Introduction
Plants are incredibly important for life on Earth. They absorb CO2 to produce oxygen, provide habitat, and regulate water cycles, and they are the most
important source of food for humans and animals. Since ancient times, plants have
also been exploited to heal human diseases. For instance, Mesopotamian populations
left written records of approximately 12 recipes for drug preparation [1]. More detailed usage in medicine of plants for medications comes from the Ebers
Papyrus (1550 BC), which cites more than 800 medications involving many ingredients,
such as pomegranate, willow, poppy, and mandrake [2]. These recipes represent the earliest documented records of medicines, even though
the isolation of the first pharmacologically active substances from one of the putative
ingredients listed in the ancient texts awaited approximately 5000 more years [3]. The isolated substance originally
named “principium somniferum”, today known as morphine, marked the beginning of the drug discovery era. Plant-derived
drugs are specialized or secondary metabolites that are grouped by their chemical
formula alkaloids, flavonoids, terpenoids, and phenolics. Secondary metabolites are
small molecules that are produced by metabolism and are dispensable for plant growth.
They play a key role in the plant–environment interaction, including plant–plant communication
and defense against herbivores, fungi, viruses, and bacteria. But they are also involved
in the response to abiotic stresses. It is widely believed that plant secondary metabolites
are pivotal for environmental adaptation by supporting the growth of symbiotic organisms
and providing protection toward biotic and abiotic stressors [4], [5], [6].
Among plant secondary metabolites, alkaloids are low-molecular-weight nitrogen-containing
organic compounds, typically alkaline, produced by most organisms, including bacteria,
fungi, plants, and animals. Alkaloids are a large group of secondary metabolites;
more than 20,000 have been identified in 20% of known vascular plants [7]. They are biosynthesized by different precursors, mostly aminoacidic, and can be
classified according to their biogenesis or their ring structure [8]. In plants, alkaloids are thought to enhance plant fitness through a broad range
of functions such as protection from ultraviolet radiation [9], oxidative stress [10], [11], or pathogens [12], as well as allelopathy toward other plants. The most characterized function is
the protection against the microorganisms, i.e., bacteria [13], [14] and viruses [15], [16], but also against insects due to their strong biological activities, that interfere
with many cell processes.
By virtue of their biological activity, alkaloids are extensively used in medicine;
therefore, this review provides a focus on the alkaloids that are commonly used in
the ophthalmology field, such as atropine and pilocarpine, and on those showing potential
in treating eye disorders such as berberine, caffeine, and reserpine. Eye disorders
leading to vision impairment or blindness affect at least 2.2 billion people worldwide
and have a negative impact on the quality of life and an enormous cost to national
health systems and productivity. Many of these disorders can be treated and the vision
impairment improved, but finding new drugs is never-ending research, and plant-derived
compounds are an incredible resource. However, the huge exploitation of plant-based
drugs requires high industrial production that cannot be fulfilled only by extraction
from plants. On the other hand, chemical synthesis can be challenging and expensive;
hence, biotechnology approaches have been developed
to meet the pharmaceutical demand, and the last part of this review wants to offer
an overview of this topic.
Exploitation of Plant Alkaloids in Ophthalmology
Atropine, a tropane alkaloid
Atropine belongs to the group of tropane alkaloids, characterized by a tropane ring
in their structure. Tropane alkaloids are present in many plants belonging to the
Solanaceae family, including Datura Stramonium, Atropa mandragora, Atropa Belladonna, and many others. All these plants were known to have potent effects on humans for
millennia, and in fact, they were used as drugs, poisons, and hallucinogens. Atropine
was isolated for the first time from the roots of Atropa Belladonna by the pharmacist Heinrich F. G. Mein in 1831 and consists of a racemic mixture of
D- and L-hyoscyamine characterized by a tropane ring group [17] ([Table 1]). Even though D- and L-hyoscyamine have distinct biological activities, atropine
formulation is pharmacologically accepted because it is stable and reliable [18]. Atropine is known to be a non-specific reversible antagonist of muscarinic
receptors, which respond to the neurotransmitter acetylcholine, and they are fundamental
for the correct functioning of the central nervous system. In the eyes, muscarinic
receptors have been found in all ocular tissues ([Fig. 1]) where they regulate the activity of the conjunctival goblet cells, iris sphincter,
and circular muscular fibers of the ciliary body, thus influencing tear production,
pupil size, accommodation of the lens, eye development and growth, and retina function
[19], [20]. The effects of atropine on the eyes have been known for a long time; Cleopatra
and Italian women during the Renaissance used extracts of Atropa belladonna as eye drops to dilate pupils and look more attractive [21]. The pupil dilatation effect, or mydriasis, is due to the relaxation of the iris
sphincteric muscle upon atropine binding to the muscarinic receptor M3
([Fig. 2]), antagonizing acetylcholine, combined with the unopposed action of the dilatator
muscle. It routinely helps with the ophthalmoscopic evaluation of the posterior part
of the eye, including the peripheral retina.
