nutraceutical Capsules™ and Veg E Wagon Shankhpushpi Powder (Bhowmik et al., 2012). Such extensive industrial uses of CP further confirms the holistic significance of this nontoxic wonder herb (Jalwal et al., 2016).

#### PHYTOMEDICINAL FORMULATIONS CONTAINING C. prostratus

The CP herb has also been used as a phyto-ingredient of a polyherbal medicinal formulation: Sankhahauli, which contains leaves of C. prostratus (15 g); seeds of Piper nigrum (3 g) and Papaver somniferum (20 g); whole plant of Prunus amygdalus (10 g), Vitis vinifera (20 g) and Coriandrum sativum (10 g). This formulation is mainly used for the management of insomnia, drug addiction and hypertension (Bhutya, 2011). Several such marketed herbal formulations containing CP are being used for the management of a variety of neurological ailments in India, for example., Divya Medha Vati, Divya Medha Kwath (Patanjali Ayurved Ltd.); BR-16A (Himalaya Drug Co. Ltd.); Dimagheen (Dawakhana Tibiya College); Shankhpushpi syrup (Unjha); Shankhavali Churna (Narnaryan Pharmacy); Brain tab and Shankhpushpi syrup (Baidyanath Pharmaceuticals) (Aggarwal et al., 2011; Sethiya et al., 2013; Amin et al., 2014).

## CHEMICAL PROFILE OF C. prostratus

All of these stated medicinal utilities of C. prostratus (CP) have been attributed to various phytoconstituents, belonging to the chemical family of alkaloids, flavonoids, coumarins and polyphenols. Among these phytoconstituents, certain compounds are known to be present at a higher concentration (almost 20% w/w) and are known as major phyto-constituents. CP plant is known to contain kaempferol, b-sitosterol, Nhexacosanol, taraxerol, taraxerone, delphinidine and hydroxycinnamic acid as the major phyto-constituents, as depicted in Table 2 (Billore et al., 2005; Amin et al., 2014). Moreover, an alkaloid, namely, Sankhpuspine has also been isolated from this plant and is known as a chemotaxonomic marker for this species (Basu and Dandiya, 1948; Saroya and Singh, 2018).

Several other hydrocarbons, namely, 1- pentyl-2-tridecanyl cyclopentyl cyclohexane carboxylate, 1,2-benzenedicarboxylic acid, 10-bromodecanoic acid, 1-octadecanesulphonyl chloride, 2-butanone, 2-pentanol, 7- hydroxyheptadecanyl-1,7, 17 tricarboxylic acid, ascorbic acid, cyclononasiloxane, cyclooctadecanyl methanol, decanoic acid, dicyclohexyl cyclo-octyl acetic acid, eicosane, heneicosane, hydroxy cinnamic acid, octatriacontyl pentafluoropropionate, pentadecyl 2-propyl ester, pentanoic acid, pentyl hexacosanoate, phthalic acid, silane, squalene, tetracyclohexanyl caproate and tridecane are also found in the extract of the CP plant (Bhalerao et al., 2014; Malik et al., 2016; Rachitha et al., 2018; Sultana et al., 2018).

In addition, CP is known to be a good source of vitamins and minerals, namely, calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sulphur, zinc, vitamin C and E (Sethiya et al., 2010; Babu et al., 2015).

## NEURO-PHARMACOLOGICAL PROFILE OF C. prostratus

#### Nootropic Activity

C. prostratus (CP) contains volatile oil; fatty alcohols; flavonoids, i.e. kaempferol; hydroxy cinnamic acid; b-sitosterol; and carbohydrates such as glucose, rhamnose, sucrose etc., which endow this plant with nootropic capabilities. Moreover, an alkaloid, namely, convolvine present in this herb has also been found to block cholinergic muscarinic receptors: M2 and M4. Convolvine also aids in potentiating the effect of another muscarinic memory enhancer, namely, arecoline, thereby imparting nootropic abilities to CP (Sethiya et al., 2009). In a study, Rawat and Kothiyal have also found that the aquomethanolic, ethanolic and petroleum ether extracts isolated from CP (50–400 mg/Kg) exhibited anxiolytic, memoryenhancing and nootropic activity as evaluated by using Elevated Plus Maze (EPM) and step-down models in mice. EPM test has mainly been used to investigate the interactions between aversive memory and anxiety responses of the mice. CP effects on EPM activity has been found to be comparable to the standard of care drug, Piracetam (Rawat and Kothiyal, 2011; Kaushik, 2017). Moreover, treatment with alcoholic extract of CP plant led to an increase in the average time-span spent by mice in the enclosed arm of plus maze model, and an escalation in the mean avoidance response on the jumping box model (Rawat and Kothiyal, 2011).

### Neuroprotective Activity

The aqueous extract of the roots of C. prostratus inhibited the activity of acetylcholinesterase (AChE) within the cortex and hippocampus of male Wistar rats, that have been intoxicated with scopolamine. CP extract also posed evident anti-oxidant activity and elevated the levels of glutathione reductase, superoxide dismutase and reduced glutathione within the cortex and hippocampus (Kaushik, 2017). Similar results have been observed in case of aluminium chloride induced neurotoxicity in rat cerebral cortex. Regular administration of the CP root extracts (150 mg/Kg) for 3 months inhibited the decline in Na+ /K+ ATPase activity and also preserved the mRNA expression levels of muscarinic acetylcholine receptor 1 (M1 receptor), choline acetyl transferase (ChAT) and nerve growth factor-tyrosine kinase A receptor (NGF-TrkA) (Bihaqi et al., 2009; Kaushik, 2017). The Na+ /K+ ATPase pump aids in maintaining the osmotic equilibrium and membrane potential in neuronal cells (Forrest, 2014). Secondly, the muscarinic receptors bind to the neurotransmitter acetylcholine, thereby facilitating the transmission of electrical signals within the central nervous system (Brown, 2018). Furthermore, the choline acetyl transferase enzyme is essential for the synthesis of neurotransmitter acetylcholine; and the nerve growth factortyrosine kinase A receptor is necessary for binding of the neuronal trophic factors, thereby ensuring the survival of the neurons (Johnson et al., 2018; Indo, 2018).

In another experiments, it has been shown that the oral administration of the aqueous extract of the CP roots (150 mg/

† Source of Chemical structures: PubChem (https://pubchem.ncbi.nlm.nih.gov/).

Kg) to scopolamine induced rats causes a marked reduction in the mRNA levels of tau protein (Bihaqi et al., 2012). Such reduction in the tau protein expression is responsible for causing an amelioration in the amyloid b-induced deficits in case of neurodegenerative disorders such as Alzheimer's disease (Vossel et al., 2010). Hence, the presence of phytoconstituents such as convolvine might be responsible for endowing CP with the abilities to regulate all the neuronal factors/proteins and enzymes, thereby showcasing its evident neuroprotective status (Sethiya et al., 2009; Ray and Ray, 2015).

#### Anxiolytic Activity

The ethanolic and chloroform extracts isolated from the aerial parts of CP showed significant anxiolytic activity as recorded using elevated plus maze test on experimental mice. There was increase in the time spent in open arms; and in the number of open arm entries upon the CP oral administration to mice at a dose of 200 mg/Kg (Bhalerao et al., 2014). Similar results have also been observed in case of the ethanolic extract of the CP flower petals at doses 200–400 mg/Kg in mice (Kaushik, 2017).

In another study, CP methanolic extract was evaluated for anxiolytic activity on Obsessive Compulsive Disorders (OCDs) in mice by employing marble burying behaviour analysis, hole board and rota-rod tests. The results have shown that the mice group treated with 200–400 mg/Kg CP methanolic extracts can modulate serotonin or dopaminergic levels, thereby harmonizing the major pathway involving serotonergic or dopaminergic receptors manifesting obsessive compulsive disorders (Subramani et al., 2013). Such anxiolytic activity can be linked with the hypotensive effect of this herb, which in turn is attributed to the presence of GABA-A-benzodiazepine agonists, such as convolamine and scopoletin (Figure 1B) (Malik et al., 2011; Amin et al., 2014; Siddiqui et al., 2014).

#### Anti-Convulsant Activity

The chloroform, ethanol and aqueous extracts of CP have been evaluated for anti-convulsant activity against strychnine induced as well as pentylene tetrazol (PTZ) induced convulsive seizures in different animal models. Five hundred mg/Kg concentration of the CP extracts have shown statistically significant (p < 0.001) protection against strychnine and PTZ induced clonic convulsions (Ratha and Mishra, 2012; Siddiqui et al., 2014). Methanolic extract of this plant (500–1000 mg/Kg) also exhibited anti-convulsant activity, as characterized by reduction in the mean recovery time of convulsions in case of maximal electroshock seizure model in mice (Kaushik, 2017).

The fundamental mechanism behind such evident anticonvulsing activity of CP might be the presence of coumarins and triterpenoids (Quintans Júnior et al., 2008). Moreover, it has also been proposed that the anti-convulsant activity of a phytomedicine is escalated by the presence of certain functional groups like, quinoline, quinazoline, thiazole, benzothiazines, oxadiazole, pyridine, pyrazole, imidazole, pyrimidine, phthalazine, triazine, triazoles, cyclopropane carboxylate, and oxime ether (Wei et al., 2015; Song and Deng, 2018). Indeed, many such functional groups have been found in the CP phyto-constituents (Table 2) for e.g., quinones, microphyllic acid, 6-methoxy coumarin, 4' methoxy kaempferol, 7-methoxy quercetin, vanillic acid, syringic acid and melilotic acid (Daniel, 2016).

#### Anti-Depressant Activity

Bhalerao and co-workers have found that the chloroform fraction isolated from the CP ethanolic extract reversed the reserpine-induced extension of immobility period of mice in Forced Swim Test (FST), and elicited a significant antidepressant effect by interaction with adrenergic, dopaminergic and serotonergic systems (Bhalerao et al., 2014). Similarly, a polyherbal formulation (Trans-01) containing C. prostratus (30%), Valeriana wallichii (45%), Plumbago zeylanica (7.5%), Boswellia serrata (15%) and Acorus calamus (3.5%) also exhibited similar anti-depressant properties as tested by employing the forced swim test (FST), tail suspension test (TST) and forced swimming stress (FSS)-induced alterations in serum corticosterone levels. In TST and FST, Trans-01 showed a dose-dependent decrease in immobility time. Moreover, Trans-01 significantly attenuated the elevated corticosteroid levels, thereby indicating a significant anti-depressant activity of this formulation (Shalam et al., 2007). CP herb is known to contain alkaloids (convolamine and scopoletin), flavonoids (kaempferol), and steroids (phytosterol and b-sitosterol). These phytoconstituents most probably act as GABA-Abenzodiazepine agonists and bind to the GABA-Abenzodiazepine receptors, thereby causing an increase in the chloride ion flux and consequent hyperpolarization of the postsynaptic membrane. Such hyperpolarization leads to a hypnotic effect and may alleviate depression (Siddiqui et al., 2014).

## Anti-Inflammatory Activity

Hydroxy-cinnamic acid is a phenyl-propanoid compound found in CP. It is known to cause a downregulation in the expression of cytokine mediators such as IL-8, MCP-1 and ICAM-1, thereby blocking the expression of cytokine-mediated adhesion molecules and therefore the fundamental process of leukocyte– endothelial cell adhesion is deterred (Billore et al., 2005; Rathee et al., 2009). Hence, the CP herb may aid in ameliorating the conditions of neuro-inflammation and consequent cognitive impairment. Indeed, oral administration of the ethanolic extract of the CP leaves at dose of 800 mg/Kg, showed significant inhibition of rat paw edema, in the Carrageenaninduced paw edema and Cotton pellet-induced granuloma animal models (Agarwal et al., 2014).

