Beyond Alkaloids: Novel Bioactive Natural Products From Lobelia Species

In this work, we reviewed the progress in the phytochemical and biological investigations of bioactive components derived from medicinally valuable Lobelia species. In the last 60 years, Lobelia has garnered significant attention from the phytochemist from around the world, majorly due to the discovery of bioactive piperidine alkaloids (e.g., lobinaline and lobeline) in the early 1950s. Later, lobeline underwent clinical trials for several indications including the treatment of attention deficit hyperactivity disorder and a multicenter phase three trial for smoking cessation. Subsequently, several other alkaloids derived from different species of Lobelia were also investigated for their pharmacological characteristics. However, in the last few years, the research focus has started shifting to the characterization of the other novel chemical classes. The major shift has been noticed due to the structurally similar alkaloid components, which essentially share similar pharmacological, physicochemical, and toxicological profiles. In this review, we present an up-to-date overview of their progress with special attention to understanding the molecular mechanisms of the novel bioactive components.


INTRODUCTION
Lobelia, named after the botanist Matthias de lobel, is a large genus of medicinally valuable flowering plants belonging to the Campanulaceae family comprising more than 450 species distributed predominantly in tropical and temperate regions of the world. Several species of Lobelia are traditionally used in different folk medicine to treat various diseases. Among them, Lobelia inflata, Lobelia nicotianaefolia, Lobelia cardinalis, Lobelia chinensis, Lobelia laxiflora, Lobelia trigona Roxb., Lobelia siphilitica L., Lobelia sessilifoilia Lamb, Lobelia polyphylla, and Lobelia pyramidalis Wall. are some of the frequently used Lobelia species. The phytochemical researches of these species led to the discovery of several novel bioactive secondary metabolites.
The earliest scientific report on the pharmacological use of Lobelia species in diseased conditions dates back to 1828 (Andrew, 1828). More than a century later (in 1950), Tondeur and Charlier reported the extraction and pharmacological activities of alkaloids from Lobelia suavibracteata (Haumann) and Lobelia giberroa (Hemsl.) Tondeur and Charlier, 1950). In the 1950s, other studies began to reveal the distinctive pharmacological properties of the alkaloid constituents derived from different Lobelia species (Lendle and Richter, 1950). One of the alkaloids that garnered major attention was lobeline 1 (Figure 1), which was primarily derived from the aerial parts of Lobelia inflata, (Kaczmarek and Steinegger, 1959;Saldanha et al., 1959). In the subsequent decades, the studies on the prospective evaluation of the use of lobeline have seen unprecedented advances, including but not limited to smoking cessation (Glover et al., 2010;Stead and Hughes, 2012), treatment of drug and alcohol abuse (Bell et al., 2009;Hart et al., 2010;Roni and Rahman, 2017), adult ADHD (Martin et al., 2018), anti-depressant (Roni and Rahman, 2015), anti-epileptic (Tamboli et al., 2012), and Parkinson's disease .
For example, in their study, Roni and Rahman noticed that adult C57BL/6J mice when treated with once daily dose of lobeline (1 mg/kg, s.c.) in the last 14 days of their exposure to chronic unpredictable stress (CUS) for 6 weeks produced antidepressant-like effects. They proposed targeting brain nicotinic acetylcholine receptors (nAChRs), brain-derived neurotrophic factor (BDNF) expression and/or cell proliferation in the hippocampus to be the most likely cause of the observed antidepressant-like effects of lobeline (Roni and Rahman, 2015). Saline was used as the negative control. Hart et al. reported that at doses of 1.0 mg/kg and 3.0 mg/kg subcutaneous injections of lobeline attenuates self-administration of intravenous (i.v.) heroin (3,6 diacetylmorphine HCl) infusions (18 μg/kg) in male Sprague-Dawley rats suggesting their use in pharmacotherapy for opioid abuse (Hart et al., 2010). In both studies, however, they did not make a comparison of the effect of lobeline with any reference standard. Martin et al. reported the results of a preclinical study in which they studied the effect of lobeline in adult ADHD (Martin et al., 2018). In their study, they subjected a total of nine adults (5 females, 4 males, ages 23-41 [31.11 ± 7.08 years]) to a 7-day protocol of oral administration of methylphenidate capsule (0, 15, or 30 mg, p.o.) followed by sublingual (s.l.) administration of lobeline tablets (0, 7.5, 15, or 30 mg, s.l.) after 1 h. Inactive doses of both sublingual and oral formulations were used as placebo. They observed that lobeline could improve working memory in a modest capacity (without significant improvement in their attention) in nine volunteers with ADHD.