Table 1 Summary of the alkaloids presented in this review, with their structure and biological
activity. The list of the plants producing the alkaloids and their applications in
the ophthalmology field are also reported.
|
Drug
|
Alkaloid Type
|
Biological Activity
|
Structure
|
Plants
|
Eye Disease
|
|
Atropine
|
Tropane alkaloid
|
Antagonist of muscarinic receptors
|
|
Family of Solanaceae
|
Myopia
|
|
Pilocarpine
|
Imidazole alkaloid
|
Agonist of muscarinic receptors
|
|
Genus Pilocarpus
|
Acute close-angle glaucoma
|
|
Berberine
|
Isoquinoline alkaloid
|
Antioxidant and anti-inflammatory
|
|
Families of
Berberidaceae, Annonaceae, Papaveraceae, Ranunculaceae,
Rutaceae
|
Diabetic retinopathy
|
|
Reserpine
|
Indole alkaloid
|
Interactor of vesicular monoamine transporters and competitive inhibitor of endogenous
monoamines
|
|
Genus Rauwolfia
|
Retinal ciliopathies
|
|
Caffeine
|
Methylxanthine alkaloid
|
Antagonist of adenosine receptors
|
|
C. arabica, C. sinensis, Theobroma plants, P. cupana, I. paraguariensis
|
Glaucoma, cataract
|
Fig. 1 Schematic representation of eye tissues (a) and cell types in the retina (b).
Fig. 2 Representation of atropine and pilocarpine mechanism of action. Both atropine and pilocarpine bind muscarinic receptors (MR), which are seven transmembrane
proteins responding to the neurotransmitter acetylcholine (Ac) and controlling the
contraction of the iris and ciliary muscles. Atropine acts as an antagonist of the
receptor; hence, it avoids the binding of Ac, inhibiting the activation of the receptor,
and the eye muscles become relaxed. This leads to pupil dilatation and thinning of
the lens that focuses on distant objects. In contrast, pilocarpine acts as an agonist
enhancing the function of muscarinic receptors leading to eye muscle contraction.
Hence, the pupil constricts, and the lens thickens, increasing the refractive power
and the focus on close objects. Moreover, pilocarpine facilitates trabecular meshwork
outflow, contributing to the reduction in IOP.
The inhibition of the M3 receptors of the ciliary muscle, which causes its relaxation,
induces cycloplegia (paralysis of the accommodation of the lens) ([Fig. 2]), which is useful for the correct evaluation of refractive errors, especially in
children, who may frequently show an excess of accommodation. The cycloplegic property
of the atropine is also applied for relieving pain caused by keratitis and anterior
uveitis by blocking the ciliary spasm. In the case of uveitis, the concomitant mydriatic
effect prevents the formation of synechiae between the iris and the cornea (anterior
synechiae) or the lens (posterior synechiae), avoiding complications such as an increase
in intraocular pressure (IOP).
The cycloplegia induced by atropine also showed beneficial effects in the treatment
of myopia. Myopia is characterized by blurred vision of distant objects, which can
be due to the increase in the axial length of the eye that causes the light to focus
in front of the retina instead of on the retina [22]. Despite lenses and laser surgery being largely used in the management of myopia,
atropine seems to be a very effective pharmacological therapy, at least for children
[23]. In fact, it is largely used in Asia, while it is less diffused in Europe because
of possible side effects such as photophobia and poor visual acuity [24]. However, many pieces of evidence suggest that atropine may additionally prevent
myopia via a non-accommodative mechanism (revised in [25]). Although the exact mechanism by which atropine reduces myopic defect has not been
clarified yet
[26], [27], many studies showed how this alkaloid affects different eye tissues.
Atropine treatment seems to revert the remodeling of the sclera occurring in myopic
eyes, consequently improving visual acuity [28], [29], [30], [31], [32] ([Fig. 1]). In the myopic eyes, the composition of the extracellular matrix of the sclera
is altered, leading to a thinning of the tissue as the axial length of the eye increases
and the myopia progresses [33]. In the retina, atropine administration causes the release of dopamine [34], which is a well-known neuromodulator for myopia. However, recent data show that
the positive effect of atropine on form-deprivation myopia in chicks is independent
of the atropine-mediated release of dopamine, given that the co-administration of
atropine with a dopaminergic antagonist does not block the
protection provided by atropine [35]. This data should be validated in other model organisms and other forms of myopia,
but they point out the importance of further dissecting the role of retinal tissue
in the atropine-mediated correction of myopia. Zhu et al., 2022, carried out a proteomics
experiment in guinea pigs to identify new proteins and pathways of retinal cells that
could be affected by atropine administration [36]. The analysis revealed that eukaryotic initiation factor 2 (EIF2) signaling and
glycolysis are significantly affected by atropine. Some pieces of evidence suggested
that these two pathways could be misregulated in myopia, but they have never been
associated with atropine treatment; hence, they could be the subjects of future investigations
to deeply understand the atropine-mediated effects on the retina.
Lastly, atropine has an effect also on the choroid, which is positioned between the
sclera and retina ([Fig. 1] a). It is highly vascularized, and its main function is to supply oxygen and nourishment
to the retina. Choroid is involved in myopia development because its thickness determines
the position of the retina and it produces factors for eye growth and sclera remodeling
[37]. Atropine administration at low concentrations (between 0.01% and 0.05%) increased
choroid thickness in children [38], [39] and adults [40], contributing to the recovery of the myopic defect.