## Anti-Oxidant Activity

The reactive oxygen species are known to deteriorate the cellular physiology of nerve cells and ultimate lead to neurodegenerative disorders. Polyphenols, flavonoids and vitamin E present in the CP plant act as reactive oxygen species (ROS) scavengers and also ameliorate the lipid peroxidation, thereby attributing towards the anti-oxidant activity of CP (Nasri et al., 2015). It has also been observed that the ethyl acetate and methanolic extract of CP have shown appreciable results (IC50 ~ 0.07 mg/mL and 0.075 mg/mL, respectively), as observed in 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Similarly, in Ferric Reducing Antioxidant Power (FRAP) analysis, aqueous CP extract has been found to be potentially active with anti-oxidant capacity of 460 ± 8 ascorbic acid equivalent/mg of the extract (Jain et al., 2011). Interestingly, the Shankhpushpi syrup and its isolated compounds (Scopoletin and Bacoside A) also exhibited evident anti-oxidant activity as evaluated by using DPPH assay with average IC50 value ranging from 0.94 - 2.39% v/v (Jain et al., 2017; Rachitha et al., 2018). Furthermore, the aqueous extract of CP roots diminished the endogenous levels of reactive oxygen species in tauopathy flies as induced by overexpression of t -protein, thereby substantiating its oxidative stress ameliorative effect (Olakkaran and Antony, 2017).

## Analgesic and Spasmolytic Activity

The ethanolic extract of CP at dose 750 mg/Kg showed statistically significant analgesic activity as compared to the standard analgesics like, morphine sulphate, when tested in hot plate method and tail-flick assays in rats (Agarwal et al., 2014). Such evident analgesic activity is cohesively attributed by flavonoids, volatile oils, alkaloids, polyphenols and organic acids by means of prevention of the formation of cyclooxygenase enzyme and prostaglandins, i.e., mediators of pain sensitization. Hence, these CP phyto-constituents ultimately aid in ameliorating the neuronal pain and headache (Aleebrahim-Dehkordy et al., 2017).

The CP ethanolic extract has exhibited spasmolytic activity in isolated rabbit ileum, isolated rat uterus, intact intestine and tracheal muscles of dog (Barar and Sharma, 1965). Such antispasmodic action is linked to the inhibition of acetylcholine production which is mainly brought about by the specific alkaloid present in this CP herb, convolvine (Amin et al., 2014).

#### Sedative Activity

Interestingly, the ethanolic and aqueous extracts of the CP aerial parts showed statistical significant potentiation of sleeping time in rats induced with thiopental sodium (Siddiqui et al., 2014). In another similar experiment, the aqueous extract of the CP leaves and flowers showed an evident barbiturate hypnosis potentiation in albino rats at a dose of 300 mg/Kg (Mudgal, 1975). Such sedative activity is directly linked to the presence of phytoconstituents like convolamine and scopoletin which act similarly to GABA-A agonists, thereby bringing about the effects of sedation (Figure 1B) (Siddiqui et al., 2014).

This CP herb has also been reported for several other pharmacological activities, including anti-diabetic, antihyperlipidemic, anti-hypertensive, anti-microbial, anti-platelet aggregation, anti-ulcer, cardio-vascular, hepatoprotective, and hypothyroidism (Barar and Sharma, 1965; Mudgal, 1975; Rizk et al., 1985; Mali, 1995; Jain et al., 2011; Ravichandra et al., 2013; Jalwal et al., 2016). Anti-diabetic activities of this plant might be attributed to the presence of tropane alkaloids which are known as potent inhibitor of a-glucosidases and R-galactosidases (Gaikwad et al., 2014; Rachitha et al., 2018). The polyphenols present in this species act as reactive oxygen species (ROS) quenchers, thereby ameliorating the oxidative stress that is generated as a diabetic manifestation. Additionally, the presence of vitamin E in this herb also aids in controlling the levels of protein oxidation and lipid peroxidation, thereby leading to an escalation in the antioxidant defense system (Cowan, 1999; Nasri et al., 2015; Domínguez-Avila et al., 2016; Foretz et al., 2018). Furthermore, the presence of GABA-A-benzodiazepine agonists, such as convolamine, scopoletin, ceryl alcohol, kaempferol, phytosterol, and b-sitosterol endow this herb with hypotensive and sedative activities (Malik et al., 2011; Siddiqui et al., 2014). More specifically, a compound, namely, 29-oxodotriacontanol, isolated from the CP herb has also been assessed to possess antimicrobial and anti-fungal activity (Amin et al., 2014). Certain flavonoids and phenyl-propanoids from CP have been shown to provide anti-platelet aggregation and anti-ulcerogenic activity by means of inhibition of cyclic nucleotide phosphodiesterase enzyme and clot retraction capabilities (Beretz and Cazenave, 1991; Tognolini et al., 2006). The anti-ulcerogenic effect was largely observed due to upregulation of mucosal defensive factors such as mucin and glycoprotein secretion, which in turn was induced by the flavonoids and steroids present in this herb (Srinivas et al., 2013). These flavonoids also pose the profound effects on the thyroid hormone regulation and deiodinase-1 inhibition, thereby endowing this herb with anti-thyroid activity (Nagarathna and Jha, 2013). Additionally, the fundamental principle responsible for the cardio-vascular activity of this herb has been proposed to be linked with it alkaloid derivative, evolvine hydrochloride, which is known to exhibit lobeline-like action on the cardiovascular system. This phytoconstituent acts as a cardiac depressant, ultimately leading to a fall in blood pressure, which gets gradually normalised (Dwoskin and Crooks, 2002; Sethiya et al., 2009).

## Safety Profile of C. prostratus

The ethanolic and aqueous extracts of the CP leaves have been evaluated for acute oral toxicity study in albino Wistar rats. The animals did not show any toxicity or behavioural changes up to the dose of 5,000 mg/Kg (Agarwal et al., 2014). Similarly, the iron oxide nanoparticles of the CP herb exhibited maximum tolerable dose up to 2,000 mg/Kg in Swiss albino mice with no clinical signs of toxicity. The histopathology of brain also did not show any aberrations or degeneration of neurons. Furthermore, no inflammation was observed in the heart and liver (Ravichandra et al., 2013). These toxicological studies, therefore, confirmed that the administration of CP is safe for the vital organs within the respective treatment durations.

#### Summary and Way Forward

The use of herbal medicines continues to escalate rapidly with about 70% of the world population still relying upon traditional medicines for their primary healthcare needs. The natural plant products have negligible toxicities, if any, and are endowed with a multitude of phytoconstituents which are responsible for their holistic therapeutic action. Cognitive dysfunction is one of the major health problem in today's world, wherein the available synthetic chemotherapeutic modalities have proven to be non-absolute and, at times toxic in nature. In such a scenario, safer herbal alternative medicines play a vital role in managing the neurological etiologies. One such cognitive booster herb is C. prostratus Forssk., commonly known as Shankhpushpi, which is mainly endowed with neuroprotective, nootropic and neuro-modulatory activities (Figure 1B). Besides, it also possesses several other therapeutic properties, such as immunomodulatory, antimicrobial, antidiabetic and cardioprotective activities. The fundamental bioactive compounds responsible for the nootropic activities of this herb have been identified as 4'-methoxy kaempferol, 7-methoxy quercetin, convolamine, scopoletin, ceryl alcohol, b-sitosterol and hydroxycinnamic acid. Additionally, this herb did not exhibit any signs of toxicity and neurodegeneration up to a dose of 2000 mg/Kg in mice, thereby indicating its safety profile. A few initial clinical trials have conducted for CP, however, more detailed and controlled clinical trials are needed to establish and validate the neuro-pharmacological profile of C. prostratus. In addition, detailed mechanistic studies are yet to be executed to unravel the underlying mechanism of action for this cognition enhancing herb. Taken together, C. prostratus is likely to be the front runner for the clinical phyto-pharmaceutical status for treatment of neurological ailments.

## AUTHOR CONTRIBUTIONS

AB conceived the presented research. PT analyzed the information, generated the artwork, and co-wrote the manuscript. AV investigated and supervised the findings of the work. AB and AV provided critical revision of this review article, and approved the manuscript for submission. All authors agreed with the final version of this manuscript.

## FUNDING

The presented research work been funded by the research funds of Patanjali Research Foundation Trust (PRFT), Haridwar, India.

#### ACKNOWLEDGMENTS

The authors are grateful to Param Pujya Swami Ramdev Ji for institutional research facilities and supports. Authors gratefully

#### REFERENCES

acknowledge the efforts of Dr. Shivam Singh and Dr. Swami Narsingh Dev at Patanjali Research Institute, for their help in data collection and processing. We are also thankful to Mr. Gagan Kumar and Mr. Lalit Mohan for their swift administrative supports and encouragements.

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Chaudhary, R. D. (1996). Herbal drug industry (Delhi, India: Eastern Publisher).

Spondias mangifera and Terminalia belerica. Eur. J. Pharm. Med. Res. 5, 310– 318. Available at https://pdfs.semanticscholar.org/696f/2e4d7280 727d2baba08bc7c6e3a7cd03ef3a.pdf

Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Balkrishna, Thakur and Varshney. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Effect of Berberine on Atherosclerosis and Gut Microbiota Modulation and Their Correlation in High-Fat Diet-Fed ApoE−/− Mice

Min Wu1† , Shengjie Yang1† , Songzi Wang2 , Yu Cao2 , Ran Zhao1,3, Xinye Li1,3 , Yanwei Xing1 \* and Longtao Liu2 \*

1 Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China, 2 Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing, China, 3 Beijing University of Chinese Medicine, Beijing, China

#### Edited by:

Youhua Xu, Macau University of Science and Technology, Macau

#### Reviewed by:

Bin Geng, Chinese Academy of Medical Sciences, China Yanfei Liu, Beijing University of Chinese Medicine, China

#### \*Correspondence:

Yanwei Xing<EMAIL_ADDRESS>Longtao Liu<EMAIL_ADDRESS>†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology

Received: 30 October 2019 Accepted: 18 February 2020 Published: 13 March 2020

#### Citation:

Wu M, Yang S, Wang S, Cao Y, Zhao R, Li X, Xing Y and Liu L (2020) Effect of Berberine on Atherosclerosis and Gut Microbiota Modulation and Their Correlation in High-Fat Diet-Fed ApoE−/− Mice. Front. Pharmacol. 11:223. doi: 10.3389/fphar.2020.00223 Atherosclerosis and its associated cardiovascular diseases (CVDs) are serious threats to human health and have been reported to be associated with the gut microbiota. Recently, the role of berberine (BBR) in atherosclerosis and gut microbiota has begun to be appreciated. The purposes of this study were to observe the effects of high or low doses of BBR on atherosclerosis and gut microbiota modulation, and to explore their correlation in ApoE−/− mice fed a high-fat diet. A significant decrease in atherosclerotic lesions was observed after treatment with BBR, with the effect of the high dose being more obvious. Both BBR treatments significantly reduced total cholesterol, APOB100, and very low-density lipoprotein cholesterol levels but levels of high/lowdensity lipoprotein cholesterol and lipoprotein (a) were only reduced by high-dose BBR. Decreased pro-inflammatory cytokines tumor necrosis factor-alpha, interleukin (IL)-1β, IL-6 and increased anti-inflammatory IL-10 and adiponectin levels were observed in the high-dose BBR group, but no decrease in IL-6 or increase in IL-10 was evident using the low-dose of BBR. 16S rRNA sequencing showed that BBR significantly altered the community compositional structure of gut microbiota. Specifically, BBR enriched the abundance of Roseburia, Blautia, Allobaculum, Alistipes, and Turicibacter, and changed the abundance of Bilophila. These microbiota displayed good anti-inflammatory effects related to the production of short-chain fatty acids (SCFAs) and were related to glucolipid metabolism. Alistipes and Roseburia were significantly enriched in high-dose BBR group while Blautia and Allobaculum were more enriched in low-dose, and Turicibacter was enriched in both BBR doses. Metagenomic analysis further showed an elevated potential for lipid and glycan metabolism and synthesis of SCFAs, as well as reduced potential of TMAO production after BBR treatment. The findings demonstrate that both high and low-dose BBR can improve serum lipid and systemic inflammation levels, and alleviate atherosclerosis induced by high-fat diet in ApoE−/− mice. The effects are more pronounced for the high dose. This anti-atherosclerotic effect of BBR may be partly attributed to changes in composition and functions of gut microbiota which may be associated with anti-inflammatory and metabolism of glucose and lipid. Notably, gut microbiota alterations showed different sensitivity to BBR dose.

