Natural Antispasmodics: Source, Stereochemical Configuration, and Biological Activity

Natural products with antispasmodic activity have been used in traditional medicine to alleviate different illnesses since the remote past. We searched the literature and compiled the antispasmodic activity of 248 natural compounds isolated from terrestrial plants. In this review, we summarized all the natural products reported with antispasmodic activity until the end of 2017. We also provided chemical information about their extraction as well as the model used to test their activities. Results showed that members of the Lamiaceae and Asteraceae families had the highest number of isolated compounds with antispasmodic activity. Moreover, monoterpenoids, flavonoids, triterpenes, and alkaloids were the chemical groups with the highest number of antispasmodic compounds. Lastly, a structural comparison of natural versus synthetic compounds was discussed.


Introduction
Antispasmodic compounds are currently used to reduce anxiety, emotional and musculoskeletal tension, and irritability. Although most of the available antispasmodic compounds are synthetic or semisynthetic, traditional uses of this group of compounds are still popular.
We collected information about natural compounds with antispasmodic activity isolated from terrestrial plants. We searched the databases of Google Scholar, PubMed, and Sci-Finder and compiled the information about 248 compounds published until December 2017. This review focuses on the antispasmodic activity of isolated compounds and activities from extracts without further purification are not discussed.

The Neurons
Nerve cells or neurons are responsible for receiving, conducting, and transmitting signals. A neuron consists of a nucleated body, a long thin extension called an axon, and several dendrites or prolongations extended from the cell body. Axons conduct signals from the nucleated body towards distant targets, while dendrites provide an enlarged surface area to receive signals from the axons of other neurons.
Signal transmission through axons is driven by a change in the electrical potential across the plasma membrane of neurons. This plasma membrane contains voltage-gated cation channels, which are responsible for generation of action potentials. An action potential is triggered by a depolarization of the plasma membrane or a shift to a less negative value.
In nerve and skeletal muscle cells, a stimulus can cause sufficient depolarization to open voltage-gated Na + channels allowing the entrance of Na + into the cell. This influx of Na + depolarizes the membrane further causing the opening of more Na + channels. To avoid a permanent influx, Na + channels are able to reclose rapidly even when the membrane is still depolarized. This function is based on the presence of voltage-gated K + channels, which are responsible for K + efflux equilibrating the membrane potential even before the total inactivation of Na + channels. In some cases, the action potential in some muscles depends on voltage-gated Ca 2+ channels.

Transmission of Signals.
The transmission of signals occurs mainly between neurons or from neurons to skeletal muscles, which are the final acceptors of electrical signals, causing a muscular contraction.

Signal Transmission between Neurons.
Neuronal signals are transmitted between neurons at specialized sites of contact known as synapses. Neurons are separated by a synaptic cleft where a release of a neurotransmitter occurs. This neurotransmitter is stored in vesicles and is released by exocytosis. Upon triggering, the neurotransmitter is released into the cleft provoking an electrical change in the postsynaptic cell by binding to the transmitter-gated ion channels. To avoid a continuous electrical change and to ensure both spatial and temporal precision of signal transmission, the neurotransmitter is rapidly removed from the cleft either by specific enzymes in the synaptic cleft or by reuptake mediated by neurotransmitter carrier proteins [1].
Neurotransmitters can also open cation channels causing an influx of Na + and then called excitatory neurotransmitters (e.g., acetylcholine, glutamate, and serotonin) or produce an opening of Cl − channels and then inhibiting the signal transmission by maintaining the postsynaptic membrane polarization [e.g., -aminobutyric acid (GABA) and glycine].

Neuromuscular Signal Transmission.
The transmission of electrical signals to muscles involves five sequential and orchestrated steps: (i) nerve electric signal reaches the nerve terminal, (ii) it depolarizes the plasma membrane of the terminal, (iii) voltage-gated Ca 2+ channels opens causing an increase in Ca 2+ concentration in the neuron cytosol, and (iv) release of acetylcholine into the synaptic cleft is triggered. Acetylcholine binds to acetylcholine receptors in the muscle plasma membrane opening Na + channels and provoking a membrane depolarization. This depolarization enhances the opening of more Na + channels causing a self-propagating depolarization. The generalized depolarization of the muscle plasma membrane activates Ca 2+ channels in specialized regions on the membrane causing Ca 2+ release from the sarcoplasmic reticulum (Ca 2+ storage) into the cytosol.
As a consequence of an increase in the Ca 2+ concentration, myofibrils in the muscle cell contract. The increase of Ca 2+ in the cytosol is transient because Ca 2+ is rapidly pumped back into the sarcoplasmic reticulum causing a relaxation of the myofibrils. This process is very fast and Ca 2+ concentration at resting levels is restored within 30 milliseconds [2].

