Bioactivity and toxicity of coumarins from African medicinal plants

Introduction: Coumarins are naturally occuring metabolites from plants and a few micro-organisms. They have been widely used in the food and drug industry in their natural or synthetic forms. Numerous coumarins possess several biological activities such as anti-inflammatory, anti-ulcers, anti-tumour, anti-microbial, anti-coagulant. The aim of this study was to assess the bioactivity, and toxicity of coumarins from African medicinal plants. Methods: We searched online databases and search engines such as PubMed, Google Scholar and Web of Science for key terms such as coumarins, toxicity, bioavailability, bioactivity with appropriate Boolean operators. Only full-length research articles published in English between 1956 to 2023 were reviewed. Results: We recorded 22 coumarins from 15 plant species from Africa. Most of the plant species (33%) were from North Africa. These were followed by East Africa at 21%, then West, and Central Africa at 18.2% each. Most of the coumarins (21.3%) were isolated from the entire plant and the leaves (19.1%) and most of them (46.7%) had some antimicrobial activity. Five coumarins viz osthole, pseudocordatolide C & calanolide, chartreusin and esculetin had either antitumor or anticancer activity. Six coumarins had varying levels and types of toxicity ranging from inhibiting blood clotting as anticoagulants, to cytotoxic effects, causing hyperventilation, tremor, & photophobia, pulmonary haemorrhage, carcinogenic activity, severe neurotoxicity, hepato- and phototoxicity. Conclusion: Several African medicinal plants are sources of various coumarins that possess several biological activities as well as toxicities. This calls for more research into their safety and efficacy because of their wide spread applications as therapeutic agents.


Introduction
Coumarins are naturally occurring metabolites in a variety of plants, micro-organisms and in some animal species (Perone, 2016).Coumarin (1,2-benzopyrone; 2H-1-benzopyran-2-one; cis-o-coumarinic acid lactone) is also known as benzopyrone or coumarinic anhydride and tonka bean camphor.It is a white crystalline solid, belonging to the class of lactones.It consists of an aromatic ring fused to a condensed lactone ring (Venugopala et al., 2013).
The chemical formula of coumarins is C 9 H 6 O 2 , and they can exist in various isomeric forms due to differences in the substitution pattern on the benzene and the pyrone ring (Wu et al., 2009).The presence or absence of particular functional groups confers the coumarin its name and properties, such as simple coumarins which are the basic coumarin metabolites with no additional substituents.These include hydroxycoumarins like scopoletin and umbelliferone, methoxycoumarins like esculetin and 7-methoxycoumarin and alkyl coumarins like daphnetin and aesculetin (Wu et al., 2009).Coumarins are variably soluble in most organic solvents or their combinations (Huang et al., 2015) but are freely soluble in ethanol, chloroform, diethyl ether and oils (Jung and Oh, 2011).
Coumarin occurs naturally in many plants and confers a pleasant spice aroma akin to vanilla (Egan et al., 1990).Coumarins were first isolated from Dipteryx odorata (Aubl.)Forsyth f (tonka beans) by Vogel in 1820 (Doctor et al., 2020).They also occur in high concentrations in some essential oils, such as Cinnamomum aromaticum (cinnamon bark oil) and Cassia fistula L. (cassia leaf oil).Coumarins have been identified in various plant species from many different families including the fruits of Vaccinium myrtillus L. (bilberry), Rubus chamaemorus L (cloudberry), Camellia sinensis (L.) Kuntze (green tea), Melilotus albus Medik.and Melilotus officinalis (L.) Lam (sweet clover), Galium odoratum (L.) Scop(sweet woodruff), Vanilla planifolia Andrews (vanilla leaves and beans), Lavendula officinalis subsp.angustifolium (lavender) and Myroxylon pereirae (L.) Harms (balsam of Peru) (Kostova et al., 2011;Kicel and Wolbis, 2012).A broad spectrum of coumarins present in both free state and as glucosides have also been found in many other plant species.Several coumarins have been identified, principally as secondary metabolites in green plants, but the exact number is hard to determine due to synthetic coumarins created regularly (Kostova, 2005).
Most commercially used coumarins were synthesized from salicylaldehyde (DeGarmo and Raizman, 1967).High-grade coumarin is still isolated from tonka beans using various methods such as hot water steeping and sub critical water extraction (Doctor et al., 2020).Coumarins are extremely variable in structure leading to various pharmacological activities (Lacy and O'kennedy, 2004).The simplicity and versatility of the coumarin scaffold makes it a convenient starting-point for a wide range of applications (Stefanachi et al., 2018;Annunziata et al., 2020).

