“Wild cannabis”: A review of the traditional use and phytochemistry of Leonotis leonurus

Author’s Accepted Manuscript “WILD CANNABIS”: A REVIEW OF THE TRADITIONAL USE AND PHYTOCHEMISTRY OF LEONOTIS LEONURUS Baudry N. Nsuala, Gill Enslin, Alvaro Viljoen www.elsevier.com/locate/jep

PII: DOI: Reference:

S0378-8741(15)30080-5 http://dx.doi.org/10.1016/j.jep.2015.08.013 JEP9678

To appear in: Journal of Ethnopharmacology Received date: 19 June 2015 Revised date: 14 August 2015 Accepted date: 15 August 2015 Cite this article as: Baudry N. Nsuala, Gill Enslin and Alvaro Viljoen, “WILD CANNABIS”: A REVIEW OF THE TRADITIONAL USE AND PHYTOCHEMISTRY OF LEONOTIS LEONURUS, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2015.08.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

“WILD CANNABIS”: A REVIEW OF THE TRADITIONAL USE AND PHYTOCHEMISTRY OF LEONOTIS LEONURUS Baudry N. Nsuala1, Gill Enslin1*, Alvaro Viljoen1,2 1

Department of Pharmaceutical Sciences and 2SAMRC Herbal Drugs Research

Unit, Faculty of Science, Tshwane University of Technology, Private Bag X680, Pretoria, 0001, South Africa. *

Corresponding author: Gill Enslin ([email protected])

Tel: +27 12 382 6303, Fax: +27 12 382 6243

ABSTRACT

Ethnopharmacological relevance: Leonotis leonurus, locally commonly known as “wilde dagga” ( = wild cannabis), is traditionally used as a decoction, both topically and orally, in the treatment of a wide variety of conditions such as haemorrhoids, eczema, skin rashes, boils, itching, muscular cramps, headache, epilepsy, chest infections, constipation, spider and snake bites. The dried leaves and flowers are also smoked to relieve epilepsy. The leaves and flowers are reported to produce a mild euphoric effect when smoked and have been said to have a similar, although less potent, psychoactive effect to cannabis. Aim of the review: To amalgamate the botanical aspects, ethnopharmacology, phytochemistry, biological activity, toxicity and commercial aspects of the scientific literature available on Leonotis leonurus. Methods: An extensive review of the literature from 1900 to 2015 was carried out. Electronic

databases

including

Scopus®,

SciFinder®,

Pubmed®,

Google

Scholar® and Google® were used as data sources. All abstracts, full-text articles and books written in English were considered. Results: The phytochemistry of particularly the non-volatile constituents of Leonotis leonurus has been comprehensively investigated due to interest generated as a result of the wide variety of biological effects reported for this plant. More than 50 compounds have been isolated and characterised. Leonotis leonurus contains mainly terpenoids, particularly labdane diterpenes, the major

1

diterpene reported is marrubiin. Various other compounds have been reported by some authors to have been isolated from the plant, including, in the popular literature only, the mildly psychoactive alkaloid, leonurine. Leonurine has however, never been reported by any scientific analysis of the extracts of L. leonurus. Conclusion: Despite the publication of various papers on L. leonurus, there is still, however, the need for definitive research and clarification of other compounds, including alkaloids and essential oils from L. leonurus, as well as from other plant parts, such as the roots which are extensively used in traditional medicine. The traditional use by smoking also requires further investigation as to how the chemistry and activity are affected by this form of administration. Research has proven the psychoactive effects of the crude extract of L. leonurus, but confirmation of the presence of psychoactive compounds, as well as isolation and characterisation, is still required. Deliberate adulteration of L. leonurus with synthetic cannabinoids has been reported recently, in an attempt to facilitate the marketing of these illegal substances, highlighting the necessity for refinement of appropriate quality control processes to ensure safety and quality. Much work is therefore still required on the aspect of quality control to ensure safety, quality and efficacy of the product supplied to patients, as this plant is widely used in South Africa as a traditional medicine. Commercially available plant sources provide a viable option for phytochemical research, particularly with regard to the appropriate validation of the plant material (taxonomy) in order to identify and delimit closely related species such as L. leonurus and L. nepetifolia which are very similar in habit. Keywords: Leonotis leonurus, “wilde dagga”, ethnonpharmacology, labdane diterpenes, marrubiin

Contents

1

INTRODUCTION

2

BOTANICAL ASPECTS 2

3

TAXONOMY

4

ETHNOPHARMACOLOGY

5

4.1

Traditional use in humans

4.2

Ethnoveterinary uses

4.3

Recreational use

BIOLOGICAL ACTIVITY 5.1

Anticonvulsant activity

5.2

Psychoactive properties

5.3

Antidiabetic activity

5.4 Anti-infective activity: antibacterial, anti-amoebic, anthelmintic, antimalarial and anti-HIV activity and toxicity.

6

5.4.1

Antibacterial activity

5.4.2

Anti-amoebic activity

5.4.3

Anthelmintic activity

5.4.4

Antimalarial activity

5.4.5

Anti-HIV activity

5.5

Anti-oxidant activity

5.6

Toxicity

PHYTOCHEMISTRY 6.1

Chemotaxonomy

7

CONCLUSION

8

ACKNOWLEDGEMENTS

9

REFERENCES

1

INTRODUCTION

In terms of the general regulations (Government Notice R766 in Government Gazette 36929 dated 14 October 2013 and Government Notice R870 in Government Gazette 37032 dated 15 November 2013) of the Medicines and Related Substances 3

Act (101/1965 as amended), the Department of Health in South Africa has required that all herbal medicines be regulated and controlled for safety, quality and efficacy. These essential attributes of any medicine are reliant on the quality of the raw materials used, which includes appropriate specifications for both active and inactive ingredients, and the application of Good Manufacturing Practices in the manufacturing process, from the procurement of raw materials, through in-process controls to finished product specifications and control procedures. Appropriate quality assurance protocols are urgently needed for indigenous medicinal plants to ensure products that are safe, effective and of good quality, both for the local and export market (South Africa, Medicines and Related Substances Act, No. 101 of 1965, as amended). An example is Leonotis leonurus, also known as wild cannabis or “wilde dagga”, which is native to South Africa and for many years has been commonly used for the treatment of numerous ailments by traditional healers (Scott et al., 2004). The traditional medicinal uses of the plant, which is administered both topically and orally, range from its use in the treatment of snake bite to its use as a purgative and vermifuge (Inouye et al., 1974). Aerial parts of the plant, both leaves and flowers, are administrated in various ways such as decoction, infusion or by smoking (Van Wyk et al., 2000). A short review of L. leonurus as a herbal medicine has been published by Mazimba (2015), in which traditional uses, phytochemistry and pharmacology are briefly mentioned. The present review focuses, in depth, on this plant, L. leonurus (L.) R.Br. (Lamiaceae) and its botanical and taxonomic aspects, ethnopharmacology, biological activity and phytochemistry, as well as elucidating aspects which require clarification and further research. L. leonurus is a broadleaf evergreen plant indigenous to Southern Africa and known for its medicinal and psychoactive properties and colloquially referred to as Cape hemp, wild “dagga”, lions’s ear or minaret flower (Hutchings et al., 1996). It is also known in Afrikaans as “wilde dagga”, “rooidagga” or “duiwelstabak”, in isiZulu as “umunyane”, in seSotho as “lebake”, in isiXhosa as “umfincafincane” and in Shona as “umhlahlampetu” (Hutchings et al., 1996). Some authors also refer to L. leonurus as “klip dagga” (Thring and Weitz, 2006), whereas others indicate that L. nepetifolia is the species referred to as “klip dagga” (Watt and Breyer-Brandwijk, 1962).

4

2

BOTANICAL ASPECTS

The name of the genus, Leonotis, is derived from a combination of the Greek (λέων) “leon” meaning lion and “otis” meaning ear, as suggested by the likeness of the flower petals to a lion’s ear (Figure 1a and b) (Plantzafrica, 2015), while etymology of the species name, leonurus has already been elucidated for the Eurasian genus Leonurus species. This genus-name of is confusingly identical to the species-name of L. leonurus. The second part of the species-name, "urus", is derived from ουρά (urá) and means "tail", "leonurus" therefore means "lion's tail". This name may have been given to the plant as an allusion to its appearance or, perhaps more probably, due to the botanical and perhaps also medicinal similarities to its European counterparts of the genus Leonurus which have very similar traditional medicinal uses. This reference in the botanical name to both “Lion’s ear” and “Lion’s tail” is probably also responsible for the confusion between L. leonurus and L. nepetifolia in the popular literature, where both species are referred to as lion’s ear or lion’s tail, although “Lion’s ear” or “wild dagga” most often refers to L. leonurus and “Lion’s tail” or “klip dagga” to L. nepetifolia. Leonotis leonurus is an easily cultivated, drought tolerant and fast growing plant that is frost hardly and blooms in the late summer to early autumn. Belonging to the Lamiaceae (mint) family, L. leonurus is a subtropical woody shrub between two and five metres in height and about 1.5 m wide (Iwarsson and Harvey, 2003; Van Wyk and Gericke, 2003). It branches from a thick woody base with a pale brown, herbaceous and densely pubescent square stem, the immature stem is greyish green. The simple, pubescent leaves of the plant are narrowly ovate to linear, the apex narrowly acute, the base narrowly cuneate, with a serrated margin, located opposite each other on the stem, petiolate and coriaceous (Figure 2). The lower surface of the leaf is more densely pubescent than the upper, while both have sessile glands. The leaves have a characteristic pungent smell, with a bright yellowgreen colour and a rough texture. The inflorescence of L. leonurus comprises of two to 11 verticils per branch, internodes 40 to 70 mm long, with conical to hemispherical verticils, 25 to 40 mm in diameter. Bracts are leaflike and bractioles are linear and densely pubescent with sessile glands, the calyx is 12 to 16 mm long, 4 mm in diameter at the mouth and pale green to yellowish. The tubular corolla is 40 to 49 mm long, orange with orange hairs, tube 26 to 30 mm long, bending forwards, 5

widening at the mouth with one to three fringes within the corolla and a hair fringe at the margin and sparsely covered with short orange hairs on the outer surface. The posterior lip of the corolla is significantly longer than the anterior lip which is deflexed and soon withering (Iwarsson and Harvey, 2003; Hurinanthan, 2009). L. leonurus is widely distributed in South Africa including the Eastern and Western Cape Provinces, KwaZulu-Natal and Mpumalanga (Figure 3). It grows along forest margins particularly on river banks, on rocky hillsides and in grasslands and is easily identified as it rises above the shrubbery mass throughout the summer season (Figure 1c). The plant is currently not threatened (Iwarsson and Harvey, 2003). 3

TAXONOMY

In a definitive monograph, Iwarsson and Harvey (2003) revise the genus Leonotis (necessary since the authors point out that the genus was last revised in 1900 by Baker) to unite all the fragmented African Leonotis research and describe four new taxa. These authors consider Leonotis leonurus (L.) R. Br the type species of the genus. Many taxa are used medicinally throughout Africa, although L. leonurus is endemic to South Africa, a number of species have been misidentified as L. leonurus, notably L. decadonta var. vestita (Briq.) Iwarsson & Y.B.Harv., although their distributions do not overlap, with L. decadonta Gürke occurring in countries to the north of South Africa. Leonotis nepetifolia (L.) R.Br. is often confused with L. leonurus in the popular literature, particularly internet sites where lion’s ear and lion’s tail, wild “dagga” and “klip dagga” are often used as synonyms (Wikimedia, 2015; Wikipedia, 2015; Drugs-forum, 2015). L. nepetifolia is distributed throughout much of the tropics and is often regarded as a weed. 4 4.1