In conclusion, atropine is an important drug in ophthalmology that deserves to be
further investigated, and it could be used as a tool to define the molecular mechanisms
underlying myopia.
Pilocarpine, an imidazole alkaloid
Pilocarpine is an imidazole alkaloid, synthesized from L-histidine [11]. It is extracted from the leaves of South American plants belonging to the Pilocarpus genus ([Table 1]), which are still used for the commercial production of this alkaloid. The medical
properties of jaborandi (the common name of Pilocarpus plants) extracts had already been known by South Americans for a long time and in
the 19th century were introduced into Europe where, in 1875, changes in eye accommodation
and pupil contraction were observed for the first time in patients treated with an
ophthalmic solution made of jaborandi extracts and glycerin [41]. Pilocarpine interacts with M3 receptors, but unlike atropine, pilocarpine is an
agonist, hence causing the activation of these receptors ([Fig. 2]). It is worth pointing out that studies on atropine and pilocarpine helped to
formulate the receptor theory of drug action. In fact, John Newport Langley, one of
the founders of this theory, showed that the administration of only a certain quantity
of atropine could counteract the effect of a determined quantity of pilocarpine on
the heartbeat and salivary secretion [42]. These experiments led him to formulate the hypothesis that the activity of these
alkaloids depends on the binding to specific molecules in the cells, which were afterward
discovered to be the muscarinic receptors, and no less important, it was learned that
the effect is dose-dependent. At the ocular level, the opposite activity of these
two alkaloids can be explained by several observations, such as pilocarpine-induced
myopia in both rabbit and guinea pigs [43], [44], while atropine reduces myopia defect. Moreover, choroidal thickness is reduced
by pilocarpine [45] and
increased by atropine, and lastly, the pupil constricts upon pilocarpine administration
[41], while atropine dilatates it.
In 1877, Adolf Weber introduced the cholinergic agent pilocarpine as a treatment for
glaucoma [46]. The term glaucoma includes a variety of forms that differ in cause, risk factors,
and symptoms, but they all have in common the reduction in the visual field caused
by the degeneration of the retinal ganglion cells (RGCs), the thinning of the retinal
nerve fiber layer composed of the axons of the RGCs, and a consequently increased
cupping of the optic disc [47]. Actually, the only modifiable risk factor for glaucoma is lowering IOP, which can
be obtained by medical treatment, surgery, or laser therapy [48]. Pilocarpine has been used in glaucoma to lower the IOP because of its effects on
the trabecular meshwork. The trabecular meshwork, located in the iridocorneal angle,
allows the drainage of the aqueous humor into the Schlemm canal, which is connected
to the blood system. The dysfunction or
the blockage of this system causes the accumulation of aqueous humor in the eye and
the elevation of the IOP, which, if not treated, causes damage to the optic nerve
typical of glaucoma.
In the type of glaucoma known as open-angle glaucoma, pilocarpine lowers the IOP because
it increases outflow through the trabecular meshwork, induced by the contraction of
the ciliary muscle and the expansion of the anterior chamber of the eye. In angle
closure glaucoma, in which the iridocorneal angle is closed and the trabecular meshwork
is obstructed, the IOP can rise dramatically and in a short time. In this case, pilocarpine
opens the trabecular meshwork and the Schlemm canal by reducing pupil size and contributing
to the decrease in IOP [49], [50], [51] ([Fig. 2]). Pilocarpine can be also used before a laser iridotomy is performed or in patients
in whom the laser iridotomy is not resolutive. However, to date, pilocarpine is not
used in the chronic treatment of glaucoma because it has several ocular and systemic
side effects (including induced myopia,
cataract development, retinal detachment, bradycardia, vomiting, salivation, and bronchial
spasm) [52], and more effective drugs exist. Furthermore, in patients with glaucoma linked to
lens abnormalities (e.g., spherophakia, phacomorphic glaucoma, and pseudoexfoliative
syndrome), pilocarpine may cause a paradoxical IOP increase because of the mycotic
effect that determines an anterior shift of the lens–iris diaphragm and the consequent
closure of the iridocorneal angle [53]. Lastly, more recently, many clinical trials have shown the benefit of low-concentrated
pilocarpine administration on patients with presbyopia [54], [55], [56], [57], which is the progressive inability to focus on close objects associated with the
elderly.
In addition to the ophthalmology field, pilocarpine is used for the treatment of several
other diseases, pointing out the valuable potential of this alkaloid.
Berberine, an isoquinoline alkaloid
Berberine belongs to the group of isoquinoline alkaloids that are biosynthesized from
tyrosine in many plants of the families Berberidaceae, Annonaceae, Papaveraceae, Ranunculaceae,
and Rutaceae (for a detailed review check [58]) ([Table 1]). B. vulgaris (barberry), belonging to the Berberidaceae family, is the most diffused source of
berberine, where it is present in bark and roots. B. vulgaris was used in traditional medicine both in Asia and Europe, and traditional Iranian
textbooks reported Berberis as a valid remedy for treating gastrointestinal diseases
[59]. Coptis chinensis, belonging to the Ranunculaceae family, was used more than 2000 years ago in China
for the same purpose [60]. In the last few years, berberine has found some applications in ophthalmology because
of its anti-diabetic properties. Berberine in combination with a
correct lifestyle helps to lower blood glucose [61], making this alkaloid a good candidate for the treatment and prevention of diabetes.