Industrial applications and uses of Coumarins
Although coumarins are widely distributed in the plant kingdom, most of the commercially used ones have been synthetically produced for many years.Coumarins have several applications such as in the perfumery, cosmetic and related industries (Givel, 2003).Large quantities of coumarin, mostly associated with vanillin, are used in the food industry for flavouring chocolates, baked goods, and in flavoured beverages (Givel, 2003).However, since 1954, synthetic vanillin use as a direct additive in tobacco products has been suspended in the United States by the FDA due to the production of hazardous compounds when burnt (Riveiro et al., 2010;Sarker and Nahar, 2017;Prusty and Kumar, 2020).Because of their unique sweet note and stability, coumarins have long been recognized as an important raw material in the fragrance industry as odourenhancers.They help to achieve a long-lasting effect when combined with natural essential oils (Doctor et al., 2020).As such, they are widely used in hand soaps, detergents, lotions and perfumes, in tobacco to enhance its natural aroma, in toothpastes, antiperspirant deodorants, creams, hair sprays, shampoos and many other household products (Perone, 2016).Large quantities of coumarins are used in rubber and plastic materials and in paints and sprays to neutralize unpleasant odours (Kirsch et al., 2016).Coumarins also have significant uses in electroplating, mostly in the automotive industry, to provide high polished quality chrome-plated steel.However, this use has been on the decline (Kirsch et al., 2016).

Medicinal applications of coumarins
Coumarins have several pharmacological properties with applications in the medical field (Skalicka-Woźniak et al., 2016), as a cardiovascular targeting drug (Najmanova et al., 2015), and as a cancer screening molecule and therapy (Lacy and O'Kennedy, 2004).Coumarins possess both immunomodulatory and antitumor activity (Riveiro et al., 2010).They have been recommended for treatment of a number of clinical conditions, including high protein oedema and brucellosis (Luszczki et al., 2009;Riveiro et al., 2010;Raja et al., 2011).Coumarins such as osthole have undergone clinical trials for treatment of lymphoedema (Farinola and Piller, 2005), following breast cancer treatment and in treatment of lung and kidney cancer and melanoma alone or in combination with cimetidine (Riveiro et al., 2010;Prusty and Kumar, 2020).Coumarins have also been  (Eason and Spurr, 1995;Hirsh et al., 2001;Sun et al., 2020).

Anthogenol
The vitamin K epoxide reductase (VKORC1), the key enzyme of the vitamin K cycle is the molecular target of coumarins (Oldenburg et al., 2007).Thus, the objective of this study was to assess the bioactivity, and toxicity of coumarins from African medicinal plants.

Methods
We performed a literature search using PubMed, Google Scholar and Web of Science.We searched for terms such as coumarins, toxicity, bioavialability, bioactivity with appropriate Boolean operators.Only full-length articles published in English between 1956-2023 were reviewed.The review was limited to African medicinal plant species only.We used only original research articles or review papers and excluded results from in silico studies.

Results
We recorded 22 coumarins that were isolated from 15 plant species (Table 1).Most of the plant species (33%) were from North Africa, followed by East Africa (21%), then West, and Central Africa at 18.2% each.Most of these coumarins (21.3%) were isolated from the whole plant.These were followed by the leaves (19.1%), then seeds, fruits and roots each at 12.8%.However, only two plant species Mammea africana Sabine and Tetrapleura tetraptera (Schumach.& Thonn.)Taub.had a more restricted native range to Africa.The rest of the plant species were more widely distributed in different parts of the world.The distribution of the plant species was checked with the Kew database at https://powo.science.kew.org.All the coumarins showed multiple bioactivities.