Keywords: berberine, atherosclerosis, gut microbiota, inflammation, lipid metabolism

## INTRODUCTION

fphar-11-00223 March 12, 2020 Time: 18:56 # 2

Atherosclerosis is the common pathological basis of many cardiovascular diseases (CVDs). Although lipid-lowering drugs, interventional therapy, and other conventional treatments have been widely used to reduce the threats to human health, atherosclerosis and its associated CVDs are still the leading cause of death worldwide (Anderson et al., 1991; Vilahur et al., 2014). Atherosclerosis is accompanied by dyslipidemias, including elevated levels of low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), triglyceride (TG), etc. (Ju et al., 2018). Statins are the first-line pharmacotherapy for dyslipidemias and effectively control lipid levels and reduce major adverse cardiovascular events (MACE) (Catapano et al., 2016; Kobiyama and Ley, 2018). However, statins have been reported to be correlated with various adverse events related to myopathy, renal disease, hepatobiliary disorders, and other events (Law and Rudnicka, 2006). In addition, atherosclerosis is considered to be a chronic inflammatory disease of the large and medium arteries (Ross, 1999; Libby, 2002; Hansson, 2005), with changes in proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α),interleukin (IL)-6, and IL-1β, and anti-inflammatory cytokines including IL-10 (Tedgui and Mallat, 2006). The role of these cytokines in the development, progression, and complications of atherosclerosis has been reviewed (Tedgui and Mallat, 2006).

Studies in the past decade have shown that the gut microbiota is associated with certain human diseases, including obesity (Boulange et al., 2016), type 2 diabetes (Qin et al., 2012), hypercholesterolemia (Martinez et al., 2009) and CVDs (Tang et al., 2017), all of which are associated with atherosclerosis (Jonsson and Backhed, 2017). The role of the gut microbiota in atherosclerosis has begun to be appreciated in recent years, mainly including the regulation of inflammation and immunity, and cholesterol and lipid metabolism by gut microbiota and its metabolites (Jonsson and Backhed, 2017; Ma and Li, 2018). These findings have highlighted the great potential preventative and therapeutic benefits for atherosclerosis that might be realized by targeting the gut microbiome.

Berberine (BBR) is an isoquinoline alkaloid that can be isolated from various medicinal plants, such as Coptis chinensis Franch and Cortex phellodendri. BBR has various biological functions including anti-inflammatory activity, improvement of cholesterol levels and lipid metabolism, and prevention of metabolic diseases. These functions are also very important in anti-atherosclerosis effect. Nutraceuticals containing BBR can significantly reduce plasma LDL-C levels of elderly statinintolerant hypercholesterolemic patients (Marazzi et al., 2011). Another study showed that when it plus low-dose statins and/or ezetimibe, most coronary artery disease (CHD) patients who were high-dose statin intolerant could achieve target LDL-C levels within 3 to 6 months (Marazzi et al., 2019).Of note, the therapeutic effects of BBR on these inflammatory and metabolic diseases, such as emergent mild diarrhea (Yue et al., 2019), ulcerative colitis (Cui et al., 2018), obesity (Xie et al., 2011; Zhang et al., 2015), hyperlipidemia (Wang et al., 2017) and diabetes (Zhang et al., 2012), appeared to be related to the regulation of the gut microbiota.

Previous studies have indicated that the anti-atherosclerotic effect of BBR may be associated with changes in the gut microbiota, specifically the increased abundance of Akkermansia, altered abundance of Firmicutes and Verrucomicrobia, and decreased serum trimethylamine N-oxide (TMAO) levels (Zhu et al., 2018; Shi et al., 2018). However, the specific linking of the role of BBR in gut microbiota with atherosclerosis and the potential mechanism are still unknown. Therefore, we investigated the correlation of the effect of BBR (high and low doses) on gut microbiota with the alleviation of atherosclerosis, including improvements in plaque area, serum lipid levels, and systemic inflammation. The results of 16S rRNA sequencing and metagenomic analysis showed that the anti-atherosclerotic effect of high and low-dose BBR may be partly attributed to the changes in composition and functions of the gut microbiota, which may be related to anti-inflammatory and the metabolism of glucose and lipid.

## MATERIALS AND METHODS

#### Animal Model

All animal experimental procedures conformed to the ARRIVE guidelines, the United States National Institutes of Health Laboratory Animals Care and Use guidelines, and the Peking University Animal Investigation Committee guidelines. Sixweek-old male ApoE−/− mice and C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME, United States) and were raised at the animal laboratory center of Peking University. The mice were housed in a specific pathogenfree environment with a room temperature at 22–24◦C, relative humidity of 50%, 12 h light/dark cycle, and freely acquirable food and water. The ApoE−/− mice were fed with HFD containing 21% saturated fat and 0.15% cholesterol (Beijing Keao Xieli Feed Co., Ltd., Beijing, China) for 13 weeks. C57BL/6J mice (n = 12) were fed a normal-chow diet as the Control group. Mice fed with HFD were randomly divided into three groups (n = 12 in each group). The Model group was treated with 0.9% sterile saline by gavage once daily. The High group was given 100 mg/kg body weight BBR hydrochloride (97% purity, Xi'an Platt Biological Engineering Co., Ltd., Xi'an, China) by gavage once daily. The Low group was given 50 mg/kg body weight BBR by gavage once daily. Berberine were administrated for 13 weeks. C57BL/6J mice were administered with 0.9% sterile saline by gavage once daily. The experiment lasted 26 weeks.

#### Plaque Lesion Analysis

Heart tissues containing the aortic arch were excised from the proximal aortic root to the iliac artery branch and placed in a 4% paraformaldehyde environment for 6 h. The atherosclerotic lesion area of aorta was evaluated by en face Oil red O staining as previously described (Shi et al., 2018). Measurement of the lesion size was based on hematoxylin and eosin staining of sections of paraffin embedded aortic root. The area and size of the lesion were analyzed by a blind observer using Image J software.

## Measurement of Serum Lipid Profiles

Blood samples were taken from the retro-orbital plexus after overnight fasting and centrifuged, and the serum was immediately stored at −80◦C. Measurements of the levels of TC, TG, LDL-C, high density lipoprotein cholesterol (HDL-C), very LDL-C (VLDL-C), lipoprotein (a) [Lp (a)], Apolipoprotein B100 (ApoB100) and Apolipoprotein A-1 (APOA-1) were made using enzymatic methods in a model AD2700 automated biochemical analyzer (Olympus, Tokyo, Japan).

### Measurement of Inflammatory Cytokines

The levels of serum inflammatory cytokines, including TNFα, IL-1β, IL-6, IL-10, and adiponectin (ADPN), were measured using ELISA kits (Abcam, Cambridge, United Kingdom) according to the manufacturer's instructions. The level of highsensitivity C-reactive protein (Hs-CRP) was measured by a nephelometry immunoassay.

## Fecal DNA Extraction and PCR Amplification

Total genomic DNA was extracted from fecal samples using Omega Stool DNA Kit (Omega Bio-Tek, Norcross, GA, United States) following the manufacturer's instructions. The extracted fecal DNA was used as template. Amplification of the V3-V4 hypervariable regions of 16S rRNA gene was performed with the forward primer 338F(50 - ACTCCTACGGGAGGCAGCAG-30 ) and reverse primer 806R(50 -GGACTACNNGGG TATCTAAT-30 ). The PCR reaction system (total 25 µL) included 12.5 µL KAPA 2G Robust HotStart Ready Mix (Kapa Biosystems, Wilmington, MA, United States), 1 µL forward and 1 µL reverse primers (5 µM), 5 µL DNA template (30 ng), and 5.5 µL doubledistilled water. The amplification procedure involved an initial denaturation at 95◦C for 5 min, 28 cycles of denaturation at 95◦C for 45 s, 50 s annealing at 55◦C, 45 s elongation at 72◦C, final extension at 72◦C for 10 min, and storage at 4◦C. PCR products were detected by 1% agarose gel electrophoresis to determine the size of the amplified target band and purified using Agencourt AMPure XP kit (Beckman Coulter, Brea, CA, United States). The library quality was assessed on the Qubit Fluorometer (Life Technologies, Carlsbad, CA, United States) and the Bioanalyzer 2100 system (Agilent, Santa Clara, CA, United States). Sequencing was performed on Illumina Miseq PE300 (Illumina Inc., San Diego, CA, United States) at Allwegene Technology Co., Ltd. (Beijing, China). All raw sequences have been submitted to the Sequences Read Archive database at the NCBI under accession number SRP226688.

## High-Throughput Sequencing and Bioinformatics Analysis

USEARCH (Edgar, 2010), QIIME v.1.9.1 (Caporaso et al., 2010) and VSEARCH (Rognes et al., 2016) software were used to process the 16S rRNA gene sequence. Paired-end Illumina reads were quality controlled by FastQC v.0.11.5 (Andrews, 2010), and USEARCH was used to join the paired-end reads and rename. Removal of barcodes and primers, and quality filter were performed and non-redundancy reads were removed. All 16S rRNA chimeric sequences were detected and removed using the UCHIME algorithm against the RDP Gold database (Edgar, 2013). The non-chimeric sequences were sorted by abundance, and clustered into operational taxonomic units (OTUs) using the UNOISE algorithm (100% similarity threshold) with low abundant sequences removed (eight sequences). The OTU table was produced using VSEARCH software and the representative sequence of each OTU was classified by RDP classifier algorithm against the SILVA128 database with a confidence threshold of 70%.

Alpha (α) diversity analysis, including the Shannon index and Chao1 index, were carried out using the vegan (Oksanen et al., 2007) package in R. The beta (β) diversity was evaluated by Principal Coordinate Analysis (PCoA) based on the Bray Curtis distance algorithm. The LEfSe algorithm was used to identify microbes that differed significantly among groups at different taxonomy levels, with a linear discriminant analysis (LDA) score threshold of 4. The Statistical Analysis of Metagenomic Profiles (STAMP) software was used to analyze significant taxa between different groups using Welch's t-test with 0.95 confidence interval permutations (Parks et al., 2014). Differentially abundant OTUs were detected using the EdgeR package and depicted as volcano plots. Representative sequences from most abundant OTUs (relative abundance > 0.002) were selected to make a maximum likelihood phylogenetic tree (ML tree) using IQ-Tree software (Nguyen et al., 2015) with the following parameters: model gtr, 5000 Ultrafast bootstrap and 5000 SH-like approximate likelihood ratio test. Then, the tree was visualized by iTOL web software (Letunic and Bork, 2019) with annotations of relative abundance of groups1 . Potential correlation between gut microbiota and serum levels of lipids and inflammatory cytokines was evaluated based on the Spearman rank correlation. Analysis and plotting were done using the vegan package.

## Library Construction and Metagenomic Sequencing

The qualified DNA extract was broken to an average fragment of 300 bp size approximately with a Covaris S220 (Covaris, Inc., MS, United States) for paired-end library construction through NEBNext Ultra II DNA Library Prep Kit (NEB, Beijing, China). Paired-end sequencing was conducted on Illumina HiSeq4000 (Illumina Inc., San Diego, CA, United States) utilizing HiSeq 4000 Reagent Kits v2 under the manufacturer's instructions.

## Sequence Quality Control and Genome Assembly

SeqPrep2 was used to separate the adapter sequences from the 3 0 and 50 end of paired-end reads. Low-quality reads with insufficient length or quality value were eliminated with Sickle3 . Reads were aligned to the host DNA sequences using the BWA4 , which removed contaminated reads with high alignment

1http://itol.embl.de

2https://github.com/jstjohn/SeqPrep

3https://github.com/najoshi/sickle

4http://bio-bwa.sourceforge.net

similarity. MEGAHIT (Li et al., 2015) was used to assemble the metagenomics data, and contigs which were over 300 bp in length were used as the final assembled result.

## Gene Prediction, Taxonomy, and Functional Annotation

MetaGene (Noguchi et al., 2006) was employed on predicting open reading frames (ORFs) of the assembled contigs. Predicted ORFs which were over 100 bp in length were translated into amino acid sequences according to the translation table of the National Center for Biotechnology Information (NCBI).

All predicted genes were clustered using the CD-HIT (Fu et al., 2012) (parameters: 95% identity, 90% coverage). The representative sequences were selected from the longest sequences in each cluster to construct the non-redundant gene catalog. SOAPaligner (Li et al., 2008) was used to map the reads after quality control to the non-redundant gene catalog with 95% identity, and the gene abundance in the corresponding samples was evaluated.