Diabetic retinopathy (DR) is a microvascular complication of diabetes and represents
one of the leading causes of irreversible blindness in the world. At clinical levels,
DR is characterized by typical neuronal and microvascular disfunction, which consists
of microaneurysms, microhemorrhages, and exudates from the damaged retinal vessels
(non-proliferative DR). In a more advanced stage (proliferative DR), the development
of areas of retinal ischemia leads to the release of vascular endothelial growth factor
(VEGF) and consequent formation of neo-vessels. Alterations of the vesselsʼ microarchitecture
may cause vitreous hemorrhages and scar tissue development that may cause predisposition
to retinal detachment and vision loss [62]. At cellular levels, persistent hyperglycemia causes the
activation of inflammatory pathways and oxidative stress that, together with vascularization,
leads to degeneration of RGCs [63]. The berberine effects on DR have been extensively studied both at cellular and
molecular levels. In the DR rat model, the retinal ganglion cells show high levels
of ROS (reactive oxygen species) and malondialdehyde, two markers of oxidative stress,
which were reduced after administration of berberine for 8 weeks. The reduction in
these two markers was accompanied by an increase in SOD (superoxide dismutase) activity
and GSH (glutathione) levels, two enzymes known to act as antioxidants and to contrast
oxidative stress [64]. Berberine effects on RGCs were also studied on the GABA (gamma-aminobutyric acid)
receptors. In fact, diabetic RGCs showed a reduction in the activity and levels of
these receptors. It was shown that feeding diabetic rats with berberine increased
the levels of GABA
receptors, decreasing RGC apoptosis [65]. In addition to RGCs, Müller cells have a prominent role in the pathogenesis of
DR. Müller cells are the main glial cells of the retina, and they provide nutritional
and regulatory support to the retinal neurons ([Fig. 1] b). In the presence of high glucose, Müller cells show increased signaling through
the nuclear factor-kappa B (NF-kB) pathway, which is the most important pro-inflammatory
pathway (a thorough description of the pathway is provided elsewhere [66]). It has been shown, both in animal and in cell culture, that berberine mitigates
the high-glucose-induced death of Müller cells, reducing NF-kB levels and the negative
effect of chronic inflammation [64]. Berberine seems to protect Müller cells from high-glucose-induced apoptosis also
modulating the AMPK/mTOR pathway [67],
which is a network that integrates intracellular and extracellular stimuli to adapt
intracellular metabolism. Among the downstream pathways regulated by this network,
macroautophagy, often referred to as autophagy (microautophagy and chaperone-mediated
autophagy are currently considered more targeted pathways) has a prominent role in
cell homeostasis. Macroautophagy is a major proteolytic pathway degrading damaged
or unwanted organelles (e.g., mitochondria) by lysosome [68]. Basal activity of autophagy is maintained by almost all cell types for general
homeostasis, but it can be triggered in response to intra- and extracellular stresses,
which finely tune the Akt/mTOR axis. However, depending on type, duration and amplitude
of the stress cells can undergo autophagy or apoptosis, and in some cases, treatments
inducing autophagy avoid cell apoptosis and vice versa [69]. In fact, although autophagy activation is commonly
viewed as a protective pathway, excessive autophagy activation can be followed by
apoptotic death, also called autophagic death. In this framework, rat primary Müller
cells showed reduced autophagy after high glucose treatment, which was linked to reduced
levels of phosphorylated AMPK and high levels of phosphorylated mTOR. However, 48
hours of berberine treatment restored the rate of phosphorylated AMPK and mTOR, inducing
autophagy and inhibiting apoptosis [67]. Nevertheless, the evidence that high glucose levels (typically > 25 mM) may impact
the autophagy signaling cascade, but also the ubiquitin-proteasome system (UPS), has
been envisaged by studies carried out in rMC1 cells, an immortalized strain of rat
Müller cells [70], [71]. In 2021, Wang and co-authors proposed that berberine improves the response of retinal
endothelial cells to insulin treatment [72]. Insulin is the main treatment for the control of glycemia in type I diabetes but
also in some cases of type II diabetes. Even though the tight control of glycemia
is critical for avoiding sight loss, some studies suggested that insulin could cause
an early but reversible worsening of DR [73]. The link between insulin and retinopathy is controversial, but it could be explained
by molecular experiments showing that insulin activates hypoxia inducible factor 1
(HIF1), which in turn induces the expression of VEGF
[74], [75]. High levels of VEGF cause vascular endothelial cell proliferation, contributing to the neovascularization
typical of the proliferative stage of DR. Given the importance of VEGF in DR, many
therapies aim to inhibit VEGF by different strategies [76]. In this regard, berberine could be a valid remedy given that it was
shown to inhibit the insulin-mediated induction of the HIF1/VEGF-pathway in endothelial
cell lines and to improve the endothelial dysfunction in the retina of type I and
II diabetic mice [72].
In addition to high glucose, high blood lipid levels are also a risk factor for DR,
and berberine reduces apoptosis caused by highly oxidized and glycated LDL, a modified
form of low-density lipoprotein [77]. Taken together, these data suggest that berberine has a protective role against
stresses that occur during DR pathogenesis, but despite all these promising results,
berberine is still suggested as a diet supplement rather than an effective therapy.