Li et al. investigated the effect of s.c administrations of lobeline hydrochloride (1 or 3 mg/kg) on male C57BL/6J mice against methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP, 30 mg/kg)-induced cell death in vivo using L-dopa (80 mg/kg) and the potent and selective dopamine reuptake inhibitor, GBR12935 (10 mg/kg) as positive controls . The s.c. injections of lobeline, GBR12935, or L-dopa were done for 11 days 30 min prior to MPTP administration. They observed that MPTP induced locomotive effects detected in behavioral tests were significantly reduced by lobeline as compared to L-dopa and GBR12935. Further, they observed that the loss of neurotoxin-induced immunoreactivity was significantly reduced in the substantia nigra and striatum regions of the brain. The overall results indicated that lobeline may protect dopaminergic neurons and alleviate the symptoms of Parkinson's disease. Saline was used as the negative control.
Tamboli et al. investigated lobeline isolated from Lobelia nicotianaefolia in chemoconvulsant-induced seizures using diazepam as a reference standard (Tamboli et al., 2012). The anticonvulsant activity of the isolated lobeline was investigated at 5, 10, 20 and 30 mg/kg, intraperitoneal (i.p.) doses in Pentylenetetrazol (PTZ) and strychnine induced seizures in Swiss albino male mice. They found that isolated lobeline induces potent anticonvulsant activity against PTZ induced seizures by enhancing the brain gamma amino butyric acid (GABA) level at 20 mg/kg i.p. within a duration of 45 min after their administration. Saline was used as the negative control.
Mechanistically, lobeline was reported binding to the α4β2 nAChR with nanomolar affinity (Brioni et al., 1997) but with less potent affinity against muscarinic and acetylcholinesterase receptors (Terry et al., 1998). The high-affinity partial agonist property of lobeline against α4β2 nAChR was later identified as the mechanism for smoking cessation (Wu et al., 2006;Billen et al., 2012). Recently, Anitha et al. shed additional light on how smoking cessation by lobeline occurs through trapping in α4β2 receptors-containing acidic vesicles and how intracellular pH homeostasis plays a crucial role in it (Govind et al., 2017). Lobeline was also demonstrated to block the blood-brain barrier (BBB) basic amine transporter (Allen et al., 2003), and act as VMAT2 ligand (Zheng et al., 2005;Zheng et al., 2006). In another study, (−)-lobeline hydrochloride monohydrate (10 or 20 mg/kg, i.p.) was reported to reduce the DNA damage and oxidative stress induced by seizures in a pilocarpine-induced seizure model on 69 adult male CF-1 mice (da Costa E Silva et al., 2018). The administrations of pilocarpine (300 mg/kg) were performed after 30 min treatment of lobeline, diazepam (positive control) or saline (negative control).
Overall, to date, more than 610 scientifically peer-reviewed research articles carrying the pharmacological investigation of the pyridine alkaloid lobeline and its structural analogs were reported underlying its medicinal importance. However, the gastrointestinal side effects (due to nicotinic effects), the lethal toxicity due to its narrow therapeutic index, and other adverse side effects such as choking and cough limits the clinical utility of lobeline (Butler et al., 2001;Raj et al., 2005;Stead and Hughes, 2012;Heinzerling, 2013).

Beyond Lobeline: Other Bioactive Alkaloids
In the early 1950s, the German phytochemist Steinegger and team reported several key discoveries on the identification and pharmacological studies of the newer alkaloids from different Lobelia species. First, they reported the identification of a crystallized alkaloid named lurenine isolated from Lobelia urens (Steinegger and Grutter, 1950). Subsequently, they also identified two new alkaloids lophilin and lophilacrin in the alkaloid fraction of Lobelia siphilitica (Steinegger and Egger, 1952). In the 1950s, other studies began to reveal the distinctive pharmacological properties of the alkaloid constituents identified in different Lobelia species (Lendle and Richter, 1950), which has further raised tremendous interest in phytochemical research. Lobinaline 2, the only alkaloid found in the Lobelia cardinalis L. and the first binitrogenous alkaloid extracted from any Lobelia species was reported having a unique nAChR binding activity (Klosa, 1953;Kaczmarek and Steinegger, 1958;Brown et al., 2016). In addition to their equipotent α4β2-and α7-nAChRs binding profile, the purified lobinaline obtained from CHCl 3 fraction of air-dried aerial portions of L. cardinalis also exhibited potent 2,2-Diphenyl-1picrylhydrazyl (DPPH) free radical scavenging properties, inhibited in vitro [(3)H]-dopamine uptake in adult male Sprague-Dawley rats striatal synaptosomes, and potent dopamine transporter inhibition (Brown et al., 2016). Reportedly, lobinaline (stock solution: 1 mg/ml) scavenge DPPH free radicals with an EC 50 of 17.98 μM when examined in DPPH (stock solution: 600 μM) free radical scavenging assay. In contrast, lobeline (stock solution: 1 mg/ml) demonstrated a weak DPPH free radical scavenging property (EC 50 228.8 μM). In vivo electrochemical studies indicated that lobinaline in a short acting and likely competitive-capacity inhibits dopamine transporter function in the dorsal striatum of urethaneanesthetized rats. Interestingly, lobinaline (1 mM) was noticed significantly increasing 45 Ca 2+ entry in SH-SY5Y cells.