Non-redundant gene catalog representative sequences were aligned to the NCBI NR database (truncating e-value to 1e−5 ) through BLASTP (Version 2.2.28 +) (Altschul et al., 1997) and further obtained taxonomic annotation. BLASTP against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was performed to determine the KEGG annotation. KOBAS 2.0 was used for functional annotation based on the comparison results.

#### Statistical Analyses

Data are expressed as mean ± SEM. Part of the 16S rRNA analysis was carried out in R software. In addition, differential levels of atherosclerotic plaques, lipids, inflammatory cytokines, and the abundance of potential gut biomarkers at the genus level were tested using one-way analysis of variance (ANOVA), followed by the Sidak multiple comparisons test. The Kruskal-Wallis rank sum test was used to test differential abundance of functional genus and KEGG pathways. A p-value < 0.05 was considered statistically significant. GraphPad Prism 8.0 (GraphPad, La Jolla, CA, United States) was used for the statistical analysis.

### RESULTS

#### BBR Attenuates HFD-Induced Atherosclerosis

Oil Red O and H&E staining showed that the atherosclerotic plaques and lesions in the Model group were higher than those in the Control group (**Figures 1A,B**). However, significant reductions in atherosclerotic lesions after BBR treatment were observed in both the High and Low groups. Quantitative analysis also showed that atherosclerotic plaque area and lesion size decreased significantly in both the High and Low groups compared with the Model group (both p < 0.001). The decrease was more significant in the High group compared to the Low group (p < 0.05 for atherosclerotic plaque area and p < 0.01 for lesion size) (**Figures 1C,D**). These data suggested that HFD feeding markedly promoted the development of atherosclerosis, while the high or low dose of BBR could ameliorate HFDinduced atherosclerosis, with the effect of high dose being more pronounced.

#### BBR Ameliorates Serum Lipid Levels in HFD-Fed ApoE−/− Mice

Model group mice had significantly increased serum levels of TC (p < 0.001), TG (p < 0.05), HDL-C (p < 0.001), LDL-C (p < 0.001), APOB100 (p < 0.001), LP(a) (p < 0.05), and VLDL-C (p < 0.001), and significantly decreased APOA-1 levels (p < 0.001) compared with the Control group mice. In addition, both high and low doses of BBR significantly reduced TC, APOB100, and VLDL-C levels (all p < 0.001), with the decrease of TC (p < 0.001) and APOB100 (p < 0.05) being significantly different between the High and Low groups. TG (p < 0.05), HDL-C (p < 0.001), LDL-C (p < 0.01), and LP(a) (p < 0.05) were only significantly reduced in the High group compared with the Model group. However, there was no significant difference in serum APOA-1 levels after treatment with high or low doses of BBR (p > 0.05) (**Figure 2**).

#### BBR Improves Systemic Inflammation

Atherosclerosis is an inflammatory disease and inflammatory cytokines play an important role in the process of atherosclerosis. Here, we found that compared with the Control group mice, serum levels of TNF-α, IL-1β, IL-6 were significantly increased, and IL-10 and ADPN levels were significantly decreased in HFDinduced atherosclerosis mice (Model group; all p < 0.001). These changes could be reversed by BBR treatment. High dose BBR significantly reduced the levels of TNF-α, IL-1β, and IL-6, and significantly increased levels of IL-10 and ADPN (all p < 0.001). However, in the Low group, only TNF-α and IL-1β levels were decreased and ADPN levels were increased (all p < 0.001), while the decrease in IL-6 and increase in IL-10 levels were not significant (p > 0.05). Moreover, the levels of these inflammatory cytokines were significantly different between the High and Low groups (p < 0.05 or p < 0.001). Serum Hs-CRP level was higher in the Model group than in the Control group (p < 0.05), while there was no significant difference between the other groups (p > 0.05) (**Figure 3**).

#### Effect of BBR on Composition of Gut Microbiota

To determine the effects of BBR on gut microbiota composition, the Illumina Miseq PE300 high-throughput sequencing platform was used to conduct 16S rRNA amplicon paired-end sequencing. After sequence quality control and denoising, a total of 1,027,879 high-quality sequences were obtained from 30 samples. After removal of singleton (> 8) and non-bacteria sequences (such as mitochondria), 1085 OTUs were obtained with an average of 25,801 sequences per sample (range: 21,577-46,467), accounting for 75.3% of the total sequences.

The OTU table was normalized to the minimum number of reads (21,577) per sample and then diversity comparison

was performed. Alpha diversity analysis revealed no significant difference in gut microbiota diversity between each group based on Chao 1 and Shannon indices (p > 0.05) (**Figures 4A,B**). PCoA based on Bray Curtis distance was used to visualize the dissimilarity in the compositions of the bacterial communities. The community compositional structure differed in the four groups, among which the High and Model groups and the Low and Model groups were significantly different (p < 0.001) (**Figure 4C**). A BBR dose-related difference was observed on PC2, in which the High group clustered separately from the Low group, and this explained 14.57% of the total variation in microbial composition. Mice treated with high or low doses of BBR showed similar microbial characteristics on PC1, which explained 22.05% of the total variation (**Figure 4C**).

Next, we investigated the differences of gut microbiota at different taxonomic levels. The compositions of gut microbiota at the phylum and genus levels in each group are shown in **Figures 4D,E**. At the phylum level, Firmicutes (75.4%) and Bacteroidetes (15.1%) were the two most dominant phyla, followed by Proteobacteria (3.3%) and Actinobacteria (2.5%) (**Figure 4D**). However, the abundance of these major phyla did not appear to differ significantly between the groups. One-way ANOVA analysis showed that only Proteobacteria and Actinobacteria had significant differences in abundance between the Control group and the High or Low group (p < 0.05), with decreased Proteobacteria abundance and increased Actinobacteria abundance after BBR treatment (**Figures 4F,G**).

We extracted all the representative sequences from OTUs with relative abundant > 0.2%. In total, 116 bacterial OTUs were selected and all the representative sequences were used to make an ML tree (**Supplementary Figure S1**). There were 116 OTUs annotated at the genus level, with color ranges identifying the phylum to which the OTUs belonged. These OTUs mainly distributed in Firmicutes and Bacteroidetes phylum, and Lachnospiraceae\_NK4A136\_group and unassigned genus. Heatmap was used to annotate the relative abundance of OTUs, and the OTUs with higher abundance were mainly distributed in Allobaculum, Bacillus, Lachnospiraceae\_NK4A136\_group, Blautia, etc. The outermost layer was added with circle annotation at the family level.

At the genus level, LEfSe analysis showed that Turicibacter and Alistipes were enriched in the High group, Allobaculum and Blautia were enriched in the Low group, and the abundance of Bilophila was relatively higher in the Control group than that in other groups (**Figure 5A**). Moreover, STAMP analysis revealed significant differences in Roseburia between the groups. Further analysis of the relative abundance of microbes that differed among groups revealed that the abundance of Turicibacter was higher, and Bilophila and Blautia were lower in the Model group than that in the Control group, but these differences

were not significant (p > 0.05). Both high and low doses of BBR increased the abundance of Turicibacter, and it was more obvious and significant at the high dose (p < 0.05) (**Figure 5B**). The abundance of Alistipes and Roseburia was only significantly enriched in the High group (p < 0.05 and < 0.01, respectively; **Figures 5C,D**). Of note, compared with the Model group, the abundance of both Allobaculum and Blautia was significantly increased when treated with low-dose BBR (both p < 0.05), but there was no significant change after high-dose BBR treatment (p > 0.05) (**Figures 5E,F**). In addition, the abundance of Bilophila decreased in the High group (p < 0.05) and increased in the Low group (p < 0.01) compared with the Model group (**Figure 5G**). These results showed that BBR significantly altered the composition of gut microbiota in HFDinduced atherosclerosis mice, and different doses of BBR showed different influence on specific microbes.

To further analyze these differences more precisely at the OTU level, we analyzed the number of significantly enriched and depleted OTUs in each group (**Supplementary Figure S2**). Compared with the Model group, 80 OTUs were enriched in the Control group, 213 OTUs were enriched in the Low group, and 228 OTUs were enriched in the High group. The number of OTUs with a significant difference increased obviously after BBR treatment, revealing an increasing change.

#### Correlation Between Gut Microbiota and Lipid Profile and Inflammatory Cytokines

As BBR significantly altered the composition of the gut microbiota and improved serum lipid levels and systemic inflammation in HFD-induced atherosclerotic mice, we further analyzed the correlation between gut microbiota and serum levels of lipids and inflammatory cytokines using the Spearman

p < 0.05, ∗∗ p < 0.01; ns, not significant.

rank correlation. We observed a significant negative correlation between serum levels of TC, HDL-C, LDL-C, APOB100 and the abundance of gut microbiota (at the genus level, in the top 25 genera), among which Alistipes showed the most significant negative correlation with lipid levels. Other potential genus-level biomarkers including Roseburia and Bilophila were also negatively correlated (**Figure 6A**). In addition, the antiinflammatory cytokines IL-10 and ADPN displayed a positive correlation with gut microbiota, and pro-inflammatory cytokines IL-1β, IL-6, TNF-α and Hs-CRP were negatively correlated (**Figure 6B**). Notably, the correlation with Alistipes was still very significant.

#### Functional Alterations in Gut Microbiota Induced by BBR

To further investigate the effect of BBR on the functional changes of the gut microbiome in HFD-induced atherosclerotic mice, we determined the extent of different KEGG pathways and genes enriched in each group (three mice per group). LEfSe analysis was performed to detect the KEGG pathways with significantly different abundance between the four groups. At KEGG level 1, environmental information processing and cellular processes were significantly enriched in the model group, while the proportion of sequences related to metabolism was remarkably reduced (LDA > 2.5, p < 0.05, **Figure 7A**). This also supports the view that atherosclerosis is a disease that is closely associated with a variety of metabolic and cellular processes (Jie et al., 2017). At level 2, lipid metabolism, glycan biosynthesis and metabolism and carbohydrate metabolism are important components under the metabolism classification. The samples from the model group displayed reduced potential for lipid metabolism (p < 0.01) and glycan biosynthesis and metabolism (p < 0.001) compared with controls, while these were both increased after treatment with high or low dose BBR (all p < 0.001, **Figures 7B,C**). Differences between the high and low dose BBR groups were not significant. In addition, there was no significant difference in carbohydrate metabolism between groups (p > 0.05). Analysis of the relative contribution of species (at the genus level) to functional attributes found that (**Figure 7D**) Bacteroides, Prevotella and Alistipes were the major contributors to these functions. Consistent with the analysis results of the 16S rRNA sequencing, a lower abundance of Alistipes displayed a decreased functional contribution in the model group compared with the control group, and the functional contribution accordingly increased after high and low dose BBR treatment, with a higher abundance of Alistipes. Other potential genera that could be classified as gut biomarkers included Roseburia, Allobaculum, Blautia, and Bilophila; all of these were in the top thirty enriched genera with important contributions to lipid and glucose metabolism.