ETHNOPHARMACOLOGY Traditional use in humans

L. leonurus boasts a large number of recorded traditional uses. For many years, the plant has been widely used, both topically and orally, for the treatment of various ailments by southern African traditional healers (Scott et al., 2004). The traditional use indications of the plant include a wide number of ailments such as piles, eczema, skin rashes, boils, itching and muscular cramps via the topical application of a decoction, in which the water soluble compounds are extracted by boiling the plant 6

material. This procedure consists of adding one tablespoon of chopped dried leaves (about 10 g) to three cups of boiling water and boiling for a further ten minutes. The decoction is then cooled overnight, strained and used as a clean liquid for both internal and external use (Mugabo et al. 2012). A decoction of L. leonurus is administered orally to treat coughs, colds and bronchitis. It has also been used to relieve cardiac complaints and asthma. Powdered leaves are compounded into an ointment and applied topically for relief of pain above the eye (Hutchings et al., 1996). Watt and Breyer-Brandwijk (1962) report that a decoction of the powdered stem or seed is administered orally for the relief of haemorrhoids and also used topically as a lotion for sores on the legs and head. The leaf decoction was used as a strong purgative and emmenagogue by the Khoisan people. Different parts of the plant, used either as decoctions or inhalations, are reported to treat the common cold, epilepsy, leprosy, high blood pressure and other 'cardiac conditions' (Duke, 2001). The leaves and flowers of L. leonurus have been frequently used in Southern Africa for the treatment of chest infection, flu, fever, dysentery, influenza, constipation, headache, epilepsy, spider and snake bites, delayed menstruation, intestinal worms, hypertension and scorpion stings (Bryant, 1966, Jäger et al., 1996; Van Wyk et al., 2000; Stafford et al., 2008). Infusions of the flowers, leaves or stems are widely used as purgatives and tonics to treat influenza, tuberculosis, jaundice, muscular cramps, skin diseases and sores. The use of L. leonurus leaves and flowers as a remedy for snakebites and to treat other bites and stings, such as bee and scorpion stings is widespread in South Africa (Hutchings et al., 1996). In treating snake bite, the ground roots of L. leonurus were often mixed with other plants such as Strychnos spinosa (roots and green fruits), and a hot infusion was given to the patient to drink as an emetic (Bryant, 1966). In South Africa, an infusion of the stem and leaves of the plant is used by the Zulu people for treatment of coughs and colds. The Zulu also use a cold infusion of the leaf as a nasal douche to relieve headache associated with high fever. Leaf infusions have been reportedly used as an asthma treatment and to alleviate viral hepatitis (Watt and Breyer-Brandwijk, 1962). In his 2013 review of the traditional use of medicinal plants in south-central Zimbabwe, Maroyi indicates that the leaves of L. leonurus, known in the vernacular as “mutodzvo”, are chewed and the sap swallowed as a treatment for ulcers. Dried leaves and flowers of L. leonurus 7

have been traditionally smoked for the relief of epilepsy and as a cure for partial paralysis (Watt and Breyer-Brandwijk, 1962; Van Wyk et al., 2000). The leaves are also frequently brewed as a minty tea that is used as a diuretic and for obesity (Watt and Breyer-Brandwijk, 1962, Hutchings et al., 1996, Van Wyk et al., 2000). The traditional uses of L. leonurus are summarized in Table 1. In addition to the traditional uses mentioned above, wild “dagga” is commercially available and marketed for its purported psychoactive effects (Hutchings et al., 1996; Van Wyk et al., 2000). The dried leaves or flowers are reported to produce a mild euphoric effect when smoked and have been said to have a similar effect to cannabis but with a less potent high (Wu et al., 2013). In South Africa, indigenous ethnic groups often smoked L. leonurus instead of tobacco. Smoking “wild cannabis” flowers reportedly results in a mental and emotional condition in which the user experiences intense feelings of well-being, elation, happiness and joy, but can also cause many side effects such as visual changes, nausea, dizziness, sedation, sweating and light headedness. It is reported to have an unpleasant taste and cause lung and throat irritation. The flowers are known to be hallucinogenic (Richard et al., 2001). 4.2

Ethnoveterinary uses

As in many developing countries, rural livestock farmers in South Africa often treat their animals using the expertise and skill that they themselves possess with respect to the medicinal properties of indigenous plants (McGaw and Eloff, 2008). A multifaceted system of traditional beliefs, learned skills, knowledge handed down from generation to generation and local practices all contribute to the ethnoveterinary uses of plants to prevent, control and treat disease. Allopathic medicine certainly has its place in managing epidemics of contagious diseases but for many commonly occurring ailments in animals, such as mild diarrhoea, intestinal parasites, wounds and skin infections, ethnoveterinary medicine is the treatment of choice for many farmers due to its ready availability and affordability. The year-round availability of plant material due to seasonal changes, inefficacy or even harmful potential of certain remedies and lack of standardisation for accurate dosing are inadequacies of ethnoveterinary medicine. Research attempts to overcome these difficulties and

8

integrate the scientific findings into the primary animal healthcare environment (Martin et al., 2001). The use of traditional herbal medicines, including L. leonurus by small-scale livestock farmers of the Eastern Cape Province of South Africa has been investigated by a few researchers, notably Masika et al., (1997 and 2000), Masika and Afolayan (2003) and found to be a common practice. L. leonurus, known locally as “UmFincafincane” (Xhosa) was reportedly used to treat eye inflammation by instilling a drop of the squeezed leaf sap daily into the eyes of the afflicted animal. Pounded roots and leaves of L. leonurus are added to the drinking water of poultry by traditional farmers in South Africa to prevent sickness, as well as to treat gallsickness (anaplasmosis) in their cattle (Hulme, 1954 as quoted by Hutchings, 1996 and McGaw and Eloff, 2008). Interestingly L. ocymifolia (Burm.f.) Iwarsson, a species that resembles L. leonurus, is also used to treat gallsickness and to prevent poultry diseases. Leonotis leonurus is also one of the plants used by farmers in the Eastern Cape Province of South Africa to control worm infestations in their livestock, particularly goats (Maphosa and Masika, 2011). A study by Maphosa and Masika (2011) found that aqueous extracts of L. leonurus, Aloe ferox Mill. and Elephantorrhiza elephantina (Burch.) Skeels were effective against gastrointestinal parasites in high doses in goats which were naturally infected with mixed gastrointestinal worms and Coccidia species. 4.3

Recreational use

Zuba et al. (2013) report that in the first years of the 21st century numerous herbal products were advertised, particularly over the internet, as legal alternatives to cannabis. L. leonurus was one of these herbal products, amongst others such as Nymphaea caerulea Savigny, Turnera diffusa Willd. ex Schult. and Zornia latifolia DC. Although these were claimed to be “pure” herbal products, in 2008 it was discovered that they were adulterated with synthetic psychoactive compounds, usually synthetic cannabinoids. Cornara et al. (2013) refer to these aromatic plant species as a “green shuttle” that have the chief role of disguising the synthetic cannabinoids as products available to produce a “legal high”, but in reality they pose a serious threat to public health when adulterated with synthetic cannabinoids. 9

5

BIOLOGICAL ACTIVITY

A range of biological activities have been reported for extracts and isolated molecules

obtained

from

L.

leonurus,

including

antibacterial,

antifungal,

antiprotozoal, enzyme inducing, anti-inflammatory activities and modulation of immune cell functions. Aqueous leaf preparations of L. leonurus have been reported to possess various properties such as anticonvulsant, anti-inflammatory, antidiabetic and antinociceptive effects in rodents which could potentially be useful in the control of painful arthritis, including arthritic inflammation (Ojewole, 2005). Analysis of an aqueous leaf extract indicated the presence of potentially useful components with anticonvulsant (Bienvenu et al., 2002), antidiabetic (Oyedemi et al., 2011a), antioxidant (Oyedemi and Afolayan, 2011) and anthelmintic activities (Maphosa et al., 2010). The flowers are reported to be hallucinogenic (Richard et al., 2001), although this is disputed by some authors (Watt and Breyer-Brandwijk, 1962; Bryant, 1966). The biological and pharmacological properties of the isolated compounds are concisely presented in Tables 2 and 3. 5.1

Anticonvulsant activity

GABA (gamma amino butyric acid) is the major inhibitory neurotransmitter in the brain, while glutamic acid is an excitatory neurotransmitter. The inhibition of GABA neurotransmission and the enhancement of the action of glutamic acid are implicated in epilepsy (Westmoreland et al., 1994; Rang et al., 1999). It has been reported that some of the compounds identified in the aqueous extract of L. leonurus, including alkaloids, did not show any anticonvulsant properties in an animal model of epilepsy (Akah and Nwaiwu, 1988). However, a study on the anti-epileptic activity of L. leonurus and the possible involvement of GABA and glutamic acid systems in this activity conducted in 2002 by Bienvenu et al. refute these results. Their investigation into the effect of an aqueous leaf extract showed that anticonvulsant activity was exhibited in mice against seizures produced by the intraperitoneal injection of pentylenetetrazole (PTZ), picrotoxin, bicuculline and N-methyl-DL-aspartic acid. The authors reported that in doses of 200 and 400 mg/kg, the plant extract prevented seizures in 37.5% and 50% of animals, respectively and significantly delayed the onset of pentylenetetrazole induced tonic seizures, when dosed at 90 mg/kg, through an as yet undetermined mechanism. However De Sarro and co-workers previously

10

revealed that pentylenetetrazole most likely induces seizures via the inhibition of gabaergic mechanisms (De Sarro et al., 1999). It is therefore likely that L. leonurus aqueous extracts exert their anticonvulsant activity by the enhancement of GAGAmediated neurotransmission in the brain. The same doses of L. leonurus crude aqueous extract (200 and 400 mg/kg) also significantly delayed the onset of tonic seizures produced by picrotoxin (8 mg/kg) although the incidence of seizures was not altered significantly. Picrotoxin induces seizures through the antagonism of the effect of GABA by the blocking of chloride channels linked to GABAA-receptors, antagonising GABA-mediated inhibition (Westmoreland et al., 1994; Rang et al., 1999). This finding suggests that L. leonurus in all likelihood acts via gabaergic mechanisms, possibly by opening the chloride channels that are associated with GABAA-receptors. Conversely, seizures induced by bicuculline (20 mg/kg), which is a selective antagonist of GABA at the GABAA-receptors, were not altered to any significant extent, by all the doses (100, 200 and 400 mg/kg) of the aqueous extracts prepared from L. leonurus. These findings suggest that L. leonurus does not exert its effects by direct action on the GABAA-receptors (Bienvenu et al., 2002). N-methylDL-aspartic acid (NMDLA) elicited seizures in all the mice in the study of Bienvenu et al. (2002), NMDLA is an agonist at NMDA receptors, mimicking the activity of glutamic acid and enhancing glutaminergic activity (Rang et al., 1999). L. leonurus extract delayed the onset of NMDLA-induced seizures, indicative of some glutaminergic involvement in the mechanism of anticonvulsant activity of the plant extract. Bienvenu et al. (2002) concluded that L. leonurus may exert its anticonvulsant effect via non-specific mechanisms, since it has been shown to delay the onset of seizures produced by compounds affecting both gabaergic and glutaminergic systems. This experiment confirms the claims by traditional medicine practitioners that the plant is effective as an anticonvulsive treatment. The same research group, Muhizi et al., (2005), later isolated, identified and evaluated two anticonvulsant compounds from L. leonurus leaves. The diterpene lactone, 20-acetoxy-9α,13α-epoxylabda-14-en6β(19)-lactone (5) protected 50% of the experimental animals (mice) from PTZ induced seizures or significantly delayed seizure onset at a dose of 400 mg/kg. A second compound, a quinone, isolated from the methanol extract, but not identified, protected 75% and 87.5% of the experimental animals (mice) from PTZ induced 11

seizures or significantly delayed seizure onset at doses of 200 mg/kg and 400 mg/kg respectively. Moreover, He et al. (2012) also investigated the anticonvulsant effect by screening an aqueous extract of L. leonurus (1 g/mL) in a binding assay at the GABAA site and an inhibition of 81% was reported. However, two compounds isolated in the study, Leonurenone A and B, were reported to be inactive at this receptor which indicates that these compounds are not in themselves responsible for the anticonvulsant activity via this mechanism. 5.2