Lobelane 8, a defunctionalized analog of lobeline exhibited 10-15 fold more potent effects than lobeline in inhibition of the vesicular dopamine (DA) uptake by VMAT2, and inhibited (+)-methamphetamine-evoked DA release (Vartak et al., 2009;Nickell et al., 2010). It was also reported to possess a fivefold binding affinity than lobeline for the dihydrotetrabenazine binding site on VMAT2 (Miller et al., 2004;Zheng et al., 2005). In a study aimed at synthesizing lobelane analogs as therapeutic agents, replacement of the phenyl groups of lobelane with quinolyl groups resulted in a water-soluble analog called quinlobelane nine that possessed stronger VMAT2 inhibition (Vartak et al., 2010).
The phytochemical research conducted over the years suggested that alkaloids are the predominant secondary metabolites (∼46%) present in most of the Lobelia species known.
These alkaloids ubiquitously contain N-methylpiperidine ring or piperidine ring along with one or two substituents at the C 2 and/or C 6 position of the ring (Felpin and Lebreton, 2004;Folquitto et al., 2019).
One of the major disadvantages of the alkaloids derived from the Lobelia species has been their extreme resemblance in the chemical structures to each other, essentially sharing similar pharmacological, physicochemical, and toxicological profiles which also include cardiotoxicity (Figure 2). The alkaloids in most of the species of Lobelia (with lobeline being the most cited in the literature) are broadly known for presenting medicinal properties against drug abuse, neurological disorder, and respiratory stimulant. The limited medicinal properties of these alkaloids can be attributed to their binding properties at the nAChR receptors. In this present manuscript, we have only considered the publications where the alkaloid was biologically tested. Unfortunately, the bioactivity characterization of a large majority of those alkaloids was not yet reported and thus excluded. Moreover, to date, more than 80 alkaloids are known to be isolated from different species of Lobelia. The readers are recommended to the following review for the updates on other isolated alkaloids (Folquitto et al., 2019).

Beyond Alkaloids: The Bioactive Components of Other Classes
Over the years, the isolation and characterization of secondary metabolites derived from Lobelia species have garnered significant attention. It is now an established fact that most species of the genus Lobelia contain different classes of secondary metabolites including but not limited to flavonoids, terpenoids, fatty acids, polyacetylenes, coumarins, neolignans, polysaccharides, and amides in varying concentrations. In recent years, the identifications of these newer compounds also presented the opportunities to address the limited bioactivity of alkaloids mentioned earlier. Due to this fact, growing numbers of literature are being published. In this section, we will present a current account of these newer novel bioactive natural products derived from Lobelia species. A special focus has been given to accounting for the molecular mechanism of these newer secondary metabolites.
Since mid-to end-20th-century investigations into the pharmacology of components derived from Lobelia species were centered onto the alkaloids. In 1961, Sasaki et al. reported the isolation of a new glucofructan "sessilifolan" from Lobelia sessilifolia Lamb. I. (Sasaki et al., 1961;Sasaki and Mikami, 1962). It was perhaps the first report identifying other secondary metabolites than alkaloids in Lobelia. In 1986, Santosa et al. reported the in vitro as well as in vivo activity of the hot water extract of Lobelia chinensis against Ehrlich ascites tumor and Sarcoma 180 cells (Santosa et al., 1986). However, in subsequent decades, not much progress has been made on characterizing the other classes of secondary metabolites.