Receptors
The autonomic nerve system controls and monitors the internal environment of the body. The input of its activity is provided by neurons that are associated with specific sensory receptors located in the blood vessels, muscles, and visceral organs (Table 1). According to the neurotransmitter secreted, these neurons are classified as adrenergic or cholinergic. The adrenergic neurons secrete the neurotransmitter noradrenalin termed also norepinephrine. Adrenergic receptors include the types and , which are further categorized as 1 , 2 , 1 , 2 , and 3 . On the other hand, cholinergic neurons secrete acetylcholine, which induces a postsynaptic event.
There are two types of cholinergic receptors, the nicotinic receptor (abundant at the neuromuscular junction) and the muscarinic receptor (abundant on smooth and cardiac muscles and glands).
There are several agonists (neurotransmitters, hormones, and others) able to bind to specific receptors and activate the contraction of smooth muscle. Upon binding the agonist to the receptor, the mechanism of contraction is based on an increase of phospholipase C. This enzyme hydrolyzes phosphatidylinositol 4,5-bisphosphate located on the membrane, producing two powerful secondary messengers termed diacylglycerol (DG) and inositol 1,4,5 triphosphate (IP3). IP3 binds to specific receptors in the sarcoplasmic reticulum, causing release of Ca 2+ within the muscle. DG together with Ca 2+ activates the protein kinase C (PKC), which phosphorylates specific proteins. In most smooth muscles, the contraction process commences when PKC phosphorylates Ca 2+ channels or other proteins that regulate the cyclic process. For instance, Ca 2+ binds to calmodulin (a multifunctional intermediate calcium-binding messenger protein), triggering the activation of the myosin light chain (MLC) kinase, which phosphorylates the light chain of myosin and together with actin carries out the process of initiating the shortening of the smooth muscle cell [147]. However, the elevation of the intracellular concentration of Ca 2+ is transient, and the contractile response is maintained by a mechanism sensitized by Ca 2+ modulated by the inhibition of myosin phosphatase activity by Rho kinase. This mechanism sensitized to Ca 2+ is initiated at the same time that phospholipase C is activated and involves the activation of the small RhoA protein bound to guanosine triphosphate (GTP). Above activation, RhoA increases the activity of Rho kinase, leading to the inhibition of myosin phosphatase. This promotes the contractile state, since the myosin light chain cannot be dephosphorylated [147].
Relaxation of smooth muscle occurs as a result of either removing the contractile stimuli or by the direct action of a substance that stimulates the inhibition of the contractile mechanism. In any circumstance, the relaxation process requires a decrease in the intracellular Ca 2+ concentration and an increase in the activity of the MLC phosphatase. The sarcoplasmic reticulum and plasma membrane remove Ca 2+ from the cytosol. Na + /Ca 2+ channels are located on the plasma membrane and help to reduce the intracellular concentration of Ca 2+ . During relaxation, other contributors that restrict the Ca 2+ entry into the cell are the voltage-operated channels and Ca 2+ receptors in the plasma membrane, which remain closed [147].