Psychoactive properties

In some countries, where cannabis is considered an illegal psychoactive plant, L. leonurus (syn. “wild cannabis” or “dagga”), has been used as an alternative. A variety of addictive drugs, including stimulants such as cocaine, act by amplifying the effects of dopamine which functions as a neurotransmitter in the brain and can play a major role in reward-motivated behaviour (Stafford et al., 2008). The use of “wild dagga” imparts a mental and emotional condition in which the subject experiences an intense feeling of well-being, elation and excitement. Some internet websites (e.g. Wikipaedia, 2015) claim that the psychoactive property of the plant is attributable to an alkaloid, leonurine (20). However, there is some doubt as to the validity of this claim since no experimental evidence has ever been published indicating that leonurine has been found to occur in L. leonurus, although it has been documented in a related species, Leonurus japonicus (Hayashi, 1962; Kong et al., 1976; Luo, 1985; Chen and Kwan, 2001). A number of synonyms occur in the literature for this plant; Leonurus sibiricus L., Leonurus japonicus Houtt., Stachys artemisia Lour., Leonurus heterophyllus Sweet, and Leonurus artemisia (Lour.) S.Y. Hu. These are in fact all a single plant species, Leonurus japonicus (Harley and Paton, 2001). Kuchta, Ortwein and Rauwald, 2012 (and cited by Qui et al. in 2014), found leonurine only present in the aerial parts of Leonurus japonicus by RP HPLC, when examining Leonurus japonicus, Leonurus cardiaca L., and Leonotis leonurus extracts. Moreover, in addition to the lack of scientific evidence for the occurrence of leonurine in L. leonurus, there is also no evidence indicating that leonurine exhibits a psychotropic activity similar to that of Cannabis. In fact, the only neuronal activity of

12

leonurine that has been experimentally verified is an effect on the GABAA receptor, similar to that found for Valerian (Rauwald et al. 2013, Rauwald et al. 2015). On the other hand, Wu et al. (2013) report that only Leoleorin C (12), which is one of the eleven compounds isolated from L. leonurus in their study, could be responsible for the psychoactive properties of the plant, after showing moderate binding affinity to the sigma-1 receptor in the radioligand binding assay (tested at a concentration of 10 µmol/L). The sigma-1 receptor is a unique ligand-regulated molecular chaperone in the endoplasmic reticulum of cells (Maurice and Su, 2009). They are involved in the modulation of various neurotransmitter systems and have a high affinity for chemically distinct classes of psychotropic drugs. There is mounting evidence to suggest that the sigma-1 receptors are implicated in higher-order brain functions, although they are also present in many peripheral organs such as the liver, heart and lung (Maurice and Su, 2009). They play an important role in the pathophysiology of neuropsychiatric diseases including schizophrenia, depression,

anxiety and

dementia (Ishikawa and Hashimoto, 2009). Research has also suggested that L. leonurus could benefit patients with social anxiety and depression through modulation of the dopaminergic system (Stafford et al., 2008). 5.3

Antidiabetic activity

Diabetes mellitus is a complex condition that is characterized by chronic hyperglycaemia and abnormality of the lipid profile leading to a series of secondary complications (Rang et al., 1991; Ravi et al., 2005). Diabetes mellitus affects the metabolism of carbohydrates, fats, proteins and electrolytes due to a deficiency of insulin or due to target organs becoming insensitive to insulin. Statistics show that the frequency of diabetes is increasing with a major impact on the population of developing countries, partially as a result of limited access to effective and affordable interventions for the disease (Marx, 2002). According to Wild et al. (2004), about 366 million people worldwide are projected to be diabetic by 2030. Many compounds, especially flavonoids and other phenolics, have been reported to enhance insulin secretion from pancreatic beta cells or to sensitize insulin receptors (Ratnasooriya et al., 2004) and scavenge free radicals that are generated in the diabetic state (Marles and Farnsworth, 1995). Several hypoglycaemic agents such as metformin and alpha-glucosidase inhibitors have been used for the treatment of 13

diabetes mellitus. However, they reportedly produce serious adverse side effects including liver problems, lactic acidosis and diarrhoea (Rajalakshmi et al., 2009). A leaf extract of L. leonurus has been reported to possess hypoglycaemic effects in a streptozotocin (STZ)-induced diabetic rat model by Scott et al., (2004) and Ojewole, (2005). A study carried out by Oyedemi et al. (2011a) also supports the hypoglycaemic potential of L. leonurus in that an aqueous extract of the leaves exhibited

anti-hyperglycaemic

and

antilipidemic

effects,

supporting

the

ethnotherapeutic usage of L. leonurus as a treatment for diabetes mellitus. An aqueous extract, dosed orally at 125, 250 and 500 mg/kg for 15 days, of L. leonurus leaves, was reported to have antidiabetic activities in STZ-induced (45 mg/kg intraperitoneal) diabetic rats. Elevated glucose, cholesterol, high density lipoprotein (HDL) and triglycerides levels, accompanied by weight loss in STZ-induced diabetic rats were reversed after treatment with the aqueous leaf extract of L. leonurus. After the treatment with L. leonurus, the weight loss of diabetic rats was reportedly close to the groups treated with glibenclamide, which is a standard hypoglycaemic drug that stimulates insulin secretion from beta cells of the Islets of Langerhans (Oyedemi et al., 2011a). Phenolics such as flavonoids and proanthocyanidins were reported to be present in the aqueous extract of L. leonurus leaves. In 2012, Mnonopi et al. conducted

an

investigation

into

the

mechanism

of

the

hypoglycaemic,

cardioprotective and anti-inflammatory effects of L. leonurus organic leaf extracts and a labdane diterpenoid, marrubiin (7), one of the constituents of the extract, originally isolated by Kaplan in 1968.The authors reported that the organic extract yielded 5% marrubiin and that both leaf extract and marrubiin (7) resulted in an increase in respiratory rate and mitochondrial membrane potential under hyperglycaemic conditions by inducing insulin secretion, increased HDL-cholesterol, and interleukin-1β and interleukin-6 levels in an obese rate model. The authors indicate that the results provide evidence that the increase in insulin secretion, attributed to the administration of the plant extract, alleviated diabetic symptoms, and that the compound responsible for this pharmacological effect was marrubiin (7) (Mnonopi et al., 2012). Flavonoids are well known in regenerating the damaged beta cells in diabetic rats found to be effective anti-hyperglycaemic agents (Chakravarthy et al., 1980; Manickam et al., 1997). Therefore the Oyedemi group’s findings proved to be in 14

accordance with that of Ojewole (2005), who also observed anti-hyperglycaemic effect of L. leonurus in streptozotocin (STZ)-induced diabetes mellitus rats and reported that various flavonoids, diterpenoids, polyphenolics and other secondary metabolites of the plant could be responsible for the observed antidiabetic effects of the aqueous leaf extract of the plant (Ojewole, 2005). 5.4

Anti-infective activity: antibacterial, anti-amoebic, anthelmintic, antimalarial and anti-HIV activity and toxicity.

Numerous studies have been conducted on the antimicrobial effects of L. leonurus due to its ethnomedicinal use for various infections and infestations as outlined in table1. 5.4.1 Antibacterial activity In 2004, Steenkamp et al. conducted a study on antibacterial effects and tissue growth stimulation and wound healing effects of the aqueous and methanol extracts of L. leonurus stems and leaves. Organisms investigated included Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli and Pseudomonas aeruginosa, and MICs values in excess of 4 mg/ml were recorded. These results are in accordance with previous studies confirming that the plant does not possess noteworthy antibacterial activity (Hutchings et al., 1996 and Kelmanson et al., 2000). Stafford et al. (2005) reported on the effect of storage on the chemical composition and antibacterial activity of L. leonurus against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae and reported that aqueous, ethanol and hexane extracts of fresh and stored leaves showed an increase in activity against E. coli after 1 year and also an increase in antibacterial activity against Bacillus subtilis and Staphylococcus aureus after 90 days of storage in exception of Klebsiella pneumoniae against which the authors reported to have lost activity after 5 years. Agnihotri et al. (2009) isolated six pure compounds from the ethanol extract of L. leonurus flowers, including dihydroxyphytyl palmitate (23), succinic acid (24), uracil (25), luteolin 7-O-glucoside (22), verbascoside (acteoside) (26) and geniposidic acid (27). None of these compounds had antimicrobial activity against Candida albicans,

15

Escherichia

coli,

Pseudomonas

aeruginosa,

Cryptococcus

neoformans,

Mycobacterium intracellulare or Aspergillus fumigatus. Jimoh et al., (2010) reported that acetone and methanol extracts of L. leonurus leaves showed antibacterial activities with a MIC range of 1-5 mg/mL using agar diffusion or dilution method against standard strains of Bacillus cereus, Staphylococcus

epidermidis,

Micrococcus

kristinae,

Staphylococcus

aureus,

Streptococcus pyogenes, Escherichia coli, Salmonella pooni, Serratia marcescens and Pseudomonas aeruginosa. However, the water extracts did not show antibacterial activity and Klebsiella pneumoniae which is commonly implicated in nosocomial infections was not responsive to leaf extracts. Naidoo et al., (2011) reported that aqueous and methanol/dichloromethane (1:1) extracts of L. leonurus leaves presented 90% growth inhibition when tested at a concentration of 1000 µg/mL against Mycobacterium tuberculosis with rifampicin used as positive control (2 µg/mL). Oyedeji et al., (2005) reported that the major essential oil components of L. leonurus were limonene (7.2-15.6%), β-ocimene (7.5-10.8%), γ-terpinene (4.0-4.7%), βcaryophyllene (15.2-19.6%), α-humulene (4.6-6.5%) and germacrene D (18.920.0%) and that the oils exhibited a broad spectrum antibacterial activity against Gram-positive Staphylococcus

(Bacillus aureus,

subtilis,

Bacillus

Staphylococcus

cereus,

Micrococcus

epidermidis)

and

kiristinae,

Gram-negative

(Escherichia coli, Pseudomonas aeruginosa, Shigella sonnei) bacteria with MIC values ranging from 0.039 – 1.25 mg/mL. 5.4.2 Anti-amoebic activity McGaw et al. (2000) report that L. leonurus had no anti-amoebic activity against Entamoeba histolytica for either the aqueous or ethanolic extracts. 5.4.3 Anthelmintic activity McGaw et al. (2000) report that L. leonurus leaf extracts exhibited activity against, Caenorhabditis elegans, a free-living nematode; hexane, ethanolic and aqueous extracts were evaluated for their effect on the reproductive ability and mortality of the parasite at concentrations of 1 and 2 mg/mL, the control was the standard treatment