Future research should be directed to better understand berberine metabolism and to
find formulations for improving its low bioavailability [78].
Reserpine, an indole alkaloid
Reserpine is an indole alkaloid present in some plants of the genus Rauwolfia
[79] ([Table 1]), consisting of evergreen plants typical of tropical regions. This alkaloid is synthesized
starting from the tryptophane. The first report of reserpine used for medical purposes
dates back to the 16th century when it was extracted from the roots of R. serpentine; then it was brought to the attention of Western medicine in the 1950 s for its effect
on blood pressure and mental condition [80]. In fact, reserpine successfully lowers blood pressure in patients with hypertension
in combination with diuretics and vasodilators [81], [82], [83]. Even though this alkaloid reduces mortality linked to hypertension, in the last
years, it has been substituted by other classes of drugs. The clinical use of
reserpine has been questioned because its assumption was linked to a depression state
in treated patients [84], but a recent revision of the literature has questioned this link [85]. The pharmacological effect of reserpine is mainly the consequence of its binding
to the vesicular monoamine transporters (VMATs), acting as a competitive inhibitor
of endogenous monoamines [86]. VMATs ensure the storage, sorting, and release of the monoamine neurotransmitters
into synaptic vesicles of the neurons, modulating the synaptic activity [87]. Hence, reserpine seems to affect blood pressure and mental state, modulating the
levels of catecholamines such as dopamine and adrenaline. Recently, reserpine raised
interest in the ophthalmology field because it seems to preserve photoreceptor integrity
in retinal ciliopathies. Ciliopathies include a wide group of diseases (e.g.,
non-syndromic retinitis pigmentosa, cone-rod syndrome, Bardet-Biedl syndrome, and
Usher syndrome) that are caused by mutations affecting cilia. A cilium is a cell organelle
made by microtubules that works as a sensor for external stimuli. In the retina, cilia
are very important for the activity of photoreceptor cells since they capture the
light stimulus starting the phototransduction. Hence, defects in these organelles
may lead to photoreceptor degeneration and severe visual impairment. Retinal ciliopathies
[88] cause irreversible blindness from a young age; hence, many researchers are focused
on finding new drugs and therapies. In 2023, Chen and co-authors [89] published the results of a screening of over 6000 existing drugs for their ability
to improve photoreceptor survival in the retinal organoids derived from a mice model
for ciliopathy [90]. Among the drugs tested, reserpine showed
the best output in terms of photoreceptor survival, which was also confirmed in organoids
derived from affected patients and in vivo animal models. The authors also found that reserpine modulates two pathways that
are critical for protein homeostasis, which is mainly regulated through the degradative
activity of autophagy and the ubiquitin-proteasome system, cited previously [89]. Clinical trials will be necessary to evaluate the real efficacy of reserpine in
retinal diseases.
Caffeine, a methylxanthine alkaloid
Caffeine is a methylxanthine alkaloid synthesized from purine nucleotides [91]. Caffeine is widely diffused in the plant kingdom, but high concentrations of this
alkaloid can be found in the seeds of Coffea plants, with Coffea arabica being the most famous source of caffeine, in the leaves of Camellia sinensis (tea plant), and also in the seeds of cacao plants and those related (Theobroma cacao, Theobroma grandiflorum, and Herrania sp.) ([Table 1]). In South America, Paullinia cupana (guarana) and leaves of Ilex paraguariensis (mate) are also well-known sources of caffeine [92]. Caffeine was first isolated in 1819, and then synthetized and characterized by
Hermann Emil Fischer in 1895 [93]. However, the stimulating properties of caffeine-based beverages have been known
since ancient times. Beverages from coffee plants
date back to 1000 BC in Ethiopia and Yemen, and in the 14th century, Arabs discovered
the process of roasting coffee seeds, spreading this beverage in Europe and the rest
of the world. Tea was used in China in 1000 BC, while cocoa beans were used as a source
of caffeine by the Mayans [92]. In addition to the social use of caffeine as a stimulating beverage, the interest
in using this alkaloid in medicine started in the 19th century, when it was used for
the treatment of asthma [92]. The confirmed molecular targets of caffeine include acetylcholinesterase, adenosine
receptor, glycogen phosphorylase, Notum, phosphodiesterase, and ryanodine receptor
(for an exhaustive review check [94]). However, the most interesting pharmacological effect of caffeine on the nervous
system is its activity as an antagonist of adenosine receptors. Adenosine receptors
are G-protein-coupled receptors that respond
to adenosine endogenous levels and act as modulators of neurotransmitter release [95]. Four adenosine receptors have been identified (A1, A2A, A2B, and A3) and all of them are confirmed targets of caffeine, making caffeine a nonselective
adenosine receptor antagonist. Despite excessive consumption of caffeine being considered
dangerous for health, especially for the cardiovascular system, this molecule has
some positive effects–for example, in pain management [96]; it is in fact often present in over-the-counter medications for headaches. To highlight
the value of caffeine in medicine, it is noteworthy to mention that caffeine citrate
is listed by the WHO as an essential drug for treating apnea in preterm infants [97]. In addition, caffeine seems to have a neuroprotective role in neurodegenerative
diseases, such as Parkinsonʼs and Alzheimerʼs
disease [98]. Hence, given these potential neuroprotective roles, it has been proposed that this
alkaloid could be used to preserve RGCs from degeneration, which is, as already mentioned,
a typical signature of glaucoma. In support of this hypothesis, Sprague–Dawley rats
fed with caffeine in drinking water showed reduced degeneration of RGCs when ocular
hypertension (OHT) is induced by laser photocoagulation of the limbal veins to mimic
human glaucoma [99]. This benefit was associated with reduced inflammation, determined as low levels
of soluble mediators of inflammation (e.g., IL1β and TNFα) and microglia reactivity, with respect to animals fed without caffeine, suggesting
that the alkaloid has anti-inflammatory properties. The anti-inflammatory properties
of caffeine in the retina were also confirmed by Conti and colleagues, who showed
how caffeine counteracts retinal degeneration in mice retina
damaged by ischemia/reperfusion treatment [100]. The idea that caffeine, acting on the adenosine receptors, could work as a neuroprotector
was also supported by data showing that chemically synthetized selective inhibitors
of the A2A receptor protect the injured retina from degeneration, decreasing inflammation
[101], [102]. Moreover, epidemiological data suggest that daily consumption of caffeine does
not elevate IOP, at least in people without a genetic predisposition for glaucoma
[103].