Short-chain fatty acids (SCFAs) are important metabolites of gut microbiota (den Besten et al., 2013) and have been reported to be closely related to inflammatory defense and antiatherosclerosis (Ohira et al., 2017; Bultman, 2018). Sequences of SCFA-producing enzymes and associated enzymes identified by reference to the gut microbiota gene catalog were determined as previously described (Claesson et al., 2012; Jie et al., 2017). We screened the metagenomic data of enzymes related to the production of acetate (acetyl-CoA synthetase, K01895), propionate (propionyl-CoA: succinate-CoA transferase, K18118;

propionate CoA transferase, K01026), and butyrate (acetyl-CoA hydrolase, K01067). Butyryl-CoA transferase was not successfully matched. We found that, compared with the model group, acetyl-CoA synthetase (K01895) was significantly enriched in the high and low dose BBR groups (all p < 0.001), indicating increased synthesis potential of acetate. This was more significant in the low dose BBR group than in the high (p < 0.001, **Figure 8A**). However, the abundance of K01026 was reduced in both the high and low dose BBR groups, which exhibited reduced potential for the synthesis of propionate compared with the model group (p < 0.05, **Figure 8B**). Another related gene, K18118, showed no significant difference between the groups (p > 0.05). Butyrate is produced through the condensation of two acetyl-CoA molecules and then reduction to butyryl-CoA, which is subsequently converted into butyrate via the classical pathway (Koh et al., 2016). Acetyl-CoA hydrolase(K01067) was observed to be more enriched in the model group compared with the high-dose (p < 0.05) and low-dose BBR groups (p < 0.001), suggesting a lower potential for butyrate synthesis in the model group (**Figure 8C**). In addition, pyruvate-formate lyase(K00656), a key enzyme in the biosynthesis of formate (Leonhartsberger et al., 2002), showed higher enrichment in the model group than in the BBR groups (all p < 0.001), which suggested that the gut microbiota in mice with atherosclerosis might produce more formate (**Figure 8D**). Previous studies have reported that formate was associated with hypertension and might be implicated in other atherosclerotic cardiovascular disease (ACVD)-related functions (Holmes et al., 2008; Goodrich et al., 2016). Spearman rank correlations (**Figure 8E**) showed that Alistipes, Allobaculum, Blautia, Turicibacter, and Roseburia exhibited a positive correlation with KEGG orthologues (KOs) related to SCFA production, while Bilophila exhibited a significant negative correlation with SCFA production (p < 0.05 or p < 0.01).

Trimethylamine N-oxide is an important pro-atherogenic metabolite produced by gut microbiota, and gut microbial enzymes were involved in the formation of its precursor trimethylamine (TMA) (Wang et al., 2011; Koeth et al., 2013). Previous studies confirmed that choline TMA lyase (K20038), betaine reductase (K21578, K21579) and L-carnitine CoAtransferase (K08298) are the main TMA-lyases involved in TMA production (Fennema et al., 2016; Zeisel and Warrier, 2017; Janeiro et al., 2018). Results at the KO level exhibited decreased abundance of betaine reductase (K21578, K21579) in both the high and low dose BBR groups compared to the model group, and was especially significantly in the low dose group (all p < 0.01, **Figures 9A,B**). The abundance of choline TMA lyase (K20038) and L-carnitine CoA-transferase (K08298) were also reduced after high or low dose BBR treatment, but were not significant (p > 0.05, **Figures 9C,D**).

FIGURE 8 | (A–D) Differential enrichment of specific gut microbial KOs for SCFAs production among four groups. \* p < 0.05, \*\*\* p < 0.001, vs Model. ### p < 0.001, High vs Low. (E) Spearman rank correlations between abundance of six biomarkers at genus level and SCFAs producing-related KOs. Red indicate being positive correlation and blue being negative.

## DISCUSSION

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In this study, we investigated the effect of BBR on atherosclerosis in HFD-fed ApoE−/− mice. We observed the changes in serum levels of lipid and inflammatory cytokines as well as compositional structure and functions of gut microbiota after treatment with different doses of BBR, and correlated these changes with atherosclerosis. BBR treatment attenuated HFD-induced atherosclerosis, decreased atherosclerotic plaques and lesion areas, and ameliorated the level of serum lipids and inflammatory cytokines. The effects of the high dose of BBR were more significant than the effects of the low dose. 16S rRNA amplicon sequencing showed that BBR significantly altered the community compositional structure of the gut microbiota, and different doses of BBR showed different changes. Further analysis of environmental factor and metagenomic function displayed the effects of BBR on inflammation, glucose and lipid metabolism, and TMAO production in gut microbiota.

Elevated lipid levels are a major risk factor for atherosclerotic CVDs. Since Kong et al. (2004) identified BBR as a new cholesterol-lowering drug (Kong et al., 2004), the therapeutic effect of BBR on dyslipidemia has been confirmed and further explored in recent years (Kong et al., 2008; Zhang et al., 2008). Studies performed in dyslipidemia patients and hyperlipidemic mice showed that BBR significantly reduced serum TC, TG, and LDL-C levels (Kong et al., 2004; Zhang et al., 2008). In addition, these results were supported by two meta-analyses (Lan et al., 2015; Ju et al., 2018), which also reported increased HDL-C levels after BBR treatment. The current results demonstrated that both high and low doses of BBR significantly reduced the levels of TC, APOB100, and VLDL-C, while TG, HDL-C, LDL-C and LP (a) levels were reduced only in the High group. Our results do not indicate an increase in HDL-C after BBR treatment. APOA-1 has an anti-atherogenic effect, and injection of APOA-1 can reduce intracellular cholesterol levels in ApoE−/− mice (Pourcet and Staels, 2016; Gaddis et al., 2018). However, there was no significant increase in reduced APOA-1 levels in the atherosclerosis mice using the high or low dose of BBR in our study.

Atherosclerosis is a chronic inflammatory disease, and inflammation is constantly induced throughout the course of the disease (Libby, 2002; Hansson, 2005; Tedgui and Mallat, 2006). The anti-inflammatory effects of BBR, such as suppressing the expression of pro-inflammatory genes and improving HFDinduced systemic inflammation, have been widely reported (Jeong et al., 2009; Zhang et al., 2012). Previous studies in HFDinduced atherosclerosis mice reported that BBR administration remarkably reduced the mRNA expression of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, in ileal and carotid arteries (Shi et al., 2018; Zhu et al., 2018). Consistent with these results, serum levels of TNF-α, IL-1β, and IL-6 were significantly decreased by the high dose of BBR, while in the low dose group, only TNF-α and IL-1β levels were decreased. In addition, anti-inflammatory cytokines IL-10 (Han and Boisvert, 2015; Metghalchi et al., 2015) and insulin sensitizing hormone ADPN (Lin et al., 2015) have been shown to have anti-atherosclerosis effects. The production of IL-10 was increased in a mouse model of colitis induced by dextran sulfate sodium after BBR administration (Hong et al., 2012). Presently, ADPN levels increased significantly after both high and low dose BBR treatment compared to the Model group, but IL-10 levels were increased only by the high dose of BBR. Combined with previous reports, the present observations support the view that the alleviation of inflammation may be an important mechanism for the reduction of atherosclerosis in BBR-treated HFD-fed mice.

The role of the gut microbiota in atherosclerosis has begun to be studied in recent years. Multiple studies have identified a strong link between the gut microbiota and atherosclerosis (Law and Rudnicka, 2006; Catapano et al., 2016; Ju et al., 2018; Kobiyama and Ley, 2018). To explore the relationship between the anti-atherosclerosis effect of BBR and gut microbiota, 16S rRNA sequencing was used to observe the changes of gut microbiota in each group. Alpha diversity analysis did not reveal a significant decrease in fecal microbial diversity after BBR treatment, which is inconsistent with other research results (Zhang et al., 2015; Zhu et al., 2018). However, in our study beta diversity analysis showed that the community compositional structure was significantly different after treatment with the high or low dose of BBR, which do support the findings of a previous study (Shi et al., 2018). Presently, Firmicutes (75.4%) and Bacteroidetes (15.1%) are the two most dominant phylum at the phylum level, accounting for 90.5%. This was similar to the composition of human gut microbiota in prior studies, with Bacteroidetes and Firmicutes constituting more than 90% of the taxa (Qin et al., 2010; The Human Microbiome Project Consortium, 2012). In our study, the higher abundance was followed by Proteobacteria (3.3%) and Actinobacteria (2.5%), which is inconsistent with the prior finding of Proteobacteria and Verrucomicrobia (Shi et al., 2018). In addition, only decreased Proteobacteria abundance in High group and increased Actinobacteria abundance in Low group was significantly observed after BBR treatment compared with the Control group. These observations might be explained by the fact that, although the species abundance or number of the microbiota varied between individuals, the gut microbiota was semblable among individuals at higher taxonomic levels, such as the phylum level (Jonsson and Backhed, 2017).

At the genus level, LEfSe and STAMP analyses revealed significant differences between groups. Previous studies have shown that animal-based diets and HFD increases the abundance of Alistipes (David et al., 2014; Wan et al., 2019). Moreover, Alistipes has demonstrated a good anti-inflammatory effect in human and animal experiments. Alistipes is an obvious microbial characteristic of patients with inflammatory bowel disease (Schirmer et al., 2018) and increased Alistipes has been directly related to the improvement of colitis induced by dextran sulfate sodium in mice (Dziarski et al., 2016). In particular, a significant correlation was described between acetic acid production and the relative abundance of Alistipes (Yin et al., 2018). In addition, the genus Roseburia was reported to produce butyric acid and metabolize dietary plant polysaccharides, and its abundance was decreased in animal-based diets (David et al., 2014; Kim et al., 2018). In our

study, the abundance of Alistipes was also increased in the HFDfed mice compared with the Control group, but not significantly, while no significant change was observed in the abundance of Roseburia. The high dose of BBR resulted in the significant increase in the abundance of Alistipes and Roseburia, which may be beneficial in the reduction of inflammation in BBR. Butyric acid and acetic acid are both SCFAs, and the role of SCFAs in relieving inflammation has been increasingly reported. For example, increased intake of SCFAs was proven to be beneficial in the treatment of colitis (Harig et al., 1989), and the antiinflammation effect might be mediated by G-protein coupled receptor 43 (Maslowski et al., 2009). The SCFA-producing genera Allobaculum and Blautia were significantly enriched and systemic inflammation decreased in BBR-treated HFD-fed rats (Zhang et al., 2012). Our study supported the latter finding. However, it is worth noting that treatment with the low dose of BBR significantly increased the abundance of Blautia and Allobaculum, while the high dose of BBR did not.

Turicibacter is a genus that is associated with disorders of glucose and lipid metabolism, and is negatively correlated with random blood glucose in diabetic fatty rats (Lai et al., 2018; Zhou et al., 2019). Turicibacter has also been associated with intestinal butyric acid (Zhong et al., 2015), which could increase the secretion and sensitivity of insulin, and has significant anti-obesity and anti-inflammatory effects (Gao et al., 2009; Li et al., 2013; De Vadder et al., 2014). A meta-analysis showed that the decrease of Turicibacter is consistent with increased inflammation in obesity (Jiao et al., 2018).  We found that both high and low doses of BBR enriched the abundance of Turicibacter, and the effect was significant and more pronounced at the high dose. This suggests that the anti-atherosclerosis effect may be related to the interference of BBR on glycolipid metabolism-related gut microbiota. Notably, the abundance of Bilophila decreased in HFD-fed mice, contrary to the prior finding that animal-based diets increased the abundance of Bilophila (David et al., 2014). Bilophila reportedly acts synergistically with HFD to promote higher inflammation and bile acid dysmetabolism, resulting in more serious glucose dysmetabolism and hepatic steatosis (David et al., 2014; Natividad et al., 2018). Presently, the abundance of Bilophila decreased in the high-dose BBR group and increased in the low-dose BBR group compared with the Model group. However, compared with normal control mice, the abundance of Bilophila was decreased after BBR treatment, although it was not obvious.

Environmental factor analysis helped us further determine the potential correlation between lipids, inflammatory cytokines, and the gut microbiota. In addition, metagenomic analysis based on functional changes of the gut microbiome showed that the potential for lipid metabolism and glycan biosynthesis and metabolism was elevated in atherosclerotic model mice after high or low dose BBR treatment. The analysis of species and functional contribution further highlighted the importance of these genus-level biomarkers for glucose and lipid metabolism, among which Alistipes contributed the most. SCFAs are known to be defensive players for inflammation and atherosclerosis (Ohira et al., 2017) and we observed an elevated potential for the synthesis of acetate and butyrate in both high and lowdose BBR groups compared to atherosclerosis model mice. Notably, the alteration of acetate was more significant in the low dose BBR group. However, the synthesis potential of propionate seemed to be decreased after BBR treatment, which was not consistent with the protective effects of propionate on hypertension, cardiovascular damage and anti-atherosclerosis (Bultman, 2018; Bartolomaeus et al., 2019). A heatmap showed that Alistipes, Allobaculum, Blautia, Turicibacter and Roseburia were positively correlated with SCFA production, while Bilophila was significantly negatively correlated. This supported the results that Bilophila acted synergistically with HFD to promote higher inflammation and more serious glucose dysmetabolism (David et al., 2014; Natividad et al., 2018). Consistently, the abundance of Bilophila decreased in the high-dose BBR group in our study. In addition, compared with the atherosclerotic mice, the abundance of TMAO-related enzymes was reduced in both the high and low dose BBR group, and betaine reductase decreased reduced more significantly in the low-dose BBR group. These results showed that different doses of BBR might have different inhibitory effects on TMAO production.