16

levamisole, 5 μg/mL. The ethanolic and aqueous extracts exhibited slight anthelmintic activity against C. elegans, whereas the hexane extract was not active. In 2011, Maphosa and Masika published their study highlighting the potential of certain herbal medicines as anthelmintics and antiprotozoals against mixed infestations of gastrointestinal nematodes in goats, showing, using the faecal egg count method, that L. leonurus aqueous leaf extract was effective at high doses, 500 mg/kg, against gastrointestinal parasites. 5.4.4 Antimalarial activity As part of the research for antimicrobial and antimalarial compounds from higher plants, Agnihotri and co-workers (2009) investigated the flowers of L. leonurus. Six pure compounds isolated from the ethanol extract of L. leonurus flowers, including dihydroxyphytyl palmitate (23), succinic acid (24), uracil (25), luteolin 7-O-glucoside (22), verbascoside (acteoside) (26) and geniposidic acid (27), were tested for antimalarial, cytotoxic and antimicrobial activities (Jain et al., 2005). Only one compound (luteolin 7-O-glucoside (22)) was reported to have antimalarial activity against two strains of Plasmodium falciparum, the D6 clone, with IC50 equal to 2.2 µg/mL and the W2 clone, with IC50 equal to 1.8 µg/mL (Kirmizibekmez et al., 2004). Chloroquine and artemisinin, the positive controls, showed IC50 values of 0.016 and 0.0048 µg/mL (for the D6) and IC50 values of 0.14 and 0.0047 µg/mL (for the W2), respectively. 5.4.5 Anti-HIV activity Klos et al. (2009) report that ethanolic extracts of L. leonurus leaves exhibited inhibitory activity in an antiviral assay in CEM.NKR-CCR5 (a human T-lymphoblastic cell line) cell cultures, showing a 33% reduction in HIV-1 p24 core protein. The aqueous extract also showed HIV-1 reverse transcriptase inhibition, but this effect was lost after dereplication for the removal of common non-specific tannins and polysaccharides. L. leonurus has thus been shown to possess anti-HIV properties, probably through HIV-1 protease inhibition, with an IC50 of 120.6 μg/mL of the crude extract that may be of therapeutic significance. L. leonurus is traditionally used to treat hepatitis, it has been found that plants used to treat hepatitis often exhibit anti-HIV activity. The ethanolic extract of L. leonurus

17

possibly contains novel antiviral compounds and a more lipid soluble extract may yield further unknown protease inhibitory compounds (Klos et al., 2009) 5.5

Anti-oxidant activity

In the past fifteen years, there has been an increased interest in finding natural antioxidants especially from plants in order to protect the human body from free radical related diseases such as diabetes mellitus, cancer, atherosclerosis, arthritis, anaemia, asthma, inflammation and neurodegenerative diseases (Kinsella et al., 1993). Free radicals are chemically unstable atoms or molecules that cause extensive damage to cells, resulting from the lack of balance between the generation of reactive oxygen species which are a group of highly reactive molecules and the anti-oxidant enzymes in the cell (Lee et al., 2004). To offer a scientific basis to the traditional treatments utilising L. leonurus, Oyedemi and Afolayan (2011) investigated the anti-oxidant activities of the plant extract both in vivo and in vitro. The results reported in this study proved that the plant is a source of anti-oxidants and could potentially protect humans from free radical related diseases. The in vivo study in Wistar rats treated with carbon tetrachloride (CCl4) for 7 days evaluated the effect of the oral administration of the aqueous leaf extract of L. leonurus at the doses of 125, 250 and 500 mg/kg body weight. The extract was reported to effectively increase the percentage inhibition of reduced glutathione (GSH), superoxide dismutase (SOD) and catalase

activities (CAT). Lipid

peroxidation was considerably reduced in rats treated with CCl4 when compared with the diabetic control rats. The authors conclude that there is evidence to support that the aqueous leaf extract of L. leonurus exhibits strong anti-oxidant activity both in vitro and in vivo, probably as a result of the high content of phenolic compounds (e.g. flavonoids and proanthocyanidins) well-known for their anti-oxidant properties. Flavonoids, such as those found in high concentrations in L. leonurus have been reported to enhance insulin secretion and scavenge free radicals that are generated in diabetic patients (Marles and Farnsworth, 1995). The in vitro anti-oxidant activity was evaluated by Oyedemi and Afolayan (2011) after determining the ferric reducing power of the anti-oxidant compounds including phenolics (high content), flavonoids, flavonols and proanthocyanidins found in water 18

extract of L. leonurus leaves at different concentrations. The ability of the extract to scavenge free radicals, as proven in anti-oxidant tests such as 2,2 diphenyl-2picrylhydrazyl (DPPH) radical scavenging assay, was also assessed. The aqueous extract of L. leonurus was reported to demonstrate strong DPPH scavenging properties; it therefore could serve as a free radical inhibitor (Oyedemi and Afolayan, 2011). 5.6

Toxicity

A number of investigators report on the toxicity of L. leonurus as part of their bioactivity studies. Maphosa et al., (2008), reported that the aqueous extract of L. leonurus shoots, evaluated in female rats, exhibited an oral LD50 of greater than 3 200 mg/kg in the acute toxicity test, while in the chronic toxicity test, significant (p<.05) changes in blood parameters were observed at dosage levels as low as 200 mg/kg and in biochemical parameters at a dosage of 400 mg/kg of the extract (p<.05). The subacute toxicity test revealed significant changes (p<.05) in haematological parameters at doses of 1,600 mg/kg of the extract. Symptoms of toxicity included decreased respiratory rate and respiratory failure, loss of righting reflex accompanied by decreased motor activity and skeletal muscle paralysis, ataxia, convulsions and coma. Maphosa et al. (2008) concluded that caution needs to be exercised in the ethnoveterinary use of L. leonurus particularly with proplonged use. The authors in their conclusion speculate on the possible clinical relevance of their findings, particularly with sustained use; the effect on haematological parameters may predispose treated animals to anaemia, the changes in white blood cell count may lead to compromise of the immune system, reduction in platelet levels could precipitate thrombocytopaenia and possible haemorrhage, the observed increase in total blood protein conversely may stimulate the immune response by increasing immunoglobulin production, the significant decrease in liver enzymes and bile pigments may be indicative of adverse effects on the liver, the decrease in the levels of urea however may indicate that the hepatic and renal systems were not adversely affected and the observed increase in organ weights may be indicative of compensatory responses to treatment induced biochemical parameters and possible toxicity. 19

Agnihotri et al. (2009) reported that none of the six compounds (dihydroxyphytyl palmitate (23), succinic acid (24), uracil (25), luteolin 7-O-glucoside (22), verbascoside (acteoside) (26) and geniposidic acid (27)) their group investigated for antimalarial activities had cytotoxic effects on mammalian kidney fibroblasts (Vero cells) up to concentrations of 4.76 µg/mL. In the same year, El-Ansari et al. (2009), in a biochemical analysis of L. leonurus reported that 70% methanol and chloroform extracts exhibited practically no toxic effects after oral administration to rats, with the LD50 values found to be in excess of 5 g/kg (500 mg/100 g) body weight. The extracts in fact possess strong hepatoprotective activity and may be useful in treating paracetamol-induced hepatotoxicity. Oyedemi et al. (2011b), however, in a study on male Wistar rats report alterations in the haematological, liver and kidney functions by aqueous extracts of L. leonurus at doses of 125, 250 and 500 mg/kg body weight, but no predisposition to cardiovascular risk, at these doses when administered daily for 21 days. The effect of the extract on haematological parameters in the Oyedemi et al. (2011b) study are reported to be not “well-defined” and the authors in their detailed discussion of their results, conclude that this may be indicative of both dose- and parameter-selective toxicity or an adaptation of the test animals to the pharmacological effects of the extract. They note that these results are in accordance with the finding of the Maphosa et al. (2008) study in female Wistar rats. The claim of the lack of cardiovascular risk is made due to the fact that Oyedemi et al. (2011b) found a decrease in the levels of major serum lipids and computed atherogenic index. The increase in liver and kidney to body weight ratios was attributed to probable swelling and/or inflammation, although increases in serum aminotransferases observed in this study, which are usually associated with enhanced membrane permeability, and the levels of other markers of plasma membrane permeability, such as ALP (alkaline phosphatase) and GGT (gamma glutamyltransferase), which were not elevated, lead to the inference that the increase in serum aminotransferases may have been due to leakage from the plasma membrane of organs other than the kidney and liver. Possible liver impairment was however indicated by the reduced levels of albumin and increased levels of bilirubin in the serum of all test animals at all doses of the extract. Although the bilirubin findings are in contrast to those of Maphosa et al. (2008), the authors attribute this disparity to the use of different plant parts (leaves 20

versus shoots), dosage differences and the different sex of the study animals. Renal function indices were once again varied, but certain parameters such as sodium and uric acid were elevated at higher doses (250 and 500 mg/kg bodyweight). The authors therefore conclude that, due to the changes in haematological and hepatic and renal function parameters, L. leonurus cannot be considered “safe” with respect to oral use in male rats. Clinical relevance of toxicity studies therefore remains to be further studied and substantiated. 6

PHYTOCHEMISTRY

A large number of phytochemical investigations have been conducted on L. leonurus and the interest in this plant continues to be high due to its numerous reported biological activities. Phytochemical studies have revealed that terpenoids (mono-, sesqui- and diterpenoids), which are known to be biologically active (Demetzos and Dimas, 2001) are the main compounds found in the plant (Purushothaman and Vasanth, 1988; Pedro et al., 1991), with labdane diterpenes being the most abundant compounds extracted from the leaves (Cragg and Little, 1962; Rivett, 1964; Kaplan and Rivett, 1968; Laonigro et al., 1979; Kruger and Rivett, 1988; McKenzie et al., 2006; Obikeze et al., 2009; Naidoo et al., 2011). Wu et al., (2013) isolated three known labdane diterpenes, known as Leoleorins A (10), B (11) and C (12), and eight new labdane diterpenes, Leoleorins D-J (13-15, 17) and 16-epileoleorin (16) from leaf extracts of L. leonurus. Marrubiin, a widely studied diterpenoid lactone, found in many of the members of the Lamiaceae, is also present in L. leonurus (Popoola et al., 2013). Other compounds reported to be present in L. leonurus are tannins, alkaloids and steroidal and triterpenoid saponins (Bienvenu et al., 2002). Also reported from phytochemical investigations of different extracts of the plant including ethanol, acetone or aqueous extracts, are an iridoid glycoside and phenolic compounds, primarily of the flavonoid category (He et al., 2012). The flowers, which have only been phytochemically investigated in the past decade, yielded flavonoids and acyclic diterpene esters from organic extracts; also reported are succinic acid (24), uracil (25), luteolin 7-O-glucoside (22), acetoside (26) and geniposidic acid (27) (El-Ansari et al., 2009; Agnihotri et al., 2009). Diterpenes (20-C) are C-13 allylic isomers mostly found in plants as mixtures with other related compounds, although di- and triterpenes rarely occur in the same plant 21