Spasmodic Compounds
The historical antecedents date from the year 1504 when South American natives inhabiting the basins of the high Amazon and the Orinoco prepared a mixture of alkaloids termed curare. This substance was placed in the tips of arrows in order to hunt (prey paralyzing) and fight in wars. Curare produces muscle weakness, paralysis, respiratory failure, and death [148]. In 1800, Alexander von Humboldt, identified that curare was made from the extracts of the species Chondrodendron tomentosum and Strychnos toxifera.
In 1935, the French physiologist Claude Bernard managed to isolate the alkaloid d-tubocurarine from the curare [149]; and one year later, it was elucidated that this compound had the ability to inhibit acetylcholine, blocking the transmission of nerve impulses to the muscles [150]. Lastly, new benzylisoquinoline alkaloids were isolated from curare by Galeffi et al. in 1977 [151, 152].
In 1822, the pharmacist Rudolph Brandes obtained an impure alkaloid from Atropa belladonna (Solanaceae), which after purification was named atropine. Interestingly, atropine was not produced as a natural compound from the plant and it was a derivative generated from the alkaloid hyoscyamine during the process of purification [153]. It is important to note that atropine has been naturally found in small quantities in other members of the Solanaceae family such as Datura stramonium, Duboisia myoporoides, and Scopolia japonica [154][155][156].
The use of the plant Papaver somniferum (opium poppy) (Papaveraceae) dates back to about 4000 BC. At present the plant is only used to extract a base material for the manufacture of other alkaloids, such as noscapine and codeine, both discovered by the French pharmacist Pierre-Jean Robiquet in 1831 and 1832, respectively [157]. In 1848, papaverine was another substance extracted from the same plant by the German chemist Georg Merck [158], which is rarely used today because of the high doses needed (approximately 6 to 12 mg). However, it is still used as a control in experimental models with the purpose of studying antispasmodic activity of plant extracts.
In the 20 th century, extracts and powders derived from A. belladonna were widely used as antispasmodics, but from the 1950s these preparations were displaced by synthetic and semisynthetic anticholinergic compounds in order to obtain a better response [159], such as the case of methocarbamol and guaifenesin. On the other hand, a series of compounds such as dantrolene, glutethimide, methaqualone, chlormezanone, metiprilone, and ethchlorvynol were introduced to replace the meprobamate, which had to be withdrawn from the market in 1960 due to problems resulting from use such as abstinence, addictions, and overdoses.
In 1962, the Swiss chemist Heinrich Keberle synthesized baclofen, which can be obtained by reacting glutarimide with an alkaline solution [160]. Glutarimide can also be found in plants such as Croton cuneatus and C. membranaceus (Euphorbiaceae) [161,162].
The arrival of the quaternary compounds of nitrogen reinforce their peripheral anticholinergic activity offering also the advantages of being poorly absorbed in the gastrointestinal tract, producing a more powerful and longer lasting sedative effect unlike atropine [1]. For example, ipratropium bromide was developed by the German company Boehringer Ingelheim in 1976 and used to treat asthma. This compound was obtained by reacting atropine with isopropyl bromide [163]. Another quaternary compound was the nbutylhyoscine bromide, which is possible to obtain by the organic synthesis of scopolamine and the cimetropium bromide found in the A. belladonna [164]. Although at present the preparations of plant mixtures are no longer used for therapeutic purposes, these compounds formed a part of and served as the basis for modern pharmacology for their applicability as antispasmodics and anesthetics.
Spasms are involuntary contractions of the muscles, which are normally accompanied by pain and interfere with the free and effective muscular voluntary activity. Muscle spasm can originate from multiple medical conditions and is often associated with spinal injury, multiple sclerosis, and stroke.
Spasticity and rigidity are caused by a disinhibition of spinal motor mechanisms. There are several scenarios where a muscle can produce a spasm: (i) unstable depolarization of motor axons; (ii) muscular contractions persist even if the innervation of muscle is normal and despite attempts of relaxation (myotonia); (iii) after one or a series of contractions, the muscle can decontract slowly, as occurring in hypothyroidism; and (iv) muscles lack the energy to relax.

Distribution of Spasmodic Compound in Nature.
Spasmodic compounds are widely distributed in nature (Table 2). Frequently, these compounds are found in animals that paralyze their preys or used for defense. Some examples include the venom of the black widow and tarantula spiders [11,165] and the venom of snakes [166]. Plants also produce spasmodic metabolites, such as strychnine, an alkaloid obtained from the tree Strychnos nux-vomica (Loganiaceae). Furthermore, microorganisms synthesize spasmodic compounds such as the neurotoxins tetanospasmin and botulinum toxin from the Gram-positive bacteria Clostridium tetani and C. botulinum, respectively. These toxins produce a toxic disorder, which is characterized by persistent spasms of skeletal muscles on spinal neurons similar to strychnine.

Mechanisms of Antispasmodic Activity of Natural Products.
Antispasmodic compounds exert their activity in different ways, such as antispasmodic activity through inhibition of the response to the neurotransmitters 5-hydroxytryptamine (5-HT) or serotonin and acetylcholine. However, other authors attribute the antispasmodic effect to (i) capsaicinsensitive neurons, (ii) the participation of vanilloid receptors [167], (iii) the activation of K + ATP channels, (iv) the blockade of Na + channels and muscarinic receptors, (v) the reduction of extracellular Ca 2+ , or (vi) the blockade of Ca 2+ channels [22,168,169]. The above is merely a reflection of the ambiguity of the studies showing the mechanisms of action of the antispasmodic compounds [36]. For example, the hydroalcoholic extract of Marrubium vulgare showed antispasmodic effect, having the ability to inhibit the Blocks the cholinergic post-synaptic response [16] neurotransmitters acetylcholine, bradykinin, prostaglandin E2, histamine, and oxytocin [170], whereas a dual effect of antidiarrheal and laxative activities was reported in Fumaria parviflora [171].