Further data sustaining the benefit of caffeine come from experiments done in animal
models of cataracts. Kronschläger and co-authors showed that caffeine administration
as eye drops before exposition to UV-B radiation reduces the formation of cataracts,
measured as lens light scattering, in a rat model [104]. These results are in agreement with in vitro data showing that caffeine supplementation in the culture media protected cultured
mouse lenses from photodamage induced by oxidant agents [105], [106]. The protective role of caffein eye drops was further examined in a rat model where
cataracts were induced by oral administration of galactose, an experimental model
that recapitulates cataract formation in diabetic patients [107]. These results showed that animals treated with caffeine eye drops five times a
day developed fewer galactose-induced
cataracts, both in short- (5 days) and long-term (25 days) experiments [107], than those in the placebo group.
Taken together, these results point out that the right amount of daily caffeine uptake
can help in preserving eye health, but further investigation will be needed to understand
if caffeine-based eye drops could be used as a proper therapy for eye diseases.
Biotechnology Approaches for the Production of Medicinal Plant Alkaloids
Both atropine and pilocarpine are present in the WHO list of essential drugs, which
are enlisted as the most efficacious and cost-effective medicines to treat severe
and life-threatening diseases. This means that a shortage of these compounds could
endanger the healthcare system with serious repercussions on peopleʼs lives worldwide.
Chemical synthesis can be very challenging for plant alkaloids and also expensive.
Hence, the demand of the pharmaceutical market is satisfied mainly by the extraction
and isolation of alkaloids directly from plants. Unfortunately, this method of production
is burdened by several drawbacks. First of all, alkaloid synthesis in plants is often
restricted to specific tissues, limiting the tissue available for alkaloids isolation.
Moreover, extraction methods are often not specific and provide a mixture of metabolites
with a low biological function and not pure alkaloid. Then, temperature fluctuations,
floodings, drought periods, and pathogen attacks
not only affect plant fitness, altering the harvestable biomass, but also influence
secondary metabolite synthesis, determining inconsistencies in the final yield of
alkaloids. Lastly, alkaloids are highly species- and genus-specific, meaning that
only a few plant species can be used for the pharmaceutical market. This means that
the heavy exploitation of these plants endangers biodiversity and their survival in
their native habitats. Hence, many lines of research looked into biotechnologies based
on the reconstitution of alkaloid biosynthetic pathways in microorganisms and in vitro culture of plant cells ([Fig. 3]).
Fig. 3 Summary of biotechnology approaches for plant alkaloid production based on microorganisms
and plant cell culture. Potentially, all plant alkaloids can be produced by genetically engineered microorganisms,
such as yeast and cell cultures. The advantage of using these approaches is that both
microorganisms and plant cells can be grown in bioreactors that provide controlled
growth conditions. In the figure, the different steps needed for the improvement of
alkaloid production have been mentioned.
The main advantage of using microorganisms for alkaloid synthesis is the possibility
of propagation in industrial bioreactors that provide controlled growth conditions,
eliminating the problem of environmental fluctuations and yield instability. Among
the microorganisms, baker yeast S. cerevisiae showed the best potential because many genetic tools are available, and it allows
a reliable expression of many plant genes, making possible the reconstitution of several
biosynthetic pathways. Even though there are no technical restrictions for yeast engineering,
the following aspects should be considered. First of all, in plants, the enzymes for
secondary metabolites are often distributed in different cellular compartments and
they are cellular/tissue-specific, meaning that their activity and/or expression could
require a specific regulatory context lacking in yeast. Second, many biosynthetic
pathways have not been fully elucidated yet. Hence, transcriptomic, genomic, and
proteomic data need to be analyzed and compared to identify the missing enzymes. Third,
it is not always possible to obtain the quantity required for industrial production,
but yeast can still be a valuable platform to produce high-quality intermediates that
can be used in the semisynthetic process to obtain the final drug. One milestone in
tropane alkaloid production was recently achieved by Srinivasan and Smolke, who optimized
the biosynthesis of hyoscyamine and scopolamine in yeast. Their system expressed 26
genes, encoding not only metabolic enzymes but also transporters to ensure the compartmentalization
of the metabolic steps in different cellular locations [108]. Afterward, the same authors were able to scale up to 172 µgr/L and 480 µgr/L of
scopolamine and hyoscyamine, respectively, solving the problem of the vacuolar compartmentalization
of the littorine intermediate by the identification and expression in yeast of functional
vacuolar
alkaloid transporters [109].