tissues, as is the case in L. leonurus, in which diterpenes and triterpenes are not found concurrently. A very large number of diterpenoids possessing a labdane (bicyclic diterpene) skeleton occur in nature, Figure 4 (Connolly and Hill, 1991). The interest in the study of labdanes may be ascribed to their wide range of biological activities. The labdane diterpenes consist of a decalin system and may contain a spirocyclic C and D epoxy ring (Wu et al. 2013). Labdane diterpenes have five stereogenic centres and occur in nature in two enantiomeric series, according to the stereochemistry on C-13 (Demetzos and Dimas, 2001). The Me-18, Me-17 and H-5 are known to be α-orientated in labdane diterpenoid structures. The first outcomes on the study of the chemistry of the diterpenes from the genus Leonotis appeared in 1962 where two compounds were isolated from the aerial parts of L. leonurus collected in South Africa and provisionally indicated as compound X (8) and compound Y (10) (Cragg and Little, 1962). In 1964, Rivett isolated the wellknown marrubiin (7) from the same species, a compound that was previously isolated from Marrubium vulgare. The dilactone containing a cyclic ether linkage, known as compound X (8), and the (,) unsaturated ketone, known as compound Y (10) (Table 2) were isolated again, and their structures were elucidated some years later by the same authors (Kaplan and Rivett, 1968). The structure of compound X (8) was confirmed by X-ray analysis (Kruger and Rivett, 1988) while the structure of compound Y (10) was proved to be correct by formal total synthesis starting from a derivative of marrubiin (7). The labdane skeleton of these two compounds and some details of their functional groups had clearly indicated that they were closely related to marrubiin (7). Some years later, two stereoisomeric premarrubiins (6) (13R) and (13S) were also isolated from L. leonurus from a sample collected in Naples, Italy (Laonigro et al., 1979). No other diterpenes were detected from the plant collected in Italy, while those from which compound X (8) had been extracted were collected in the Eastern Cape, South Africa (McKenzie, 2006). L. leonurus has been traditionally used for what have may be interpreted as central nervous system effects, including that it is mildly narcotic as it has been used as a substitute for Cannabis and that the leaves are traditionally smoked in South Africa to relieve epilepsy and partial paralysis (Watt and Breyer-Brandwijk, 1962 and cited by Stafford et al., 2008). These reports however remain anecdotal and are yet to be substantiated, despite claims on internet websites that the plant contains the 22

alkaloid, leonurine. Continuing research reports yielded many other compounds from L. leonurus, but leonurine (20) (Table 3), which is a mildly psychoactive guanidino alkaloid, was documented only to have been isolated from related species (He et al., 2012). Kuchta et al., in 2012 confirmed that no leonurine (20) could be detected in the aerial parts of L. leonurus using a newly developed sensitive and highly reproducible novel RP-HPLC analytical method. Bienvenu et al. (2002) determined the chromatographic profile of L. leonurus leaves using high performance liquid chromatography (HPLC) and identified the groups of chemical compounds present in the aqueous extract using the standard compendial phytochemical tests. The authors noted that the different standard chemical tests used showed positive reactions for alkaloids, saponins (of both steroid and/or triterpenoid groups) and tannins, and negative reactions for anthraquinones, cardiac glycosides and reducing sugars. This species had earlier also been reported to contain tannins, quinones, saponins, alkaloids and triterpenoids (Laonigro et al., 1979). A wide number of labdane diterpenoids had been previously extracted, most notably premarrubiin (6) (Table 2) (Laonigro et al., 1979), a structure closely related to leonurun (5) (Table 2), which is a novel labdane diterpenoid from L. leonurus identified by McKenzie in 2006. This compound had been isolated from an acetone extract of the leaves and its structure was determined from spectroscopic data and the relative stereochemistry from single-crystal X-ray diffraction analysis (McKenzie et al., 2006). Bienvenu et al. (2002) had isolated marrubiin (7) (Table 2) as the main diterpenoid lactone from L. leonurus leaf extracts, but its precursor, premarrubiin (6), was not found by this group. Moreover, the pharmacological effect of marrubiin (7) isolated from the plant was not known (Van Wyk et al., 2000), but later studies revealed its antidiabetic potential (Scott et al., 2004 and Ojewole, 2005, Oyedemi et al. 2011a, Mnonopi et al., 2012). Marrubiin (7) is reported to exist in high concentrations in many of the traditionally important Lamiaceae species and has been demonstrated to exhibit pharmacological activity with high safety margins in various inflammation models. Subsequent research has determined that marrubiin (7) is the compound responsible for many of the therapeutic properties observed for L. leonurus and most of the Marrubium genus, also a member of the Lamiaceae. Wide-ranging pharmacological studies have demonstrated that marrubiin (7) exhibits 23

numerous activities including antinociceptive, analgesic, antioxidant, antigenotoxic, cardioprotective, vasorelaxant, gastroprotective, antispasmodic, immunomodulating, antioedematogenic, and antidiabetic properties (Popoola et al., 2013). Perdo et al., 1991, isolated the essential oils from the sepals of L. leonurus by hydrodistillation and reported the isolation of 33 mono- and sesquiterpenes. Monoterpenes constituted 30.4 % of the oil; the major monoterpenes were α-pinene (12.6%), limonene (5.6%), (Z)-β-ocimene (4.8%) and p-cymene (4.4%). Sesquiterpenes constituted 59.4% of the oil; major components were β-caryophyllene (30.8%), caryophyllene oxide (8.4%) and α-humulene (7.8%). In 2005, Oyedeji conducted a comparative study of the essential oil composition and antimicrobial activity of L. leonurus where the essential oils, isolated from the aerial part of the plant growing in the Eastern Cape (South Africa), were analysed by GCMS. The authors reported that the major constituents of the plant were limonene (7.2-15.6%), β-ocimene (7.5-10.8%), γ-terpinene (4.0-4.7%), β-caryophyllene (15.219.6%), α-humulene (4.6-6.5%) and germacrene D (18.9-20.0%). Contrary to Bienvenu’s findings, Oyedemi et al. (2011a) reported that alkaloids, tannins and saponins were not detected after quantitative analysis of polyphenolic compounds in the water extract of L. leonurus leaves but yielded high flavonoid content. Obikeze et al., (2009) reported for the first time the presence of a novel diterpene, (13S)-9,13-epoxylabda-6(19),15(14)dioldilactone (EDD) (9) (Table 2), isolated from a methanol extract of L. leonurus leaves collected in Cape Town, for which the structure was elucidated using infra-red (IR), nuclear magnetic resonance (NMR), mass spectroscopy (MS) and X-ray diffraction analysis. The compound exhibited an effect on the cardiovascular system after being administrated intravenously in anaesthetized normotensive male Wistar rats by producing a vasoconstrictive effect accompanied by bradycardia (Obikeze et al., 2009). The new diterpenoid obtained after fractionation of the organic extract of L. leonurus leaves presented spectral data with a number of similarities with compound X (8) mentioned previously. EDD (9) and compound X (8) are positional isomers but the main structural difference between the two compounds is that the lactone carbonyl group in EDD (9) is at the

24

C-14 position while in compound X (8), the lactone carbonyl group is at the C-15 position. Moreover, Agnihotri et al. (2009) undertook an investigation of the ethanol extract of L. leonurus flowers and described the isolation, structural elucidation and biological activities of a new compound, 1,2,3-trihydroxy-3,7,11,15-tetramethylhexadecan-1-ylpalmitate (23), which is a diterpene ester (Table 3 and figure 5) and five other reported compounds which include succinic acid (24), uracil (25), luteolin 7-Oglucoside (22), verbascoside (acteoside) (26) and geniposidic acid (27). These compounds were characterized by comparison of their physical and spectral data. The structures of all compounds were resolved by spectroscopic methods including 1D and 2D NMR spectroscopy (Agnihotri et al., 2009). El-Ansari et al. (2009) in their study on the phytochemical and pharmacological properties of L. leonurus flowering aerial parts, collected from cultivated plants in the National Research Centre in Dokki, Egypt, report the isolation and chemical characterization of apigenins, in addition to labdane diterpenes reported by other groups, prior to Agnohotri (2009) and subsequently He et al., (2012), but remain the only research group to report the apigenins and novel labdanes, which include glycosides; Apigenin, 5,7-Dihydroxy-2(4-hydroxyphenyl)-4H-1-benzopyran-4-one (28), Chrysoeriol, Luteolin 3’-methyl ether (29), 6-Methoxyluteolin-4’-methyl ether (30), Apigenin 6-C-α-arabinoside-8-C-βglucoside (31), Vitexin, Apigenin 8-C-β-glucoside (32), Apigetrin, Apigenin 7-O-βglucoside (33), and Apigenin 7-O-(6''-O-p-coumaroyl)-β-glucoside (35). He et al. (2012) confirmed the presence of luteolin 7-O-glucoside (22) and luteolin (21) previously isolated by Agnihotri et al. (2009) and Leonurenone A (1) by Naidoo et al. (2011). The group was able to isolate Leonurenones A–C (1-3), two known labdanes, luteolin 7-O-β-glucoside (22) and luteolin (21), after repeated purification procedures which included HPLC and flash column chromatography on an aqueous extract of L. leonurus leaves. A leaf aqueous extract of the plant yielded Leonurenone A (1), Leonurenone B (2) (Table 2 and figure 5), luteolin glycoside (22) and luteolin (21). However, repeated chromatography of an acetone extract of the same plant afforded Leonurenone C (3), 9,13:15,16-diepoxylabdane-6β,15α-diol (14), previously reported by Naidoo (2011) a year earlier, and nepetaefolin (4) (Table 2 and figure 5) which was also found in Leonotis nepetifolia (He et al., 2012).

25

The presence of 9,13:15,16-diepoxylabdane-6β,15α-diol (14), luteolin glycoside (22) and luteolin (21) in this species is known (El-Ansari et al., 2009; Naidoo et al., 2011). However, He et al. (2012) reported the presence of nepetaefolin (4) in L. leonurus for the first time in 2012. Von Dreele et al. (1975) also proposed that the absolute configurations of Leonurenone A (1), and 9,13:15,16-diepoxylabdane-6β,15α-diol (epimeric mixture of (14)) were in agreement with the absolute configurations of other known labdanes given the concomitant occurrence of Leonurenone A (1), 9,13:15,16-diepoxylabdane-6β,15α-diol (epimeric mixture of (14)) and nepetaefolin (4) for which the crystal structure had been previously reported (Von Dreele et al.,1975). El-Ansari et al. (2009) in their study on the phytochemical and pharmacological properties of L. leonurus flowering aerial parts, collected from cultivated plants in the National Research Centre in Dokki, Egypt, report the isolation and chemical characterization of apigenins, in addition to labdane diterpenes reported by other groups. In collaboration with National Institute of Mental Health Psychoactive Drugs Screening Program, Wu et al. (2013) conducted a study of an acetone extract of L. leonurus leaves and isolated and elucidated the structures of eight new labdane diterpenes (Leoleorin D, E, F, G, H, I, J (1-3, 13-15,17) and 16-epi-leoleorin F (16)) and three known labdane diterpenoids namely, Leoleorin A (Compound Y) (10), Leoleorin B (11) and Leoleorin C (12) which had been reported earlier by Kaplan and Rivett (1968) and Naidoo (2011) (Table 2 and figure 4). However, Leoleorin E (14) also known as 9,13:15,16-diepoxylabdane-6β,15α-diol had been previously reported by Naidoo (2011) and He (2012), while Leoleorin C (12) was also isolated by Naidoo (2011). Previous phytochemical analysis of organic extracts of the flowers of L. leonurus reportedly provided flavonoids (El-Ansari et al., 2009) and acyclic diterpene esters (Agnihotri et al., 2009), while the leaves were found to contain mostly the labdane diterpenoids (Kaplan and Rivett, 1968; Kruger and Rivett, 1988; Ojewole, 2005; McKenzie et al., 2006; Obikeze et al., 2009; Naidoo et al., 2011) and polyphenolic compounds (Ojewole, 2005; Oyedemi and Afolayan, 2011). In this 2013 study, Wu and co-authors stated that Leoleorin D, E, F, G, H, I, J (1-3, 13-15,17) and 16-epi-leoleorin F (16) were obtained as new compounds in relatively 26

high concentrations and that their presence was possibly overlooked in previous investigations due to variations in harvest time, geographical location, climate and other ecological factors (Wu et al., 2013). However Leoleorin G (Leonurenone B) (2), Leoleorin H (Leonurenone C) (3) and Leoleorin I (Leonurenone A) (1) had already been reported by He et al. (2012) in the same year. Kuchta et al., in 2013 published their results of their study in which the betaine, stachydrine, was detected and quantified by HPTLC and 1H-qNMR analyses from Leonurus cardiaca, Leonurus japonicus and L. leonurus extracts. This compound, which displays interesting biological activities, is a chief constituent of the Chinese herb, Leonurus heterophyllus Sweet and is used in clinics practising traditional Chinese medicine to promote blood circulation. Yin et al., 2010, report that their cell culture study indicated that stachydrine ameliorated human umbilical vein endothelial cell injury induced by anoxia-reoxygenation, with the putative mechanism being cited as related to the inhibition of tissue factor expression. Recently, Narukawa et al. (2015) reported two new diterpenoids among the eight compounds isolated from the aerial parts of L. leonurus. These compounds are known

as

14α-hydroxy-9α,13α-epoxylabd-5(6)-en-7-on-16,15-olide

compound that showed a very similar

(18),

a

13

C-NMR spectrum to Leoleorin G (2) and 13ξ-

hydroxylabd-5(6), 8(9)-dien-7-on-16, 15-olide (19), a compound similar to compound X (8) at C11 and C16 (Table 2).