Gastrointestinal
Model. The small intestine is characterized by its large surface area as a result of its circular folds, villi, and microvilli. It is the longest part of the GI system (approximately 5 meters) and comprises about 5% of its initial length, which corresponds to the duodenum (characterized by the absence of the mesentery) and then the jejunum (around 40% of the intestinal length), ending with the ileum. It is the organ of absorption of nutrients and digestion in organisms. These functions are carried out mainly in the duodenum and jejunum.
The main types of bowel movement are the segmentation and peristaltism. The segmentation is most frequent in the small intestine and consists of contractions of the circular muscle layer in very close areas. Contractions last for 11-12 and 8-9 contractions per min in the duodenum and ileum, respectively. When this segmentation is rhythmic, the contractions are alternated with relaxation. This type of movement results in a mixed effect of the chyme (acidic fluid that passes from the stomach to the small intestine) with the digestive secretions, allowing an optimal contact with the intestinal mucosa. In the case of peristalsis, contractions of successive sections of the circular smooth muscle cause the movement of the intestinal contents in anterograde form. The short peristaltic movement also takes place in the small intestine, but less frequently than the segmentation movements. Peristaltic waves rarely cross more than 10 cm of intestine and, due to the low frequency of propulsion of the chyme, it is in this zone where digestion and absorption are preferably carried out.
Peristalsis is regulated mainly by the nervous action of the myenteric plexus (major nerve supply to the gastrointestinal tract that controls GI tract motility) in the intestinal wall.
The diversity of experimental models used for the testing of antispasmodic compounds is large. These models mainly use isolated organs or live animals. Once the organ is extracted from the animal, the intestinal motility is assessed with the administration of a substance. The use of extracted organs can be sustained for hours when placed in a physiological solution, such as Ringer, Jalon, Tyrode, and Krebs [172].
The most used organs to perform the studies are guinea pig ileum, duodenum, heart, trachea, and jejunum. The same organs can be also extracted from rabbit, mouse, rat, and hamster ( Table 3). The preparation of ileum is preferred because it evaluates the spasmolytic activity. However, although the jejunum contracts spontaneously, it allows evaluating the spasmolytic activity directly and without the use of an agonist [173].
Some advantages of performing ex vivo experiments are as follows: (i) different substances can be evaluated in fresh tissues without absorption factors, metabolic excretion or interference due to nerve reflexes; (ii) it is possible to quantify the effect produced by a precisely determined drug; and (iii) it is easier to obtain dose-effect curves, such as the smooth muscle where the contraction obtained under the influence of a spasm or in tissue homogenates is measured by determination of the enzyme activities [172,174].

Guinea Pig Ileum and Rat Stomach.
The ileum is removed and cut in strips of approximately 2 cm long and then placed in a bath filled with an isotonic solution as mentioned earlier. Electrophysiological studies are performed by graphically recording the contractions with the aid of a transducer, which is calibrated 30 min before the treatment begins. A range of 0.01 to 0.03 M is generally used to determine dose response curves of the antispasmodic substance [175].
In rats, the stomach is removed and the corpus and fundus are cut in strips of approximately 5 mm x 15 mm and placed on a prewarmed warm solution as mentioned before.

Compounds Used to Elicit a Spasmodic Activity.
The main compounds used are acetylcholine, atropine, BaCl 2 , carbachol, histamine, KCl, and serotonin.
Acetylcholine is a postganglionic neurotransmitter in the parasympathetic neurons that innervate the intestine. The response to acetylcholine is regulated by activation of the two types of muscarinic receptors: M2 and M3 [176]. The activation of these receptors causes contractions by increasing the intracellular concentration of Ca 2+ via IP3 [176]. Atropine is a competitive reversible antagonist of muscarinic acetylcholine receptors M1, M2, M3, M4, and M5.
KCl increases the voltage-operated Ca 2+ channel activity by increasing intracellular free Ca 2+ in smooth muscle [180]. Serotonin is also an important neurotransmitter mainly stored in the digestive tract, affecting the secretory and motor activities. At high concentrations, it acts as a vasoconstrictor by contracting endothelial smooth muscle directly or by potentiating the effects of other vasoconstrictors [181,182].