Subarnas et al. identified a novel antidepressant bioactive component, β-amyrin palmitate 10 in an extract of the leaves of Lobelia inflata (Subarnas et al., 1992). Further studies indicated that the antidepressant activity of β-amyrin palmitate 10 was related to the activation of the noradrenergic system by releasing [3H]norepinephrine from newly synthesized pools, with mianserin-like action (Subarnas et al., 1993b;Subarnas et al., 1993c). In a forced swimming test, β-amyrin palmitate at 10 mg/kg reduced the tetrabenazine-induced (100 and 200 mg/kg) increase in the duration of the immobility of mice whereas it has shown no effect when treated with alpha-methylpara-tyrosine (500 mg/kg). Similarly, at 5 and 10 mg/kg, it has decreased the duration of immobility in mice treated with 6hydroxy-dopamine (50 µg/mouse) and desipramine (15 mg/kg) without showing any effect to the treatment of mice with nomifensine plus 6-hydroxydopamine (Subarnas et al., 1993b). Physicochemically, beta-amyrin palmitate is a water-insoluble, extremely weak basic compound belonging to the triterpenoids compound class which contains six isoprene units. Other studies reported β-amyrin palmitate exhibiting different pharmacological properties such as induction of hypoactivity by inhibiting alpha 1-adrenoceptors (Subarnas et al., 1993a), anti-diabetic and anti-hyperglycemic activity by blocking the entry of glucose from the intestine (Nair et al., 2014), and antibacterial against Gram-positive and negative bacteria and antifungal activities (Abed et al., 2016).
The authors noticed that lobelitin A-D triggers dosedependent positive signals (monitored for 1 h) in dynamic mass redistribution (DMR) agonist assay in varying concentration. For example, at 200 pm, lobelitin A, B, and D were recorded exhibiting the largest DMR signal whereas at about 100 pm, lobelitin C exhibited a weaker signal at the maximum concentration of 250 µM. Based on these results, the authors suggested lobelitin A, B and D as the full agonist and lobelitin C as a partial agonist of GPR35. The structures and the apparent EC 50 and IC 50 values of lobelitin A-D are shown in Figure 3 and Table 1. GPR35 has been implicated in several diseased conditions including hypertension, coronary artery disease, and cancer, and is an area of active research (Divorty et al., 2015;Wei et al., 2017;Quon et al., 2020).
A neutral polysaccharide (LCPS) 18 was isolated from Lobelia chinensis lour. The monosaccharide composition of LCPS was observed as fructan composed of 2,1-linked-β-D-fructofuranosyl residue and α-D-glu-copyranosyl residue with α-D-Glcp-(1→ [1)-β-D-Fruf-(2] 15 glycosidic linkage. LCPS was identified as an inulin-type fructan with 2.6 kDa molecular weight with an effective therapeutic option in the treatment of obesity. Oral administration of this inulin alleviated obesity and reduced body weight in high-fat diet-induced mice . Specifically, the anti-obesity effects of LCPS evaluated in vivo in male Kunming mice treated with normal chow diet (D12450 J), high fat diet (D12492) and high fat diet plus LCPS (300 mg/kg/ day) for eight weeks showed 67% lower body weight gain as compared to high fat diet treatment. High fat diet plus glibenclamide (5 mg/kg/day) was used as a positive control which has shown 77% effect.
Ishimaru et al., isolated two new polyacetylene compounds lobetyolin 20 and lobetyol 21 from the hairy roots of Lobelia inflata (Ishimaru et al., 1991). Recently, He et al. studied the effect of lobetyolin on colorectal cancer (He et al., 2020). They found that lobetyolin induces colon cancer cells by inhibiting the ASCT2-mediated glutamine metabolism. Briefly, they have  reported that lobetyol induces anti-carcinoma effect in MKN45 cells and activates MAPK pathways associated with G 1 /S cell cycle arrest in a time-and dose-dependent manner (Shen et al., 2016). Specifically, the anti-proliferative effect of lobetyol in MKN45 cells was evaluated in an MTT assay at a varying concentration and time ranging from 50 µg/ml, 75 µg/ml, to 110 µg/ml and 12 h, 24 h, to 48 h, respectively. Lobetyol demonstrated an IC 50 of 71.47 ± 4.29 µg/ml at 48 h. Further, in MKN45 cells, an increasing dose of lobetyol ranging from 0, 50, and 75 µg/ml, to 110 µg/ml led to an increase in apoptotic population by 5.5%, 13.74%, 27.32%-31.57%, respectively. Two other polyacetylene isolobetyol 22 and lobetyol 21 isolated from Lobelia chinensis exhibited moderate cytotoxic activities against MSTO-211H and NCI-H292 cell lines (treated for 48 h). Isolobetyol exhibited the IC 50 values of 12.36 and 9.31 μM against the two cell lines; while the lobetyol showed the IC 50 values of 11.76 and 9.64 μM, respectively (Yang et al., 2014). In contrast, positive control (cisplatin) showed an IC 50 of 4.91 and 7.89 μM against MSTO-211H and NCI-H292 cell lines, respectively. However, the authors did not mention at which concentrations the compounds were tested.