6.1

Chemotaxonomy

L. leonurus produces a number of diterpene secondary metabolites. Terpenoids have been useful as an aid to define a species, detect hybridization in natural populations, confirm the presence of geographical races and confirm generic and tribal limits. The well-developed GC analysis of plant volatile compounds, like the terpenoids, offers a method for the qualitative and quantitative analysis of these complex compounds (Demetzos and Dimas, 2001). Although the Leonotis group has been studied extensively, little chemotaxonomic work appears to have been published; this would be particularly useful in identifying the species of Leonotis as well as determining possible hybridization within populations and geographical 27

variation. In 2011, Naidoo et al. published a paper in which they suggest that the labdane diterpenoids isolated in their research differ from those previously reported in that they do not possess a lactone moiety between C4 and C6, and have only been found in Leonotis (Lamiaceae) and the genus Vitex which is now also included in the Lamiaceae. Most of the compounds isolated up to date have been terpenoids, and the labdane terpenoids so far isolated from L. leonurus have closely related structures usually with the differences being the presence or absence of a C4-C6 lactone, a 9,13 epoxy ring, a spirocyclic epoxy ring on C13, unsaturation at C5-C6, the presence of hydroxy groups on C6 in some compounds and the unsaturation or hydroxyl substitutions of the spirofuran D ring (Wu et al., 2013). These compounds may act as chemical markers for the characterisation of Leonotis species. Other compounds however, are quite unrelated in their structures (such as diterpene esters). It has been noted that Leonotis species are rich in terpenoids, especially the diterpenoid labdane lactones, and some researchers have also noted possible geographical differences in the phytochemical constituents of L. leonurus (Laonigro et al., 1979; McKenzie et al., 2006).

7

CONCLUSION

A large number of phytochemical investigations have been carried out on L. leonurus due to its wide range of reported biological properties (Demetzos and Dimas, 2001). The plant has a wide range of traditional uses in South Africa, as well as elsewhere in Africa. L. leonurus is indigenous to South Africa and its reported use in traditional medicine in Zimbabwe, Mauritius and as far afield as Egypt raise concerns about the possible confusion of this species with other closely related members of the Lamiaceae, such as L. nepetifolia and L. decadonta or possibly L. ocimyfolia. The former is a well-known annual weed that has appeared in Europe and the Indian sub-continent. L. decadonata has been reportedly confused with L. leonurus in areas of central and eastern Africa where it is indigenous. L. ocimyfolia has a much broader distribution from north-eastern to southern Africa. All three of these taxa are quite similar in appearance, particularly the inflorescence (Iwarsson and Harvey, 2003). In addition L. leonurus is commercially available and is marketed chiefly for its 28

psychoactive effects. Some internet websites attribute this effect to leonurine, which is reported to be present in L. leonurus but which has never been verified by rigorous scientific investigation. This confusion may have originated in the mis-identification of the species because the presence of leonurine in related species has been well researched and documented (Chen and Kwan, 2001, Kuchta et al., 2012, Bienvenu et al., 2002). The phytochemistry of the furan-diterpenoids from the aerial parts of Leonotis and Leonurus has been comprehensively reviewed (Piozzi, 2007), there is still however the need for definitive research and clarification of other compounds, including alkaloids and essential oils from L. leonurus and other related genera, as well as from other plant parts, such as the roots which are extensively used in traditional medicine. The traditional use by smoking also requires further investigation as to how the chemistry and activity are affected by this form of administration. Research has proven the psychoactive effects of the crude extract of L. leonurus (Bienvenu et al., 2002) but the psychoactive compounds still need to be isolated, characterised and their biological activity confirmed by suitable bioassays. To further complicate the issue, deliberate adulteration of L. leonurus with synthetic cannabinoids has been reported recently, to facilitate the marketing of these illegal substances. The broad traditional use, as well as the newly discovered adulteration of this herbal medicine, further emphasize the necessity for refinement of appropriate quality control processes to ensure safety and quality. L. leonurus has shown many promising pharmacological activities in bioassays and in animal experiments; often these have been on the crude extract, but some researchers have taken the next step and conducted the assays on the isolated and sometimes characterised compound, for example the anticonvulsant study of Muhizi et al., (2005) in mice in which 20-acetoxy-9α,13α-epoxylabda-14-en-6β(19)-lactone and an unidentified quinone were found to be the active compounds. Much work is therefore still required on aspects of quality control to ensure safety, quality and efficacy of the product supplied to patients as the plant is widely used in South Africa as a traditional medicine to treat a diverse range of ailments. Commercially available plant sources provide as a viable option for phytochemical research particularly with regard to the appropriate validation of the plant material

29

(taxonomy) in order to identify and delineate closely related species such as L. leonurus and L. nepetifolia which are very similar in habit. 8

ACKNOWLEDGEMENTS

The support of Tshwane University of Technology, South African Medical Research Council and the National Research Foundation (South Africa), is gratefully acknowledged. Our appreciation is extended to Sandie Burrows who prepared the line drawing (Figure 2) for this paper.

9

REFERENCES

Agnihotri, V.K., ElSohly, H.N., Smillie, T.J., Khan, I.A., Walker, L.A., 2009. Constituents of Leonotis leonurus flowering tops. Phytochemistry Letters 2, 103-105. Akah, A.P., Nwaiwu, J.I., 1988. Anticonvulsant activity of root and stem extracts of Calliandra portoricensis. Journal of Ethnopharmacology 22, 205-210. Bienvenu, E., Amabeoku, G.J., Eagles, P.K., Scott, G., Springfield, E.P., 2002. Anticonvulsant activity of aqueous extract of Leonotis leonurus. Phytomedicine 9, 217-223. Bryant, A.T., 1966. Zulu medicine and medicine men. Struik: Cape Town (originally published in 1909 in the Annals of the Natal Museum). Burger, A., Kabatembe, J., 2008. Cardioactivity of 2 mg/ml Leonotis leonurus in the Langendorff rat heart not due to [K+] and [Mg]. Planta Medica 74, 991. Chakravarthy, B.K., Gupta, S., Gambir, S.S., Gode, K.D., 1980. Pancreatic beta cell regeneration. A novel antidiabetic mechanism of Pterocarpus marsupium Roxb. Indian Journal of Pharmacology 12, 123-127.

30

Chen, C.X., Kwan, C.Y., 2001. Endothelium-independent vasorelaxation by leonurine, a plant alkaloid purified from Chinese motherwort. Life Science 68, 953960. Connolly, J.D., Hill, R.A., 1991. Dictionary of Terpenoids. Chapman and Hall, London. First edition. Cornara, L., Borghesi, B., Canali, C., Andrenacci, M., Basso, M., Frederici, S, Labra, M., 2013. Smart drugs: green shuttle or real drug? International Journal of Legal Medicine 127, 1109-1123. Cragg, G.M.L., Little, G.E., 1962. The extractives of Leonotis leonurus. Journal of the South African Chemistry Institute 15, 29-30. De Sarro, A., Cecchetti, V., Fravolini, V., Naccari, F., Tabarrini, O., De Sarro, G., 1999.

Effects

of

novel

pentylenetetrazole-induced

6–desfluoroquinolones seizures

in

and

mice.

classic

quinolones

on

Agents

and

Antimicrobial

Chemotherapy 43, 1729-1736. Demetzos, C., Dimas, S., 2001. Labdane-type diterpenes: Chemistry and biological activity. Studies in Natural Products Chemistry 35, 235-292. Drugs Forum. Available from: https://drugsforum.com/forum/showthread.php?t=23149 (Accessed 16 May, 2015). Duke, J.A., 2001. Handbook of medicinal herbs. Boca Raton: CRC press.2nd edition. London New York Washington, D.C. El-Ansari, M.A., Aboutabl, E.A., Farrag, A.R.H., Sharaf, M., Hawas, U.W., Soliman, G.M., El-Seed, G.S., 2009. Phytochemical and pharmacological studies on Leonotis leonurus. Pharmaceutical Biology 47, 894-902. Harley R., Paton A., 2001. Leonurus japonicus Houtt. (Labiatae): the correct name for a common tropical weed. Kew Bulletin 56, 243-244. Hayashi, Y., 1962. Studies on the ingredients of Leonurus sibiricus L. II.Structure of leonurine (2). YakugakuZasshi 82, 1025-1027.

31

He, F., Lindqvist, C., Harding, W., 2012. Leonurenones A-C: Labdane diterpenes from Leonotis leonurus. Phytochemistry 83, 168-172. Hulme, M.M., 1954. Wild Flowers of Natal. Shuter& Shooter, Pietermaritzburg. Hurinanthan, V., 2009. Immune modulatory effect of Dichrostachys cinerea, Carpobrotus dimidiatus, Capparis tomentosa and Leonotis leonurus. M.Sc. dissertation, Durban, DUT. Hutchings, A., Scott, A.H., Lewis, G., 1996. Zulu Medicinal Plants. An inventory, Natal, South Africa: University of Natal Press 266-267. Inouye, H., Takeda, Y., Uobe, K., Yamauchi, K., Yabuuchi, N., Kuwano, S., 1974. Purgative activities of iridoid glucosides. Planta Medica 25, 285-288. Ishikawa, M., Hashimoto, K., 2009. The role of sigma-1 receptors in the pathophysiology of neuropsychiatric diseases. Journal of Receptor, Ligand and Channel Research 3, 25-36. Iwarsson, M., Harvey, Y., 2003. Monograph of the genus Leonotis. Kew Bulletin 58, 597-645. Jäger, A.K., Hutchings, A., Van Staden, J., 1996.Screening of Zulu medicinal plants for prostagladin-synthesis inhibitors. Journal of Ethnoparmacology 52, 95-100. Jäger, A.K., Van Staden, J., 2000.The need for cultivation of medicinal plants in Southern Africa. Outlook on Agriculture 29, 283-284. Jain, M., Khan, S.I., Tekwani, B.L., Jacob, M.R., Singh, S., Singh, P.P., Jain, R., 2005. Synthesis, antimalarial, antileishmanial, and antimicrobial activities of some 8quinolinamine analogues. Bioorganic and Medicinal Chemistry 13, 4458-4466. Jimoh, F.O., Adedapo, A.A., Afolayan, A.J., 2010. Comparison of the nutritional value and biological activities of the acetone, methanol and water extracts of the leaves of Solanum nigrum and Leonotis leonurus. Food Chemistry and Toxicology 48, 964-971.