Antioxidant properties of coumarins
Osthole, esculin, dihydroxycoumarin and many other coumarins have shown beneficial biochemical profiles in relation to pathophysiological processes dependent upon reactive oxygen species due to the possession of amine, and hydroxy groups (Kostova et al., 2011).A study by Abdizadeh et al. (2020) evaluated the photoprotective activity of some hydroxycoumarins as compared with p-aminobenzoic acid (PABA) as a model sun screen.The activity could be related to their antioxidant actions which could minimize skin photoaging (Abdizadeh et al., 2020).Another study by Mitra et al. (2013) compared the antioxidative activities of seven hydrocoumarins with those of α-tocopherol for the oxidation of tetralin and linoleic acid in a homogeneous solution.Hydrocoumarins exhibited a higher induction period than α-tocopherol in both systems.However, the rate of oxygen absorption during the induction period for the latter was slower than that of the hydrocoumarins in both systems.In addition, 6,7dihydroxy-4,4-dimethylhydrocoumarin showed less cytotoxicity toward human fibroblasts than 2, 6-di-t-butyl4-methylphenol (Kostova et al., 2011).

Antimicrobial activity of coumarins
Although different studies have confirmed the antimicrobial activities of various coumarins from plants (Vaou et al., 2021;Ojala et al., 1999;Karakaya et al., 2019), such studies are lacking of African medicinal plants.Some coumarin antibiotics of natural origin, although not necessarily from African plant species have been developed into modern antibiotics.These include NOVOBIOCIN ™ and CLOROBIOCIN ™ which are inhibitors of DNA gyrase.They both have a broad spectrum towards Gram-positive bacteria, including methicilin resistant strains of staphylococci species (Raja et al., 2011;Matos et al., 2012).This shows the potential of African plant species as a source of coumarin based drugs.Due to some limitations of these metabolites particularly with regard to solubility, toxicity and development of resistance, a novel series of coumarin analogues has been synthesized (Kirsch et al., 2016).The ester or carboxylic acid on the coumarin ring was found to be important for the inhibitory activity against Gram-positive and Gram-negative bacteria (Chiang et al., 2010;Annunziata et al., 2020).The presence of phenolic hydroxyl groups and or carboxylic acid was found necessary for enhanced activity against Helicobacter pylori (Venugopala et al., 2013).

Anti-inflammatory action of coumarins
Several coumarins like esculetin, dicoumarol isolated from plants or of synthetic origin possess significant anti-inflammatory and or analgesic activities (Kirsch et al., 2016).In a Quantitative Structure-Activity Relationship (QSAR) study for lead optimization in the design of coumarins as potent non-steroidal antiinflammatory agents, the coumarin positions C4-and C7contributed to the high activity (Poumale et al., 2013).Natural products such as esculetin, fraxetin, inhibit lipoxygenase and cyclooxygenase enzymic systems and neutrophil-dependent superoxide anion generation (Kontogiorgis and Hadjipavlou-Litina, 2005).
Angelica  (Wagstaff, 1991).Calanolide A and B are semi-synthetic coumarins on market used as combination therapies for HIV.Psoralen was found to be a very potent anti-HIV drug during research but was discontinued for its high hepatotoxicity in mice (Sancho et al., 2004;Guo et al., 2020).Phenprocoumon emerged as a novel lead template possessing weak HIV protease inhibitory activity (Hassan et al., 2016).TRIOXSALEN ™ was then developed and marketed as chemical derivative of psoralen despite its photosensitization of the skin (Wishart et al., 2018).

Antitumor/anticancer properties of coumarins
Osthole, umbelliferon, esculetin and geiparvarin all showed anti-tumor activity.Hydroxycoumarin might be a useful adjuvant for melanoma therapy because of its selective antiproliferative agents that mediate apoptosis in renal carcinoma cells, through modulation of mitogen-activated protein kinases attributed to a nitro group in the aromatic ring (Kostova, 2005;Jung and Oh, 2011).In an attempt by Zhang et al. (2021) to develop novel antitumor candidates, a series of coumarin sulfonamides and amides derivatives were designed and synthetized.The majority of these metabolites showed good cytotoxic activity against MDA-MB-231 and kB cell lines (Zhang et al., 2021).Menezes and Diederich, (2019) conducted an extensive review on the role of natural coumarins as anticancer agents.They showed how coumarins modulate several targets such as carbonic anhydrase, Hsp90, histone deacetylase and topoisomerase enzyme in cancer cells leading to apoptosis.