Our results suggested that high and low-dose BBR induced the alteration of abundance and function in certain gut microbiota that may be related to inflammation and glucose and lipid metabolism. These findings partially support the view that BBR may at least partly improve systemic inflammation and lipid level by regulating gut bacteria related to inflammation and glucolipid metabolism, thereby reducing HFD-induced atherosclerosis. Indeed, evidence is accumulating that gut microbiota and its metabolites, such as SCFAs, TMAO, and secondary bile acids, play a role in atherosclerosis by regulating inflammation and the metabolism of lipid, cholesterol and glucose (Barrington and Lusis, 2017; Jonsson and Backhed, 2017; Kasahara et al., 2017; Ma and Li, 2018). SCFAs have anti-atherogenic effect, in addition, it can inhibit the fat synthesis in enterocytes and adipocytes, and suppress biosynthesis of cholesterol and LDL formation in the liver, besides their anti-inflammatory effects (Chistiakov et al., 2015). The involvement of SCFAs in atherosclerosis has been reviewed (Brown and Hazen, 2018). The proatherogenic metabolite TMAO has been associated with the increased risk of atherosclerosis (Wang et al., 2011; Tang et al., 2013). Elevated TMAO levels contributed to foam cell formation and platelet hyperreactivity and inhibited reverse cholesterol transport in macrophages (Wang et al., 2011; Koeth et al., 2013; Zhu et al., 2016). Notably, previous study has shown that BBR treatment can significantly reduce serum TMAO levels (Shi et al., 2018).

Our study has some limitations. Considering the fact that sample quantity is relatively small, the persuasiveness of the results is limited. Although we explained the relationship between the regulation effect of BBR on gut microbiota and the reduction of atherosclerosis based on 16S rRNA sequencing and metagenomic analysis, further fecal bacteria transfer experiments are still needed to clarify the direct link of BBR on gut microbiota with atherosclerosis. This study was conducted in ApoE−/− mice fed with HFD. In the future study, we should extend to clinical

atherosclerosis patients to further well explore the relationship of effect of BBR on gut microbiota and atherosclerosis.

Nonetheless, our study increases the accumulating evidence that BBR can protect against atherosclerosis induced by HFD. Previous studies have shown that BBR could effectively reduce plasma LDL-C levels in patients with CHD and/or hypercholesterolemia who were statin intolerant (Marazzi et al., 2011, 2019). Studies performed in hyperlipidemia mice and metaanalyses also supported that BBR improved dyslipidemia (Lan et al., 2015; Ju et al., 2018). In addition, the anti-inflammatory effects of BBR, including the reduction of HFD-induced systemic inflammation and the suppression of proinflammatory gene expression, have been widely confirmed (Jeong et al., 2009; Zhang et al., 2012). However, the mechanism of these effects is still unclear due to the low BBR absorption rate. Recent studies suggested that the anti-atherosclerotic effect of BBR may be related to changes in gut microbiota, but the specific association and potential mechanism are still unknown (Shi et al., 2018; Zhu et al., 2018). The present study may provide a novel explanation. Our study ulteriorly supports the anti-atherogenic effect of BBR by targeting the gut microbiota at both the species and function levels. In some extent, this suggests the potential of BBR and certain gut microbiome as an alternative or adjuvant treatment strategy for atherosclerotic patients who are intolerant to conventional drugs (such as statins) or under an increased risk of adverse effects.

#### CONCLUSION

In conclusion, both high and low dose BBR can improve serum lipid and inflammation levels and alleviate atherosclerosis in a mouse model of HFD-induced atherosclerosis. This antiatherosclerotic effect of BBR may be partly attributed to changes in composition and functions of gut microbiota which may be related to anti-inflammatory activity and the metabolism of glucose and lipid. In addition, different doses of BBR had different effects, among which the attenuation of atherosclerosis was more obvious at the high dose of BBR, but the alterations of composition and functions in gut microbiota showed different sensitivity to BBR dose.

#### REFERENCES

### DATA AVAILABILITY STATEMENT

The datasets generated for this study can be found in the Sequences Read Archive database at the NCBI under accession number PRJNA579048.

#### ETHICS STATEMENT

The animal study was reviewed and approved by the Animal Investigation Committee of Peking University.

#### AUTHOR CONTRIBUTIONS

MW, SY, LL and YX designed the experiment and wrote the manuscript. SW and YC aided in the design of the illustrations. RZ and XL commented on the manuscript. All authors approved the manuscript for publication.

### FUNDING

The work was supported by the Be ijing Natural Science Foundation (7172185) and the National Natural Science Foundation of China (Grant Nos. 81202805, 81973689, and 81573821).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar. 2020.00223/full#supplementary-material

FIGURE S1 | Most abundant OTU members of the gut bacterial microbiome (Top 116).

FIGURE S2 | Differentially enriched and depleted OTUs between each group compared with the Model group. (A) Control vs Model; (B) Low vs Model; (C) High vs Model. Each point in the figure represents an individual OTU, of which the red points for significant enriched OTUs, green points for significant depleted OTUs, gray points for OTUs with no significant difference. The number labeled in the figure is the number of OTUs that significantly enriched or decreased.

element-binding protein-2 and induction of adiponectin in mice. Circulation 131, 1861–1871. doi: 10.1161/circulationaha.115.015308

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Wu, Yang, Wang, Cao, Zhao, Li, Xing and Liu. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fphar-11-00223 March 12, 2020 Time: 18:56 # 16

# Herbal Medicine for Cardiovascular Diseases: Efficacy, Mechanisms, and Safety

Abdullah Shaito1 , Duong Thi Bich Thuan2 , Hoa Thi Phu2 , Thi Hieu Dung Nguyen3 , Hiba Hasan4 , Sarah Halabi 5 , Samar Abdelhady 6 , Gheyath K. Nasrallah7\*, Ali H. Eid7,8\* and Gianfranco Pintus 9,10\*

#### Edited by:

Yue Liu, Xiyuan Hospital, China

#### Reviewed by:

Guanwei Fan, Tianjin University of Traditional Chinese Medicine, China Yanfei Liu, Beijing University of Chinese Medicine, China

#### \*Correspondence:

Ali H. Eid<EMAIL_ADDRESS>Gianfranco Pintus<EMAIL_ADDRESS><EMAIL_ADDRESS>Gheyath K. Nasrallah<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology

Received: 04 November 2019 Accepted: 19 March 2020 Published: 07 April 2020

#### Citation:

Shaito A, Thuan DTB, Phu HT, Nguyen THD, Hasan H, Halabi S, Abdelhady S, Nasrallah GK, Eid AH and Pintus G (2020) Herbal Medicine for Cardiovascular Diseases: Efficacy, Mechanisms, and Safety. Front. Pharmacol. 11:422. doi: 10.3389/fphar.2020.00422 1 Department of Biological and Chemical Sciences, Lebanese International University, Beirut, Lebanon, 2 Department of Biochemistry, University of Medicine and Pharmacy, Hue University, Hue City, Vietnam, 3 Department of Physiology, University of Medicine and Pharmacy, Hue University, Hue City, Vietnam, 4 Institute of Anatomy and Cell Biology, Justus Liebig University Giessen, Giessen, Germany, 5 Biology Department, Faculty of Arts and Sciences, American University of Beirut, Beirut, Lebanon, 6 Faculty of Medicine, Alexandria University, Alexandria, Egypt, 7 Department of Biomedical Sciences, College of Health Sciences, Qatar University, Doha, Qatar, 8 Department of Pharmacology and Toxicology, American University of Beirut, Beirut, Lebanon, 9 Department of Medical Laboratory Sciences, University of Sharjah, Sharjah, United Arab Emirates, 10 Department of Biomedical Sciences, Faculty of Medicine, University of Sassari, Sassari, Italy

Cardiovascular diseases (CVDs) are a significant health burden with an ever-increasing prevalence. They remain the leading causes of morbidity and mortality worldwide. The use of medicinal herbs continues to be an alternative treatment approach for several diseases including CVDs. Currently, there is an unprecedented drive for the use of herbal preparations in modern medicinal systems. This drive is powered by several aspects, prime among which are their cost-effective therapeutic promise compared to standard modern therapies and the general belief that they are safe. Nonetheless, the claimed safety of herbal preparations yet remains to be properly tested. Consequently, public awareness should be raised regarding medicinal herbs safety, toxicity, potentially lifethreatening adverse effects, and possible herb–drug interactions. Over the years, laboratory data have shown that medicinal herbs may have therapeutic value in CVDs as they can interfere with several CVD risk factors. Accordingly, there have been many attempts to move studies on medicinal herbs from the bench to the bedside, in order to effectively employ herbs in CVD treatments. In this review, we introduce CVDs and their risk factors. Then we overview the use of herbs for disease treatment in general and CVDs in particular. Further, data on the ethnopharmacological therapeutic potentials and medicinal properties against CVDs of four widely used plants, namely Ginseng, Ginkgo biloba, Ganoderma lucidum, and Gynostemma pentaphyllum, are gathered and reviewed. In particular, the employment of these four plants in the context of CVDs, such as myocardial infarction, hypertension, peripheral vascular diseases, coronary heart disease, cardiomyopathies, and dyslipidemias has been reviewed, analyzed, and critically discussed. We also endeavor to document the recent studies aimed to dissect the cellular and molecular cardio-protective mechanisms of the four plants, using recently reported in vitro and in vivo studies. Finally, we reviewed and reported the results of the

recent clinical trials that have been conducted using these four medicinal herbs with special emphasis on their efficacy, safety, and toxicity.

Keywords: herbal medicine, cardiovascular diseases, atherosclerosis, hypertension, medicinal plants, antioxidants, oxidative stress, inflammation

#### INTRODUCTION

Cardiovascular diseases (CVDs) are diseases of the heart or blood vessels. CVDs register a global annual toll of more than 17 million deaths. As a result, CVDs remain the world's most common cause of death and are a major economic and health burden, worldwide. The World Health Organization (WHO) reported that CVDs account for 31% of annual global deaths (World Health Organization, 2017). In Europe, CVDs account for 45% of all deaths according to the European Cardiovascular Disease Statistics 2017 (Martinet et al., 2019). The American Heart Association's current statistics estimate that around half of the population of the USA has a form of CVD (Benjamin et al., 2019).

CVDs are a variety of diseases including peripheral vascular diseases, coronary heart disease (CHD), heart failure, heart attack (myocardial infarction), stroke, cardiomyopathies, dyslipidemias, and hypertension, among others (Figure 1) (Toth, 2007; Reiner et al., 2019). CVDs majorly originate from a vascular dysfunction, which then leads to organ damage. For example, the heart can suffer a heart attack, or the brain can suffer a stroke due to vascular impairment. Major culprits in vascular impairment include atherosclerosis, thrombosis, and high blood pressure (BP). Common risk factors for CVDs include smoking, unhealthy diet, diabetes mellitus, hyperlipidemia, elevated levels of low-density lipoprotein cholesterol (LDL), suppressed levels of high-density lipoprotein cholesterol (HDL), and hypertension (Figure 1) (World Health Organization, 2017).

CVDs prevention is favored by a healthy vascular endothelium. A healthy endothelium exhibits vasodilatory, anti-atherogenic, and anti-inflammatory properties (Celermajer, 1997). Several risk factors for CVDs lead to endothelial cell (EC) dysfunction, which has been implicated as a key event in the pathogenesis of atherosclerosis, coronary vasoconstriction, and, probably, myocardial ischemia. Interestingly, EC dysfunction is a reversible phenomenon, which opens the door for CVD therapies based on its reversion (Figure 1) (Celermajer, 1997).