32

Kaplan, E.R., Rivett, D.E.A., 1968. Structures of compounds X and Y, two labdane diterpenoids, from Leonotis leonurus. Journal of the Chemical Society C: Organic, 262-266. Kelmanson, J.E., Jäger, A.K., Van Staden, J., 2000. Zulu medicinal plants with antibacterial activity. Journal of Ethnopharmacology 69, 241–246. Kinsella, J., Frankel, E., German, B., Kanner, J., 1993. Possible mechanisms for the protective role of antioxidants in wine and plant foods. Food Technology 47, 87-89. Kirmizibekmez, H., Calis I., Perozzo, R., Brun, R., Doenmez, A.A., Linden, A., Rueedi, P., Tasdemir, D., 2004. Inhibiting activities of the secondary metabolites of Phlomis brunneogaleata against parasitic Protozoa and plasmodial enoyl-ACP reductase, a crucial enzyme in fatty acid biosynthesis. Planta Medica 70, 711-717. Klos, M., Van de Venter, M., Milne, P.J., Traore, H.N., Meyrer, D., Oosthuizen, V., 2009. In vitro anti-HIV activity of five selcted South African medicinal plant extracts. Journal of Ethnopharmacology 124, 182-188. Kong, Y.C., Yeung, H.W., Cheung, Y.M., Hwang, J.C., Chan, Y.W., Law, Y.P., Ng, K.H., Yeung, C.H., 1976. Isolation of the uterotonic principle from Leonurus artemisia, the Chinese motherwort. American Journal of Chinese Medicine (GardCity, N Y) 4, 373-382. Kruger, G.J., Rivett, D.E.A., 1988. Diterpenoids of Leonotis species. Part 7. The crystal and molecular structure of compound X a labdane from Leonotis leonurus. South African Journal of Chemistry 41, 124-125. Kuchta, K., Ortwein, J., Rauwald, H.W., 2012. Leonurus japonicus, Leonurus cardiaca, Leonotis leonurus: A novel HPLC study on the occurrence and content of the pharmacologically active guanidino derivative leonurine. Pharmazie 67(12), 973979. Kuchta, K., Volk, R.B., Rauwald, H.W., 2013. Stachydrine in Leonurus cardiaca, Leonurus japonicus, Leonotis leonurus: detection and quantification by instrumental HPTLC and 1H-qNMR analyses. Pharmazie 68(7), 534-540.

33

Laonigro, G., Lanzetta, R., Parrilli, M., Adinolfi, M., Mangoni, L., 1979. The configuration of the diterpene spiroethers from Marrubium vulgare and from Leonotis leonurus. GazzellaChimica Italiana 109, 145-150. Lee, J., Koo, N., Min, D.B., 2004. Reactive oxygen species, aging, and antioxidative nutraceuticals. Comprehensive Reviews in Food Science and Food Safety 3, 21-33. López-Lázaro, 2009. Distribution and biological activities of the flavonoid luteolin. Medicinal Chemistry 9, 31-59. Luo, S.R., 1985. Separation and determination of alkaloids of Leonurus sibiricus. Zhong Yao Tong Bao 10, 32-35. Mabona, U., Van Vuuren, S.F., 2013. South African medicinal plants used to treat skin diseases. South African Journal of Botany 87, 175-193. Manickam, M., Ramanathan, M., Jahromi, M.A.F., Chansouria, J.P.N., Ray, A.B., 1997. Antihyperglycemic activity of phenolics from Pterocarpus marsupium. Journal of Natural Products 60, 609-610. Maphosa, V., Masika, P.J., Adepapo, A.A., 2008. Safety evaluation of the aqueous extract of Leonotis leonurus shoots in rats. Human and Experimental Toxicology 27, 837. Maphosa, V., Masika, P.J., Bizimenyera, E.S., Eloff, J.N., 2010. In-vitro anthelminthic activity of crude aqueous extracts of Aloe ferox, Leonotis leonurus and Elephantorrhiza elephantina against Haemonchuscontortus. Tropical Animal Health Production 42, 301-307. Maphosa V., Masika, P.J., 2011. In vivo validation of Aloe ferox (Mill). Elephantorrhiza elephantina Bruch.Skeels. and Leonotis leonurus (L) R.Br as potential anthelmintics and antiprotozoals against mixed infections of gastrointestinal nematodes in goats. Parasitology Research, 110,103-108. Maroyi, A., 2013. Traditional use of medicinal plants in south-central Zimbabwe: review and perspectives. Journal of Ethnobiology and Ethnomedicine 9: 31.

34

Martin, M., Mathias, E., McCorkle, C.M., 2001. Ethnoveterinary medicine: An annotated bibliography of community animal healthcare. ITDG Publishing. London, United Kingdom. Marx, J., 2002. Unravelling the causes of diabetes. Science 296, 686-689. Marles, R.J., Farnsworth, N.R., 1995. Antidiabetic plants and their active constituents. Phytomedicine 2, 137-189. Masika, P.J., Sonandi, A., van Averbeeke, W., 1997. Perceived causes, diagnosis and treatment of babesiosis and anaplasmosis in cattle by livestock farmers in communal areas of the central Eastern Cape Province, South Africa. Journal of the South African Veterinary Association 68, 40-44. Masika, P.J., van Averbeeke, W., Sonandi, A., 2000. Use of herbal remedies by small-scale farmers to treat livestock diseases in central Eastren Cape province, South Africa. Journal of the South African Veterinary Association 71, 87-91. Masika, P.J., Afolayan A.J., 2003. An ethnobotanical study of plants used for the treatment of livestock diseases in the Eastern Cape Province, South Africa. Pharmaceutical Biology 41, 16-21. Maurice, T., Su, T.P., 2009. The pharmacology of sigma-1 receptors. Pharmacology and Therapeutics 124, 195-206. Mazimba, O. 2015. Leonotis leonurus: A herbal medicine review. Journal of Pharmacognosy and Phytochemistry 3, 74-82. McGaw, L.J., Jäger, A.K., Van Staden, J., 2000. Antibacterial, anthelmibntic and anti-amoebic

activity

in

South

African

medicinal

plants.

Journal

of

Ethnopharmacology 72, 247-263. McGaw, L.J., Eloff, J.N., 2008. Ethnoveterinary use of Southern African plants and scientific evaluation of their medicinal properties. Journal of Ethnopharmacology 119, 559-574. McKenzie, J.M., Green, I.R., Mugabo, P., 2006. Leonurun, a novel labdane diterpenoid from Leonotis leonurus. South African Journal of Chemistry 59, 114-116.

35

Mnonopi, N., Levendal, R.A., Coleman, M., Frost, C.L., 2011. The cardioprotective effects of marrubiin, a diterpenoid found in Leonotis leonurus extracts. Journal of Ethnopharmacology 138, 67-75. Mnonopi, N., Levendal, R.A., Mzilikazi, N., Frost, C.L., 2012. Marrubiin, a constituent of Leonotis leonurus, alleviates diabetic symptoms. Phytomedicine 19, 488-493. Mugabo, P., Khan, F., Burger, A., 2012. Effect of Leonotis leonurus aqueous extract on the isolated perfused rat heart. Journal of Medicinal and Aromatic Plants 2, 281292. Muhizi, T., Green, I.R., Amabeoku, G.J., Bienvenu, E., 2005. An anticonvulsant diterpene lactone isolated from the leaves of Leonotis leonurus (L) R. Br. East and Central African Journal of Pharmaceutical Sciences 8, 54-61. Mulholland, D.A., 2005. The future of ethnopharmacology: A southern African perspective. Journal of Ethnopharmacology 100, 124-126. Naidoo, D., Maharaj, V., Crouch, N.R., Ngwane, A., 2011. New labdane-type diterpenoids from Leonotis leonurus support circumscription of Lamiaceae s.l. Biochemical Systematics and Ecology 39, 216-219. Narukawa, Y., Komori, M., Niimura, A., Noguchi, H., Kiuchi, F., 2015. Two new diterpenoids from Leonotis leonurus R. Br. Journal of Natural Medicines 69, 130-134. Nishimura, H., Sasaki, H., Inagaki, N., Masao, C., Chen, Z., Mitsuhashi, H., 1991. Nine phenethyl alcohol glycosides from Stachys sieboldii. Phytochemistry 30, 965969. Obikeze, K.C., McKenzie, J.M., Green, I.R., Mugabo, P., 2009. Characterization and cardiovascular effects of (13S)-9α,13α-epoxylabda-6β(19),15(14)diol dilactone, a diterpenoid isolated from Leonotis leonurus. South African Journal of Chemistry 61, 119-122. Ojewole, J.A.O., 2005. Antinociceptive, anti-inflammatory and antidiabetic effects of Leonotis leonurus (L) R. Br. [Lamiaceae] leaf aqueous extract in mice and rats. Methods and Findings in Experimental and ClinicalPharmacology 27, 257-264.

36

Oyedemi, S.O., Afolayan, A.J., 2011. In vitro and in vivo antioxidant activities of the aqueous extract of Leonotis leonurus. International Journal of Pharmacology 7, 248256. Oyedemi, S.O., Yakubu, M.T., Afolayan, A.J., 2011a. Antidiabetic activities of aqueous leaves extract of Leonotis leonurus in streptozotocin induced diabetic rats. Journal of Medicinal Plants Research 51, 119-125. Oyedemi, S.O, Yakubu, M.T., Afolayan, A.J., 2011b. Effect of aqueous extract of Leonotis leonurus (L.) R. Br. leaves in male Wistar rats. Human and Experimental Toxicology 295, 377-384. Oyedeji, O.A., Afolayan, A.J., 2005. Comparative study of the essential oil composition and antimicrobial activity of Leonotis leonurus and Leonotis ocymifolia in the Eastern Cape, South Africa. South African Journal of Botany 71, 114-116. Pedersen, J.A., 2000. Distribution and taxonomic implications of some phenolics in the family Lamiaceae determined by ESR spectroscopy. Biochemical Systematics and Ecology 28, 229-53. Pedro, L.G., Barroso, J.G., Marques, N.T., Ascensão, L., Pais, M.S., 1991. Composition of the essential oil from sepals of Leonotis leonurus R. Br. Journal of Essential Oil Research 3, 451-453. Piozzi, F., Bruno, M., Rosselli, S., Maggio, A. 2007. Structure and biological activity of the furan-diterpenoids from the genera Leonotis and Leonurus. Heterocycles 74, 31-52. Plantzafrica. Available from: http://www.plantzafrica.com/plantklm/leonotisleon.htm (Accessed 14 January 2015). Popoola, K., Elbagory, M., Ameer, F., Hussein, A., 2013. Marrubiin: A review. Molecules 18, 9049-9060. Purushothaman, K.K., Vasanth, S., 1988. Chemical studies on Leonotis - a review. Indian Drugs 25, 484-491.