Also, berberine was successfully produced in yeast [110], [111] after many years of attempts to produce intermediates. Han and Li were able to improve
the initial yield of 1.68 µg/L, obtained from glucose and by the expression of 15
enzymes, to the final yield of 1.08 mg/L by fermentation and the engineering of rate-limiting
enzymes [111]. Moreover, the authors suggest yeast also as a platform to overcome the limit of
low bioactivity and bioavailability of berberine.
On the other hand, plant cells can also be grown in bioreactors providing renewable
materials for metabolite production. Even though this system is used for the industrial
production of some plant metabolites, such as the alkaloids berberine and scopolamine
[112], it shows some weaknesses and limitations. First, cell cultures growing for a long
time are susceptible to mold and bacteria contaminations; hence, cultures should be
kept in highly controlled environments. Second, to provide enough oxygen for plant
growth, bioreactors are equipped with systems, such as rotation, stirring, and air
suppliers, that ensure the correct air circulation [113]. However, these systems create mechanical stresses for the cell culture, which can
be easily damaged, slowing proliferation and reducing production. Third, while microorganisms
release metabolites into the media, plants tend to store them in the cells, making
the recovery of
metabolites more difficult. Fourth, the alkaloid synthesis in plants is often tissue-specific;
for example, many biosynthetic enzymes for tropane alkaloid biosynthesis are expressed
in roots [114]. To overcome this issue, it is possible to use differentiated cultures, such as
hairy roots. Hairy root cultures can be generated, adjusting the mixture of hormones
to favorite roots development (e.g., more auxin than cytokines), but the most efficient
procedure is infecting wounded plants with Agrobacterium rhizogenes. During the infection, this bacteria transfers the Rol genes into the plant genome, wherein the product of this gene stimulates root development,
which can be then grown in liquid media for large-scale production. Similarly to hairy
root cells, cultures of the aerial parts of plants can be made to produce metabolites
that usually accumulate in the shoots [112]. However, hairy root cultures are
more diffused than the shoot ones because they do not need particular light conditions
for the proliferation in bioreactors, making their growth less demanding. The last
limitation of plant cell culture is that not all plant species can be easily cultivated
in vitro and used for tissue regeneration, and this can be a problem, considering that many
alkaloids are highly genus-specific. Hence, many researchers constantly develop new
species-specific protocols for plant regeneration that consist of growing shoots and
roots at different concentrations of plant growth regulators such as auxin and cytokines.
Despite the advantages of cell cultures, the final alkaloid yield is often far away
from the industrial demand. To improve this aspect, one strategy can be the overexpression
of key enzymes of the alkaloid synthesis pathway, which has been successfully used
for tropane alkaloids, for which the biosynthetic pathway has been extensively studied.
The review written by Gong and colleagues offers a complete overview of this topic
[115].
Another interesting method to improve alkaloid accumulation is through elicitors.
Elicitors are commonly defined as biotic and abiotic agents that trigger stress responses
in plants [116]. Given that many secondary metabolites are produced in response to stresses, elicitors
can be added to the media of cell culture to stimulate metabolite production. Some
microorganisms, such as fungi and bacteria, act as biotic elicitors of plant alkaloids,
but molecules that work as signaling modulators such as the hormone jasmonic acid
(JA) can also be used.
Conclusions
Vision impairment and loss are medical conditions that affect millions of people all
around the world, numbers that are anticipated to be doubled by 2050, at least in
the USA, for the four main eye diseases, which are glaucoma, DR, cataract, and macular
degeneration.
Blindness, but also mild visual impairment, has an incredible cost for society, not
only considering the direct medical care but also as loss of productivity. Moreover,
eye disorders highly affect quality of life, and they contribute to other conditions
such as psychiatric problems (such as depression and anxiety) and cognitive dysfunctions,
especially in the elderly. As described in this review, plant-based drugs are commonly
used to treat eye conditions, with alkaloids playing an important role, given their
plethora of biological activities ([Table 2]). Even though plant alkaloids have been largely exploited for pharmaceutical purposes,
it is important to continue the efforts to identify effective drug treatments and
to develop new formulations. This can be achieved only by an intense and active collaboration
between different areas of research. First, plant biologists are necessary for the
identification of the plant species exploitable for the
purpose of deeply studying their physiology. Second, biochemists need to work on effective
extraction methods for obtaining pure metabolites. Alkaloids show an incredible diversity
in the structures, consisting in basic nitrogen atoms and rings; hence, specific extraction
methods from raw materials need to be optimized depending on the alkaloid of interest.
Once the molecule has been identified, researchers in the cell biology field have
to test these compounds in proper cell lines and animal models to evaluate the cytotoxicity
and the effects on the molecular pathways that are relevant to the ophthalmology field.