Coumarins as enzyme inhibitors
Some natural and synthetic coumarins are cholinesterase inhibitors, which are a promising approach for the treatment of Alzheimer´s disease and possible therapeutic applications in the treatment of Parkinsons´s disease (Anand et al., 2012).A series of umbelliferon metabolites and synthetic coumarins were evaluated as inhibitors of monoaminooxigenase (MAO) and steroid 5α reductase type I in cell culture systems (Anand et al., 2012).Ether derivatives demonstrate MAO-B selectivity, and sulfonic ester derivatives demonstrate MAO-A selectivity by QSAR studies (Koyiparambath et al., 2021).Calanolide-A drugs are marketed for their ability to block activity of cytochrome P450 3A4, while ethyl biscoumacetate is a glutamine synthase inhibitor (Wishart et al., 2018;Sai et al., 1999).

Effect of coumarins on cardiovascular system
Many coumarins have different effects on blood.Most notable is warfarin, which has anti-coagulant properties (Hosseini et al., 2015).Warfarin drugs are used for the management of thromboembolism and pulmonary embolism with atrial fibrillation (Wishart et al., 2018).Ethyl biscoumacetate is a semi-synthetic compound used as anticoagulant alternative to warfarin due to fewer side effects (Sun et al., 2020).Esculin and its metabolites are used as vasoprotective agents (Kostova, 2005).Moreover, other coumarins like biscoumarin/bicoumarin have similar activity to ethyl biscoumacetate and esculin (Sun et al., 2020;Najmanova et al., 2015).

Toxicity of coumarins
There is a large body of data on the toxicity of coumarin in experimental laboratory animals.Coumarins have hepatotoxic and carcinogenic properties in vivo in rodents and other mammalian species.These included adenomas and carcinomas of the liver, lungs and bile ducts and adenomas of the kidney and liver in rats and mice respectively.Carcinomas were found only at doses higher than 100 mg/kg body weight per day (Lake, 1999;EFSA, 2004).In humans, coumarins were approved for clinical use since the 1970s for treating various venous and lymphatic oedemas, as well as tumours including renal cell carcinoma (Marshall et al., 1994).However, their use was eventually discontinued and they were recalled from the market following the development of severe hepatotoxicity in patients after treatment (Cox et al., 1989;WHO, 1995).Coumarins are rapidly absorbed from the gastrointestinal tract after oral administration and extensively metabolized by the liver in the first pass, with only 2%-6% reaching the systemic circulation intact (Guo et al., 2020).Coumarins such as furanocoumarins are toxic to humans.Methoxypsoralen derivatives are potent photosensitizers.They can be activated by near-UV light becoming mutagenic, phototoxic and carcinogenic (Ojala et al., 1999;Ceska et al., 1997).Osthole had an acute intraperitoneal LD 50 in mice of 710 mg/kg bw manifesting toxicity as hyperventilation, tremors, and photophobia.In subchronic studies, orally administered osthole in Wistar rats at 5-50 mg/kg bw for 45 days, caused pulmonary haemorrhage and mild reno and hepatoinflammation (Shokoohinia et al., 2017).Esculetin showed a low acute toxicity with an oral LD 50 of over 2000 mg/kg bw, the intraperitoneal LD 50 being 1,450 mg/kg bw (Tubaro et al., 1988).Orally administered psoralen in rats (80 mg/kg bw) caused cholestatic liver injuries (Wang et al., 2019).

Conclusion
Several African medicinal plants are sources of various coumarins which possess several biological activities.Coumarins generally have several therapeutic applications.Most of the coumarins in this review (46.7%) had some antimicrobial activity.Six coumarins: osthole, imperatorin/ umbelliferon, pseudocordatolide C & calanolide, chartreusin and esculetin had either antitumor or anticancer activity.They had varying levels and types of toxicity ranging from inhibiting blood clotting as anticoagulants, cytotoxic effects, causing hyperventilation, tremor, and photophobia.Such plants need to be conserved because of their potential as sources of coumarins and other drugs.This calls for more research into their safety and efficacy because of their wide spread applications as therapeutic agents.

TABLE 1
Bioactivity of coumarins from African medicinal plants.

TABLE 1 (
Continued) Bioactivity of coumarins from African medicinal plants.

TABLE 2
Toxicity of coumarins derived from African plants.