Antispasmodic Activity of Natural Compounds
Compounds isolated from terrestrial plants have shown the ability to function as antispasmodic compounds. The chemical group with the highest number of members of antispasmodic compounds is the monoterpenoid group (41 compounds) followed by flavonoids (35 compounds), alkaloids (with 33 compounds), and triterpenes with 31 ( Figure 1). Although we summarize in Table 3 248 compounds, in most of the cases the mechanism behind their activity has not been elucidated.

Mutagenicity
Studies related to the mutagenicity of antispasmodics are very scarce. This topic has been underestimated when testing the bioactivities of ethnomedicinal plants. Probably the most useful method to determine the mutagenicity of natural products or plant extracts is the Ames method [183]. This test is based on the rate of mutations detected in genetically modified strains of Salmonella typhimurium. Moreover, this test has also been developed to detect mutagenicity of metabolized compounds in the liver. In this situation, a mixture of liver 8 BioMed Research International KCl in guinea pig ileum IC [27] (S)-(+)-Carvone                Orphenadrine Skeletal muscle relaxant that is used for the treatment of acute muscle aches, pain, or spasms.

Phenylpropanoids
Baclofen GABA B Spinal cord injury, cerebral palsy, and multiple sclerosis Idrocilamide Prevents release of intracellular Ca 2+ Skeletal muscle relaxant and muscular pain enzymes (S9 microsomal fraction) is used to mimic the metabolites that will be produced in the liver [184]. Few studies have been performed to determine the mutagenicity of natural products with antispasmodic activity. For example, the flavonoids quercetin and luteolin were tested using the Ames method and the appearance of point mutations in four of the tested bacterial strains was shown [185]. In another study, the extracts of the plants Brickellia veronicaefolia, Gnaphalium sp., Poliomintha longiflora, and Valeriana procera were studied. Compounds isolated from these plants are listed as antispasmodic compounds ( Table 3). Results of the mutagenicity test indicated that Gnaphalium sp., Poliomintha longiflora (used in the Mexican cuisine and as a traditional medicine), and Valeriana procera induced mutagenesis in the tested bacterial strain [186].

Chemical Similarities between Natural and Synthetic Antispasmodic Compounds
To determine whether or not there is an analogy between synthetic (Table 4) and natural antispasmodic compounds, the structures of both groups were compared. Results showed that no similarities were found except for alkaloids, amines, and amino acids. One of the main differences is that commercial alkaloids are methylated in their nitrogen to make them positive, increasing their solubilities because of salt formation. In contrast, natural products have no positive nitrogen, rendering the molecule neutral and pH dependent. Thus, the compound may or may not be protonated, resulting in a change in its solubility and consequently a change on the targeting tissues.
The comparison can perhaps be focused on the distribution of charges rather than by functional groups or families of compounds, emphasizing the electron distribution. For example, a physical characterization such as the heat of formation, the surface electrostatic potential, the molecular weight, the surface tension, the refractive index, the lipophilicity, and others has been used to characterize the structure-activity relationship of alkaloids extracted from the Amaryllidaceae family [187]. These alkaloids were selected because of their ability to inhibit the effect of the acetylcholinesterase enzyme.
Of special interest is the natural compound salvinorin A isolated from the Mexican hallucinogenic Salvia divinorum (Lamiaceae) used in the traditional medicine as an antidiarrheal. It has been reported that this compound inhibited the intestinal motility through the activation of other receptors such as -opioid receptors (KORs). Upon inflammation of the gut, the cannabinoid C, B 1 , and KOR receptors are upregulated. It appears that salvinorin A interacts in the cross-talk between these receptors with a reduction of the inflammation as demonstrated in murine and guinea pig models [188,189].
Analysis of the similarities between synthetic and natural antispasmodic structures is depicted in Table 5.

Conclusions
A large number of natural products with antispasmodic activities have been reported. Although the use of plants in traditional medicine is still relevant, it is necessary to perform new studies to elucidate the mechanism of action  of antispasmodics. Moreover, more information about cytotoxicity and mutagenesis should be explored to ensure that these compounds are safe for consumption. The findings of this study corroborated the need for safety studies on plants extensively used for primary health care in countries such as Mexico. Such studies must be carried out before continuing with the widespread use of some species, which may provoke long-term and irreversible damage.

Conflicts of Interest
The authors declare no conflicts of interest.