Recently, inflammation has been confirmed as a risk factor for CVDs, especially during atherosclerosis and coronary artery disease. High levels of high-sensitivity C-reactive protein (hs-

FIGURE 1 | Pathological processes involved in the development and progression of CVDs. Several risk factors can predispose to CVDs. These can include hypertension, smoking, dyslipidemia stemming from an unhealthy diet, or endocrinopathies like diabetes mellitus, hypothyroidism, and aging. The risk factors can lead to pathological alterations most of which can be due to endothelial dysfunction or VSMC alterations. Endothelial dysfunction or VSMC alterations increase the risk of developing atherosclerosis and hypertension. Atherosclerosis and hypertension are themselves CVDs risk factors and enhancers for the development of other CVDs like myocardial infarction, coronary artery diseases, or stroke. VSMC, vascular smooth muscle cell; ECM, extracellular matrix; NO, nitric oxide; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; Ox-LDL, oxidized low-density lipoprotein.

CRP) and/or interleukin-6 (IL-6) are associated with higher absolute cardiovascular risk (Ridker et al., 1997; Ridker et al., 2000), where the CANTOS study, for the first time, established reduced rates of cardiovascular events following an antiinterleukin-1 beta (IL-1b) based therapy, independent of cholesterol levels (Ridker et al., 2017). Furthermore, common CVDs risk factors, such as diabetes or hypertension, can predispose to CVDs by the mediation of inflammation (Dokken, 2008; Aday and Ridker, 2018).

In the case of atherosclerosis, for example, inflammation can cause EC functional impairment. Dysfunctional ECs allow the accumulation of low-density lipoprotein (LDL) particles in the vessel wall intima where they become modified into oxidized LDL. Oxidized LDL can then activate the dysfunctional ECs to expose cell adhesion molecules (VCAM-1 and ICAM-1) that bind to and recruit inflammatory leukocytes (T-cells and monocytes) into the subendothelial space (Davies et al., 1993; Moore and Tabas, 2011). These inflammatory blood cells secrete interleukins and cytokines, produce reactive oxygen species (ROS) and thus form an inflamed microenvironment within the arterial wall. The inflamed microenvironment promotes vascular smooth muscle cell (VSMC) proliferation, matrix build-up, and lipid deposition, leading to the formation of an atherosclerotic plaque. The monocytes can reach the intima of the vessel, differentiate into macrophages, and uptake oxidized LDL to become foam cells (Tabas et al., 2007; Moore and Tabas, 2011; Douglas and Channon, 2014; Saleh Al-Shehabi et al., 2016; Martinet et al., 2019). Gradual intimal thickening takes place over the years and continues to expand causing decreased or complete occlusion of blood flow to organs, ultimately resulting in CVDs, such as myocardial infarction or stroke (Maguire et al., 2019). In addition, VSMCs proliferation leads to narrowing of the arterial lumen and dysregulation of the vasotone (Douglas and Channon, 2014). Usually several atherosclerotic plaques form in the intima and one of these may end up undergoing a necrotic breakdown, leading to acute luminal thrombosis, blood vessel occlusion, and cardiovascular complications, including myocardial infarction, unstable angina (chest pain caused by heart muscle ischemia), sudden cardiac death, or a stroke (Virmani et al., 2002). As a result, atherosclerosis is not only a risk factor but also a major contributor to CVD incidence. Around 50% of all deaths in developed countries are due to atherosclerosis (Tedgui and Mallat, 2006).

Hypertension also referred to as high BP, is a CVD and a major risk factor and contributor to other CVDs and other diseases (2017). Hypertension is an independent predisposing factor for heart failure, coronary artery disease, stroke, retinopathy, nephropathy, and peripheral arterial diseases (Sawicka et al., 2011; NCD Risk Factor Collaboration, 2017). Most of these diseases are associated with high mortality and morbidity (Abegaz et al., 2017). Additionally, hypertension is the single most significant risk factor for atherosclerosis, and any clinical outcome of atherosclerosis thereof (Sawicka et al., 2011). Hypertension is a "silent killer" as it does not show symptoms until later stages of the disease (Sawicka et al., 2011). Because of this, it is not surprising that hypertension affects 1.4 billion people and accounts for about 9.4 million deaths per year (Cooper et al., 2017; Egan et al., 2019). Lastly, hypertension prevalence is estimated to have a 30% worldwide increase by 2025 (Kearney et al., 2005).

The American Heart Association Hypertension Guidelines define hypertension as a persistent elevation of BP in the arteries [systolic BP (SBP) higher than 130/diastolic BP (DBP) higher than 80 mm Hg] (Muntner et al., 2018). If an elevated BP is left unmanaged, it can induce arterial remodeling; the walls of small vessels thicken, and the vessels lose their elasticity and become narrower. This process is called arteriosclerosis and can lead to "target organ damage" (TOD) (Triantafyllidi et al., 2010; Fan et al., 2017). TOD affects several organs such as the brain, kidney, or retina and may lead to death (Mensah, 2016; Abegaz et al., 2017). Arteriosclerosis can be witnessed in coronary vessels where it may cause a myocardial infarction (Rakugi et al., 1996). In the brain, arteriosclerosis can cause vessel lumen narrowing, vessel wall hardening, and blood clot formation, potentially causing a stroke (Johansson, 1999). Strokes have effects on cognitive and physical behaviors, and may result in dementia, paralysis, or death (Abegaz et al., 2017). Nephrosclerosis of the kidney is also due to arteriosclerosis which stiffens the nephron, ultimately affecting renal filtration and causing electrolyte imbalances (Bidani and Griffin, 2004; Lim et al., 2016). Overall, TOD proceed through hypertension-induced microvascular injuries in the cases of retinopathy and nephropathy and through hypertension-induced macrovascular injuries in the cases of stroke and myocardial infarctions (Nadar, 2011).

BP regulation, and therefore hypertension, depends on two main factors: cardiac output and systemic vascular resistance. Increased cardiac output or vascular resistance elevate BP. Cardiac output is majorly affected by sodium intake, renal function, and mineralocorticoids. Vascular resistance is affected by the sympathetic nervous system (SNS), rennin–angiotensin system (RAS), humoral factors, and local autoregulation (DiBona, 2013). SNS and RAS exert their effects mainly by eliciting vasoconstriction and inducing sodium retention. Humoral mediators can be vasoconstrictors like endothelin, angiotensin II, catecholamines, or vasodilators, for instance nitric oxide (NO), prostaglandins, and kinins (Oparil et al., 2003). Other factors that can modulate BP include blood flow velocity, blood viscosity, vascular wall stiffness, oxidative stress of VSMCs or ECs, VSMCs proliferation and shape changes, and EC health (Rodrigo et al., 2011).

Despite advances in CVD management and treatment, CVDs still claim more lives than the combination of all cancer forms (Mozaffarian et al., 2015). As a result, in recent years there has been major enforcement on CVD prevention (Reiner et al., 2019). Therefore, new treatment options are urgently warranted for all types of CVDs, considering the continued burden stemming from CVDs is still substantial.

#### HERBAL AND PLANT PRODUCTS AS MEDICINAL DRUGS

Traditional medicine and ethnomedicine, defined as the study of the traditional medicines practiced by various ethnic groups, are as old as human history. Traditional medicine historically relied on natural resources as medications. Historically, herbs, generally defined as any form of plant or plant product (Tachjian et al., 2010), and plant extracts formed the basis of the first drugs used in traditional medicine systems of many cultures and civilizations. Plants and herbs have always been a common source of medications, either in the form of traditional extracts or as pure active compounds (Fabricant and Farnsworth, 2001). Evidently, nature is a very important source for finding new drugs that leads to the treatment of diseases. Famous drugs from herbal and plant sources include aspirin from the Salix alba L. tree, digoxin (cardiac glycoside) from Digitalis purpurea, ephedrine from Ephedra sinica, lovastatin from Monascus purpureus L., taxol from Taxus brevifolia, reserpine from Rauvolfia serpentina, and many others (Harvey, 2000; Frishman et al., 2009; Cragg and Newman, 2013). Interestingly, reserpine is still an effective treatment for hypertension (Weber et al., 2014). Notably, the discovery of antimalarial drugs, quinine from the bark of Cinchona species and artemisinin from Artemisia annua L., represent a typical example of how ethnomedicine can guide drug discovery (Cragg and Newman, 2013).

The earliest records of drugs of natural origin, found in Mesopotamia (from around 2600 BCE), describe the use of approximately 1000 plant-derived compounds. The best record of using natural extracts in therapy is the Egyptians' Ebers Papyrus (from 1500 BCE), which documents more than 700 natural drugs, mainly of plant origin. The Chinese Materia Medica record (BCE 1100) describes 52 natural medicinal preparations, and the Indian Ayurvedic record (BCE 1000) describes more than 800 natural medicinal extracts (Cragg and Newman, 2013; Otvos et al., 2019). Hippocrates also applied phytotherapy, or healing with herbs, in his treatments (Otvos et al., 2019).

In 1985 WHO estimated that around 65% of the world population mostly depended on plant-derived traditional medicines (Farnsworth et al., 1985). People in different countries have come to use identical or comparable plants or herbal preparations for the prevention and/or treatment of physical and mental illnesses. Traditional Medicine Centers of the WHO identified 122 compounds to be commonly used in the Center's host countries. Interestingly, the 122 compounds have been reported to derive from only 94 plant species and are used for similar ethnomedical treatments in the different host countries (Farnsworth et al., 1985). Examples of such compounds include galegine, from Galega officinalis L., the base for the synthesis of metformin and similar bisguanidine-type antidiabetic drugs, and papaverine from Papaver somniferum which is the base for making the antihypertensive drug verapamil (Fabricant and Farnsworth, 2001). Commercially, drug production from natural products such as herbs is a viable commodity, where 39% of the 520 new drugs approved between 1983 and 1994 were natural compounds or derived from natural compounds and 60–80% of antibacterial and anticancer drugs were derived from natural products in that same period (Harvey, 2000).

Despite the many successes of using natural products for drug production, advances in combinatorial chemistry (in the late 1980s) shifted the focus of drug discovery efforts from natural products to synthesis at the laboratory bench (Cragg and Newman, 2013). This is mainly because natural product-based drug discovery and development is a complex endeavor demanding costly and highly integrated interdisciplinary approaches (Davison and Brimble, 2019; Otvos et al., 2019). Nonetheless, currently the use of natural products as drugs or as drug discovery platforms is "well and alive" (Newman and Cragg, 2016). In fact, traditional herbal and plant-derived extracts are becoming main stream as advances in scientific research are showing their importance in the prevention and treatment of diseases (Frishman et al., 2009).

Numerous and chemically diverse secondary metabolites have been purified from plant bioactives and have been optimized for exerting a biological effect, nonetheless, they are still away from exhaustive investigation for clinical use. However, recent published scientific evidence, technological advances, and research trends clearly point that naturally-derived compounds will be major sources of new drugs (Davison and Brimble, 2019; Otvos et al., 2019). This has provided a driving cause for the renewed popularity of traditional herbal and plant-derived medications among researchers, despite developments in combinatorial chemical synthesis and the production of modern synthetic drugs (Frishman et al., 2009).

Another reason for the regained interest in medicinal plant products is that, in their attempts to control diseases amid scarce socioeconomic resources, rural communities in developing countries have found resort in traditional herbal and plantderived remedies. This is due to several factors, but in particular to the fact that plant-based medicines are a cheaper alternative with fewer side effects (Frishman et al., 2009; Tabassum and Ahmad, 2011). Herbal and plant remedies are not only economical, but they also contain thousands of bioactive components that have known therapeutic applications (Pan et al., 2013). Additionally, because herbs are viewed as food products, they are not subject to the same surveillance and regulation as conventional drugs. Moreover, herbal remedies are viewed by patients as being natural and therefore safe. However, more research efforts are required to validate the efficacy and the safety profile of such medicaments since many have adverse outcomes that can sometimes have life-threatening effects. It should also be noted that there is concern regarding herb–drug interaction (Tachjian et al., 2010; Anwar et al., 2016; Yuan et al., 2016).

One more reason for the revived interest in natural products is that the biological activity and structural diversity of natural products are unmatched by any available synthetic drug screening library (Davison and Brimble, 2019). Natural products have been selected by nature for bioactivity, over millions of years. As a result, natural product screening libraries need not be superfluously big, as is the case with synthetic drug screening libraries. In addition, natural products need only minor structural changes to optimize their drugability (Harvey, 2000; Gerry and Schreiber, 2018). As such, natural products offer "privileged scaffolds" and serve as biologically "pre-validated platforms" for the design of compound candidate drug libraries (Davison and Brimble, 2019). Recent progress has focused on improving the potency, selectivity, and pharmacokinetics of bioactive natural products through structural modifications, which has led to the production of novel drug-like lead compounds. These structural changes are often required, as natural products usually show unfavorable toxicities and pharmacokinetics, limiting their clinical potential (Gerry and Schreiber, 2018; Davison and Brimble, 2019). Overall, natural products have been the single most productive source of drug leads even though little of nature's biodiversity has been tested for biological activity yet (Harvey, 2000).