Even though pharmaceutical research still relies on animal models, progress has been
made to identify in vitro cell cultures that can be used in pre-clinical studies. For example, the organoids
obtained from retinal iPSC derived from patients [89] were shown to be a valid system for drug screening, and it allowed the identification
of reserpine as a possible treatment for retinal disease.
Table 2 Experimental evidence showing the effects of plant alkaloids on cellular pathways
affected in eye diseases.
|
Alkaloid
|
Experimental model
|
Alkaloid effect
|
References
|
|
Abbreviations: BJ–Balb/CJ; FDM–form deprivation myopia; RGC–retinal ganglion cells;
DR–diabetic retinopathy; HREC–human retinal endothelial cells; iPSC–induced pluripotent
stem cells; UPS–ubiquitin proteasome pathway; OHT–ocular hypertension.
|
|
Atropine
|
BJ mice;
White Leghorn chicks;
FDM guinea pigs;
In vitro Scleral fibroblasts
|
Sclera remodeling
|
[28], [29], [30], [31], [32]
|
|
Male White Leghorn chickens
|
Release of dopamine from retina
|
[34]
|
|
Studies in children and young adults
|
Increase in choroid thickness
|
[39], [40], [117], [118], [119], [120], [121]
|
|
Pilocarpine
|
Study in healthy adults
|
Decrease in choroid thickness
|
[45]
|
|
Study in healthy and glaucoma patients;
Mice model
|
Dilatation of Schlemmʼs canal
|
[50], [51]
|
|
Berberine
|
RGC of DR rat model
|
Reduction in oxidative stress
|
[64]
|
|
RGC of DR rat model
|
Increase in RGC survival
|
[64], [65]
|
|
Primary rat retinal Müller cells
|
Reduction in high-glucose-mediated activation of NF-kB pathway
|
[64]
|
|
Primary rat retinal Müller cells
|
Modulation of autophagy through AMP/mTOR pathway
|
[67]
|
|
HREC cell line
|
Inhibition of the insulin-mediated induction of the HIF1/VEGF-pathway
|
[72]
|
|
Cultured human Müller cells
|
Reduction in cell apoptosis mediated by highly oxidized and glycated LDL
|
[77]
|
|
Reserpine
|
iPSC-derived retinal organoids;
retina of rd16 mouse model
|
Modulation of UPS and autophagy pathway to preserve photoreceptor integrity
|
[89]
|
|
Caffeine
|
Sprague–Dawley rats, Male C57BL/6 J mice
|
Reduction in inflammation and RGC apoptosis
|
[99], [100]
|
Another important aspect to consider in drug development in ophthalmology is the route
of administration, which can vary according to the disease to treat and its localization
(e.g., corneal vs. retinal diseases). The most frequently used is topical administration,
which has the advantage of being noninvasive but cannot ensure the effective concentration
of the drug in the posterior chamber of the eye. Less used are systemic administration
and intravitreal injection. The former has the disadvantage of presenting systemic
side effects and poor bioavailability in ocular tissues, while the latter, despite
the local high availability of drugs directly injected in the posterior chamber of
the eye, is a very invasive procedure for the patients. For these reasons, the establishment
of new formulations that optimize retention time and posology (e.g., slow-release
formulations), absorption, and drug delivery will be necessary.
Lastly, a full exploitation of plant alkaloids in medicine can be obtained only through
a deep characterization of the different biosynthetic pathways that allow the identification
of the rate-limiting plant enzymes and important intermediates of reaction. This is
critical for the creation of synthetic toolboxes that can be used for the engineering
of metabolic pathways in industrial production. In recent years, microorganisms and
plant cell cultures have been successfully used as a platform for this purpose, but
further optimization will be necessary to fulfill the pharmaceutical market. This
process includes the maximization of the enzyme activity and the capacity to obtain
a reproducible yield of metabolites over time.
In conclusion, the finding of sustainable and reliable methods for alkaloid production
will increase their diffusion in medical practice with great benefit for human and
animal health.
Literature research method
Pubmed was used as main browser for searching the literature; the research was focused
mainly over the last 10 years (from 2013 to September 2023), but older publications
were further considered if they were particularly relevant or when the research retrieved
no significant results. The terms [eye disease] AND [plant alkaloid] were used to
identify the main alkaloids used in ophthalmology. Then, the following terms were
used for the role of the alkaloids presented in ophthalmology: [atropine] AND [myopia];
[atropine] AND [sclera]; [atropine] AND [choroid]; [pilocarpine] AND [glaucoma]; [pilocarpine]
AND [myopia]; [diabetic retinopathy] AND [berberine]; [reserpine] AND [retinal disease];
[caffeine] AND [retinal disease]. For the part regarding alkaloid production, the
following terms were used: [yeast] AND [atropine]; [yeast] AND [reserpine]; [yeast]
AND [pilocarpine]; [yeast] AND [berberine]; [plant culture] AND [atropine]; [plant
culture] AND [reserpine]; [plant culture]
AND [berberine]; [plant culture] AND [pilocarpine].
For historical background, Google was used, and the following terms were used: “atropine
history”, “caffeine history”, “berberine history, “pilocarpine history”, and “reserpine
history”.