In another report, Joshi et al. analyzed the essential oil composition of the aerial parts of Lobelia pyramidalis Wall and evaluated their antimicrobial activity (Joshi et al., 2011).  Serial doubling dilutions of the essential oil were prepared from 0.10 to 200 mg/ml and incubated in Mueller Hinton broth for the bacterial strains at 37°C for 24 h. The minimal inhibitory concentration and inhibition zone of 3.12 mg/ml and 18 mm, respectively was determined against the bacterial strain S. aureus. The authors suggested natural terpenoid perilla ketone could be the responsible chemical constituent. However, no further attempt was made to isolate and characterize the compound. In a recent study, Ma and colleagues identified 208 metabolites of Lobelia chinensis by gas chromatography-mass spectrometer (GC/MS) and conducted a network pharmacology-based investigation of their anti-diabetic mechanism (Ge et al., 2020). Their study revealed that 5-hydroxymethylfurfural 23 acts on GSK3B, TNF, and MAPK1 and affects the insulin resistance signaling pathway as the major mechanism of action. Also, acacetin might act on INSR, DPP4, and GSK3B. No biological evaluation was performed to validate the network pharmacology approach's presented findings, which remains one major drawback of this work.
We have also recently investigated the neuroprotection mechanism of the pharmacokinetically favorable, experimentally validated active compounds derived from the 17 Lobelia species (Table 2) based on network pharmacology and molecular modeling (Zheng et al., 2020). In this work, we also studied the chemical diversity of the validated natural compounds identified in the genus of the Lobelia species including the major components of Lobelia chinensis studied by Ma and Colleagues (Ge et al., 2020).
Fascinatingly, the aglycone part of the flavonoid such as diosmetin and 18 other aglycones constituted the densest cluster indicating their profound presence in the Lobelia species. The representative chemical structure of the top 10 clusters is shown in Figure 4B. We have also not performed the biological evaluations. However, in this review, we intended to summarize the experimentally determined chemical constituents of Lobelia and draw a comparison in their chemical structures (as shown in Figure 4).

Data Sources and Study Selection
The literature search was performed using PubMed and Scifinder over the period from the beginning of the database until October 2020. Initially, all the literature associated with the search term "Lobelia" were collected. In addition, the databases were also directly searched using the botanical name (e.g., "Lobelia chinensis"). In addition, as a third search strategy, the databases of the major research journals including Science Direct, Frontiers, Wiley, American Chemical Society, MDPI, Springer-Nature, and Taylor and Francis were also searched manually. Articles that have reported only isolation and identification of chemical constituents without biological activities were excluded. No unpublished literature data or conference proceedings were included. Except for a recent systematic review by Folquitto et al. (Folquitto et al., 2019), other articles that described reviews or systematic reviews were excluded. The systematic review by Folquitto et al. was used as an additional strategy to validate the references for the phytochemical details.

CONCLUSION
Undoubtedly, Lobelia is an important genus with profound medicinal value. A lot of work has already been done in understanding the pharmacological role of the alkaloids derived from different Lobelia species. Yet, growing investigations on the newer chemical classes indicate that there is certainly an untapped opportunity to obtain bioactive compounds with diverse chemical and pharmacological properties. In this work, we tried to provide the readers the most up-to-date account of the progress made so far, especially toward understanding the molecular mechanism and pharmacological characterizations of the novel bioactive components. Considering the progress being made in the last few years, we believe that this is a timely review to provide the scientific basis for future research aimed at studying the therapeutic potential of the newer chemical compounds derived from the Lobelia species.

AUTHOR CONTRIBUTIONS
QZ developed the idea and the review framework, and wrote the first draft of the manuscript. YW surveyed the literature, collected and validated the data. SZ provided the supervision of the overall work, and did the final editing. All authors contributed to corrections and editing of the subsequent revisions. All authors approve and agree with the content.