We therefore address and expose the general rationale for using medicinal herbs in the therapy of diseases in general and CVDs in particular. Then, we move to discuss the medicinal potentials of four traditional herbs (Ginseng, Ginkgo biloba, Ganoderma lucidum, and Gynostemma pentaphyllum) for the treatment of CVDs, which are getting increasing popularity due to their commercial commodity in many markets worldwide and to their proven therapeutic potential in several settings including cardiovascular conditions. We describe and critically discuss their therapeutic benefits in terms of molecular, cellular, and metabolic properties in the context of CVDs. In addition, we highlight the major clinical trials in which these four herbs have been used, with an emphasis on their efficacy and safety.

### MODERN MEDICINE MANAGEMENT OF ATHEROSCLEROSIS AND HYPERTENSION

Current health care guidelines emphasize prevention to minimize the risk of CVDs (Reiner et al., 2019). This is carried out by addressing the major CVD risk factors and trying to minimize their adverse outcomes. In atherosclerosis, most therapeutic approaches aim to control hypertension and hyperlipidemia or modulate hemostasis in order to avoid thrombotic complications (Weber and Noels, 2011). Hypercholesterolemia is a major contributor in atherosclerosis, so current conventional therapeutic approaches rely significantly on lowering LDL levels using statins (Ridker et al., 2017; Aday and Ridker, 2018; Reiner et al., 2019). In cases where statin therapy does not yield a significant reduction in LDL levels, an LDL-absorption inhibitor can be used, alone or in combination with statins depending on patient response. Clinical trials have clearly shown that such therapies are effective in lowering CVD risk (Reiner et al., 2019). Recently, pro-protein convertase subtilisin/kexin type 9 (PCSK9) inhibitors were approved by the regulatory bodies as a drug that can lower LDL level, and are recommended for use in patients with heart problems, where statins were not effective at lowering LDL levels (Ridker et al., 2017; Aday and Ridker, 2018). The CANTOS clinical trial (2017) has provided evidence that in patients with elevated inflammation (hsCRP > 2 mg/L), a combination therapy of statins and canakinumab (IL-1b antibody) may be necessary to lower atherosclerosis risk (Ridker et al., 2017; Aday and Ridker, 2018; Reiner et al., 2019). Prior to the recommendations of the CANTOS study, conventional therapy regimens have neglected the role of inflammation in atherosclerosis (Weber and Noels, 2011). It is very important to highlight that complementary and alternative medicine (CAM), including herbal remedies, have already tackled the inflammatory arm of atherosclerosis much earlier than the results of the CANTOS study (Frishman et al., 2009; Orekhov et al., 2013; Al-Shehabi et al., 2016), giving a hint as to why American patients visited CAM providers much more than primary care physicians (Eisenberg et al., 1998; Tachjian et al., 2010). Of relevance to this discussion is that herbal remedies are the most common type of CAM among CVD patients (Yeh et al., 2006; Tachjian et al., 2010).

Modern therapy regimens for hypertension involve controlling BP elevations in hypertensive patients. This usually requires the use of multiple antihypertensive drug agents in the majority of these patients (Guerrero-Garcia and Rubio-Guerra, 2018). Multiple classes of antihypertensive agents are available thus offering a practitioner the ability to prescribe highly effective drug combinations in order to reduce BP and protect target organs (Stewart et al., 2019). This combination therapy is of distinctive importance in resistant hypertension, which is highly prevalent worldwide (Noubiap et al., 2019; Samaha et al., 2019).

The major drug classes available for the management of hypertension are thiazide diuretics, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor II blockers, and calcium channel blockers (Susalit et al., 2011; Munoz-Durango et al., 2016). Vasodilators, aldosterone antagonists, b-blockers, a-blockers, renin inhibitors, and central-acting agents are other agents that are occasionally used (Omboni and Volpe, 2018). These agents lower BP in patients and reduce their risk of hypertension-related CVD events, but do not prevent them thereby justifying the use of hypertension combination therapies (Rizvi, 2017).

Despite the availability of the aforementioned medications in modern-day health care systems, high BP is managed in only 34% of the patients (August, 2004; Wang and Xiong, 2012). Such an aspect appears to be mainly related not only to the cost of antihypertensive agents (Susalit et al., 2011), but also to their availability and accessibility (Wang and Xiong, 2012), their unwanted side effects (Susalit et al., 2011; Wang and Xiong, 2012), and their low patient compliance with the required dose (August, 2004). For these factors hypertension patients seek CAM medications, especially herbal-based medicaments to treat their CVDs in general and hypertension in particular (Yeh et al., 2006; Tachjian et al., 2010; Al Disi et al., 2016).

### HERBAL MEDICINE MANAGEMENT OF ATHEROSCLEROSIS AND HYPERTENSION

Herbal extracts and their derivatives can favorably modulate and ultimately ameliorate the molecular events that contribute to hypertension or atherosclerosis, the two major contributors to CVDs incidence. Herbal remedies contain numerous bioactives and, thus, have multi-modal cellular mechanisms of action. In fact, herbal remedies can have antioxidant, vasorelaxant, antiinflammatory, anti-proliferative, or diuretic effects. Herbal remedies can also prevent VSMC phenotypic switching, inhibit endothelial dysfunction, platelet activation, lipid peroxidation, ROS production, and macrophage atherogenicity. Because of such a wide range of molecular and cellular targets, herbal preparations can be used to treat and manage a range of CVDs. For example, Salvia miltiorrhiza (Red sage), an annual sage traditionally used in Chinese medicine, has been used to treat a plethora of CVDs including CHD, myocardial infarction, atherosclerosis, and angina pectoris. The active compounds are mainly utilized as the dried root of the plant rhizome named Danshen (Gao et al., 2012). The plant bioactive compounds are the lipo-soluble Tanshinones and the water-soluble Phenolics (Ren et al., 2019). S. miltiorrhiza extracts have shown strong antioxidant capabilities with a high ability to scavenge free radicals, which seems the base of its strong cardio- and vascular-protective potential (Zhao et al., 2006).

Salvianolic acid B, one of the pure compounds that can be extracted from S. miltiorrhiza, is effective against fibrosis and ischemia–reperfusion injury (Lay et al., 2003). Danshen has a protective effect against homocysteine-induced adverse effects, where homocysteine imbalance is a high-risk factor for vascular diseases (Chan et al., 2004). In combination with Pueraria montana var. lobata. (Kudzu), Danshen has showed potent anti-hypertensive effects (Ng et al., 2011). In one clinical trial, Danshen capsules (1000 mg twice daily for 12 weeks) were able to significantly reduce SBP and pulse rate in patients with uncontrolled mild to moderate hypertension and under conventional antihypertensive treatment. It has also been found to be well-tolerated and considered to be safe in patients with hypertension (Yang et al., 2012).

Astragalus membranaceus (Synonym Astragalus propinquus Schischkin. in the Missouri Botanical Garden plant list), another Chinese herb, contains Astragaloside IV, which is the plant major bioactive compound widely used as an antioxidant and for protection against ischemic-associated CVDs (Zhang et al., 2006). A. membranaceus extract has been found to maintain cardiac functions by improving energy metabolism and inhibiting the production of free radicals in a myocardial ischemia reperfusion rat model (Zhou et al., 2000). By decreasing the levels of the oxidative stress marker malondialdehyde (MDA), maintaining superoxide dismutase (SOD) activity, and reducing free radicals-induced myocardial cell injury, A. membranaceus can also improve cardiac function and provide cardioprotection in a myocardial ischemic rat model (Ma et al., 2013). A. membranaceus extract also has angiogenic effects in the ischemic injury rat model (Zhang et al., 2011). Astragaloside IV has been found to provide a positive inotropic effect improving left ventricular ejection in patients with congestive heart failure (CHF) (Luo et al., 1995). The polysaccharide of A. membranaceus has also been shown to reduce insulin resistance and to possess anti-obesity and hypolipidemic effects (Mao et al., 2009).

Allium sativum (Garlic) is a classic example of herbs used in CVDs management and is quite known for its multifaceted properties against CVD-associated conditions such as hypertension, oxidative stress, inflammation, and hyperlipidemia (Ashraf et al., 2013; Jeong et al., 2016; Thomson et al., 2016). Indeed, by reducing total cholesterol and LDL levels, decreasing the content of lipid in arterial cells and inhibiting VSMCs proliferation, garlic can be used to manage atherosclerosis and hyperlipidemia (Sun Y. et al., 2018). Owing to its endothelial NO synthase (eNOS) modulated vasorelaxation ability, Crataegus oxyacantha (Synonym Crataegus rhipidophylla Gand. Common name Hawthorn) is another example of herbs commonly used to manage hypertension (Brixius et al., 2006). Another herb, Crocus sativus (Saffron), can block Ca2+ channels via endothelium-independent mechanisms providing another vasodilator mechanism, in addition to its eNOS activating ability (Razavi et al., 2016). Among other medicinal plants Hibiscus sabdariffa (roselle), is known to reduce BP using its ability to inhibit ACE (Ojeda et al., 2010), while Camellia sinensis (Tea) extracts can reduce hypertension by significantly increasing brachial artery flow-mediated dilation (FMD) (Ras et al., 2011). Rosemary (Rosmarinus officinalis) exhibits neuroprotection by acting against ischemic stroke-associated cerebral insufficiency, which is characterized by a reduction of localized blood flow in the brain. Through its anti-inflammatory properties, rosemary can decrease the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) as well as that other pro-inflammatory enzymes and mediators (Seyedemadi et al., 2016). The use of herbal plants extends to include CHF and atrial arrhythmias. Digitalis, extracted from the dried leaves of the common foxglove, is a potent inhibitor of Na+ /K+ -ATPase and can cause depolarization leading to smooth muscle contraction and vasoconstriction and hence can strengthen muscle heart contractions (Liu et al., 2016).

Given all these restorative abilities, it is not surprising that herbal remedies are being absorbed into evidence-based medicine for the prevention and/or treatment of CVDs. Table 1 lists common herbal remedies and the form of CVDs that they can help alleviate.

Although herbs have been widely used in both traditional and modern medicine, a limited number of reviews that gather them and comprehensively focus on their mechanisms of action and safety in the context of CVDs are present. Many plant-based compounds appear to have cardiovascular protective effects, nevertheless, among the most effective compounds are flavonoids, terpenoids, saponins, and polysaccharides. These highly effective compounds are major components of four of the most recognized herbal preparations namely: Ginseng, Ginkgo biloba, Ganoderma lucidum, and Gynostemma pentaphyllum, which we decide to cover in this review.

TABLE 1 | Some herbal remedies traditionally used for the treatment of different forms of CVDs.

The herbal remedies refer to the plant extract, unless otherwise indicated where the remedy may be a purified or partially purified active ingredient of the plant extract. Data were obtained from published data in (Valli and Giardina, 2002; Frishman et al., 2009; Al Disi et al., 2016; Al-Shehabi et al., 2016; Samaha et al., 2019).

#### GINSENG

Ginseng is an anciently cultivated plant (2000 years ago) partly due to its ritual use (Figure 2). Ginseng use in traditional medicine goes back to 20 centuries ago (Kim, 2012), but its use in Western medicine dates back to the early 20th century by two British physicians F. Porter Smith and G.A. Stuart who were exploring Chinese herbal remedies at the time (Shih-Chen et al., 1973). Ginseng habitats include Asian countries such as Korea, China, Japan, and Vietnam, and North American countries, mainly Canada and the United States. Korean red ginseng (KRG; Panax ginseng C.A. Mey.), Chinese ginseng (Panax notoginseng Burkill; F.H.Chen.), American ginseng (Panax quinquefolium L.), and Japanese ginseng (Panax japonicas C.A. Mey.) represent the most commonly used ginsengs.