37

Qui, S., Sun, H., Zhang, A-H., Xu, H-Y., Yan, G-L., Han, Y., Wang, X-J. 2014. Natural alkaloids: basic aspects, biological roles and future perspectives. Chinese Journal of Natural Medicines 12, 0401-0406. Rajalaksmi, M., Eliza, J., Cecilia, E., Nirmala, A., Daisy, P., 2009. Antidiabetic properties of Tinospora cordifolia stem extracts on streptozotocin induced diabetic rats. African Journal of Pharmacy and Pharmacology 3, 171-180. Rang, H.P., Dale, M.M., Ritter, J.M., 1999. Pharmacology, 4th edition. Edinburgh: Churchill Livingstone 464-480. Ratnasooriya, W.D., Hettiarachchi, H.D.I., Jayakody, R.A.C., 2004. Cassia fistula and hypoglycemia. Australlian Journal of Medical Herbalism 16, 8-13. Rauwald, H.W., Kuchta, K., Savtschenko, A., Brückner, A., Rusch, C., Appel, K., 2013. GABAA receptor binding assays of standardized Leonurus cardiaca and L. japonicus extracts as well as their isolated constituents. Planta Medica 79: PN52. Rauwald, H,W,, Savtschenko, A., Merten, A., Rusch, C., Appel, K., Kuchta, K., 2015. GABAA receptor binding assays of standardized Leonurus cardiaca and Leonurus japonicus extracts as well as their isolated constituents. Planta Medica 2015 [Epub ahead of print]). Ravi.K., Rajasekaran, S., Subramanian, S., 2005. Antihyperlipidemic effect of Eugenia jambolana seed kernel on streptozotocin induced diabetes in rats. Journal of Pharmacology 560, 201-205. Richard, E.S., Albert, H., Christian, R., 2001. Plants of the Gods. Healing Arts Press 46. Rivett, D.E.A., 1964. Isolation of marrubiin from Leonotis leonurus. Journal of the Chemical Society, 1857-1858. Scott, G., Springfield, E.P., Coldrey, N., 2004. A pharmacognostical study of 26 South African plant species used as traditional medicines. Pharmaceutical Biology 42, 186-213. South Africa. 1965. Medicines and Related Substances Act, No. 101 of 1965, as amended (online). Available from: 38

http://www.hpcsa.co.za/Uploads/editor/UserFiles/downloads/legislations/acts/medici nes_and_related_sub_act_101_of_1965.pdf (accessed: 19 May 2015). Stafford, G.I., Jäger A.K., Van Staden, J., 2005. Effect of storage on the chemical composition and biological activity of several popular South African medicinal plants. Journal of Ethnopharmacology 97, 107-115.Stafford, G.I., Pedersen, M.E., Van Staden, J. Jäger A.K., 2008. Review on plants with CNS effects used in traditional South African medicine against mental diseases. Journal of Ethnopharmacology 119, 513-537. Steenkamp, V., Mathivha, E., Gouws, M.C., Van Rensburg, C.E.J., 2004. Studies on antibacterial, antioxidant and fibroblast growth stimulation of wound healing remedies from South Africa. Journal of Ethnopharmacology 95, 353–357. Tadahiro, T., Yuji, N., Noriyasu, H., 1999. Studies on the constituents of Leonotis nepetifolia. Chemical and Pharmaceutical Bulletin 47, 284-286. Thring, T.S.A., Weitz, F.M., 2006. Medicinal plant use in the Bredasdorp/Elim region of the Southern Overberg in The Western Cape Province of South Africa. Journal of Ethnopharmacology 103, 261-275. Van Wyk, B.E., Van Oudshoorn, B., Gericke, N., 2000. Medicinal Plants of South Africa, 2nd edition. Briza Publications Pretoria, South Africa. Van Wyk, B., Gericke, N., 2003. Peoples Plants - A Guide to Useful Plants of South Africa. Pretoria: Briza Publications. ISBN-1-875093192. Von Dreele, R.B., Pettit, G.R., Ode, R.H., Perdue, R.E.Jr, White, J.D., Manchand, P.S., 1975. The crystal and molecular structure of the unusual spiro dihydrofuran diterpene, nepetaefolin. Journal of the American Chemical Society 97, 6236-6240. Watt, J.M., Breyer-Brandwijk, B.N., 1962. Medicinal and poisonous plants of Southern and Eastern Africa, 2nd edition. Edinburgh: Churchill Livingstone514-520. Westmoreland, B.F., Benarroch, E.E., Dube, J.R., Regan, T.J., Sandok, B.A., 1994. Medical neurosciences. Rochester: Mayo Foundation 307-312. Wikimedia commons. Available from: http://commons.wikimedia.org/wiki/Leonotis_leonurus (Accessed: 16 May, 2015). 39

Wikipedia. Available from: http://en.wikipedia.org/wiki/Leonotis_nepetifolia (Accessed: 16 May, 2015). Wikipedia. Available from: http://en.wikipedia.org/wiki/Leonotis_leonurus (Accessed: 2 June 2015). Wild, S.G., Roglic, A., Green, R., King, H., 2004.Global prevalence of diabetes. Estimated for the year 2000 and projection for 2030. Diabetes Care 27, 1047-1054. World Health Organization, 1998. Obesity: Preventing and managing the global epidemic: a report of a WHO consultation on Obesity. WHO Regional Publications, Geneva, Series No 19. World Health Organization, 2000. Obesity: Preventing and managing the global epidemic. Western Pacific Region, Series No 894. Wu, H., Li, J., Fronczek, F.R., Ferreira, D., Burandt, C. L. Jr., Setola, V., Roth, B.LK., Zjawiony, J.K., 2013. Labdane diterpenoids from Leonurus leonurus. Phytochemistry 91, 229-235. Yin, J., Zhang, Z.W., Yu, W.J., Liao, J.Y., Luo, X.G., Shen, Y.J., 2010. Stachydrine, a major constituent of the Chinese herb Leonurus heterophyllus Sweet, ameliorates human umbilical vein endothelial cells injury induced by anoxia-reoxygenation. American Journal of Chinese Medicine 38(1), 157-171. Zuba, D., Geppert, B., Sekula, K., Zaba, C., 2013.[1-(Tetrahydropyran-4-ylmethyl)1H-indol-30yl]-(2,2,3,3,-tetramethylcyclopropyl)methanone:

a

new

synthetic

cannabinoid identified on the drug market. Forensic Toxicology 31, 281-291.

40

Table 1.Traditional uses, preparation and mode of administration of Leonotis leonurus Conditions

Plant materials used/Methods

Administration route

References

Leaves prepared as a decoction or infusion

Topical application of the cooled decoction as a lotion

Scott et al., (2004)

Ethnomedicinal uses Skin conditions; rashes, eczema, itching, boils, haemorrhoids, sores

Hutchings et al., (1996) Sores on the legs and head

Powdered stem and/or seeds prepared as a decoction

Mabona and Van Vuuren, (2013) Watt and BreyerBrandwijk, (1962)

Spider and snake bites, scorpion stings

Leaves and flowers prepared as an infusion

Oral administration

Bryant, (1966) Jäger et al., (1996) Van Wyk et al., (2000) Stafford et al., (2008)

Haemorrhoids

Powdered stem and/or seeds prepared as a decoction

Oral administration of the decoction

Watt and BreyerBrandwijk, (1962)

Respiratory conditions; cough, colds, bronchitis, influenza, asthma

Leaves prepared as a decoction or infusion

Oral administration or inhalation

Bryant, (1966) Hutchings et al., (1996) Jäger et al., (1996) Duke, (2001) Van Wyk et al., (2000) Stafford et al., (2008)

41

Watt and BreyerBrandwijk, (1962)

Fever

Leaves and flowers prepared as a decoction

Oral administration

Jäger et al., (1996) Van Wyk et al., (2000) Stafford et al., (2008)

Tuberculosis

Leaves and flowers prepared as an infusion

Oral administration

Hutchings et al., (1996)

Leprosy

Leaves and flowers prepared as a decoction

Oral administration and inhalation

Duke, (2001)

Viral hepatitis

Leaves prepared as an infusion

Oral administration

Watt and BreyerBrandwijk, (1962)

Headache

Leaves and flowers prepared as an infusion

Oral administration

Jäger et al., (1996)

Headache with fever

Nasal douche

Cold infusion of leaves

Watt and BreyerBrandwijk, (1962)

Pain above the eye

As an ointment made from powdered leaves

Topical application of the ointment

Hutchings et al., (1996)

Epilepsy

Leaves and flowers prepared as an infusion

Oral administration

Watt and BreyerBrandwijk, (1962)

Partial paralysis

Jäger et al., (1996) Van Wyk et al.,(2000) Stafford et al., (2008) Cardiovascular system Cardiac conditions,

Leaves prepared as a decoction or infusion

Oral administration

Hutchings et al., (1996) Duke, (2001)

Hypertension

Jäger et al., (1996)

42

Van Wyk et al., (2000) Stafford et al., (2008) Muscular cramps

Leaves prepared as a decoction

Oral administration

Scott et al., (2004)

Gastrointestinal conditions,

Leaves and flowers prepared as an infusion

Oral administration

Watt and BreyerBrandwijk, (1962)

Dysentery

Jäger et al., (1996) Van Wyk et al., (2000) Stafford et al., (2008) Anthelmintic: Vermifuge, intestinal worms

Leaves prepared as a decoction

Rectal administration as an enema, oral administration of an infusion

Watt and BreyerBrandwijk, (1962) Jäger et al., (1996) Van Wyk et al., (2000) Stafford et al., (2008)

Constipation

Leaves and flowers prepared as an infusion

Oral administration

Watt and BreyerBrandwijk, (1962)

Delayed menstruation, emmenagogue

Leaves and flowers prepared as an infusion

Oral administration

Jäger et al., (1996) Van Wyk et al., (2000) Stafford et al., (2008)

Jaundice / hepatitis

Leaves and flowers prepared as an infusion

Oral administration

Hutchings et al., (1996)

Diuretic

Leaves brewed as a decoction

Oral administration

Van Wyk et al., (2000) Hutchings et al., (1996) Watt and Breyer-

43

Brandwijk, (1962)

Obesity

Leaves brewed as a decoction

Oral administration

Van Wyk et al., (2000)

Leaf sap squeezed directly into the eye of the afflicted animal

Topical

Masika et al., (1997)

Ethnoveterinary uses Eye inflammation

Masika et al., (2000) Masika and Afolayan, (2003)

Prevention of poultry diseases

Pounded roots and leaves

Added to drinking water

Hulme, (1954)

Gallsickness (anaplasmosis) in cattle

Pounded roots and leaves

Added to drinking water

Hulme, (1954)

Worm infestations in livestock, especially goats

Leaf decoction

Oral administration

Maphosa and Masika, (2011)

Table 2. Labdane diterpenoids from Leonotis leonurus Compound, molecular weight, molecular formula

Type of extraction

Biological and pharmacological properties

44

References

Leonurenone A or Leoleorin I (9,13-epoxy-15,16-dihydroxylabd-5-en-7-one) (1)

Aqueous/acetone extract of leaves by percolation at room temperature; followed by column fractionation using a gradient water:methanol solvent system.

No anticonvulsant activity exhibited in a binding assay at GABAA site. Inhibition of radioligand binding at the 5-HT1A receptor in a battery of G-protein-coupled assays.

Bienvenu et al., (2002) He et al., (2012) Wu et al., (2013)

Aqueous/acetone extract of leaves by percolation at room temperature; followed by column fractionation using a gradient water:methanol solvent system.

No anticonvulsant activity exhibited in a binding assay at GABAA site. Inhibition of radioligand binding at the D1-dopamine receptor in a battery of G -protein-coupled assays. Inhibited binding at the M3acetylcholine and Sigman-1 receptors.

Bienvenu et al., (2002) He et al., (2012)

Aqueous/acetone extract of leaves by percolation at room temperature; followed by column fractionation using a gradient water:methanol solvent system. Aqueous extract of leaves; Organic extract of leaves, 1:1 methanol:dichloromethane.

Inhibition of radioligand binding at the 5-HT1A and D1-dopamine receptor in a battery of Gprotein-coupled assays.

He et al., (2012) Wu et al., (2013)

L. leonurus: Aqueous/acetone extract of leaves by percolation at room temperature; followed by column fractionation using a gradient water:methanol solvent system.

Compound extracted from L. leonurus but no biological assay done.

He et al., (2012)

Note: The ethanolic extract of L. nepetifolia showed activity in sarcocarcinoma screening.

Von Dreele et al., (1975)

Acetone extract of leaves by percolation at room temperature followed by silica gel column chromatographic fractionation (eluent hexane:ethylacetate, 7:3).

Positive inotropic activity.

McKenzie et al., (2006)

Not reported.

Not reported.

Rivett, (1964)

C20H32O4 336.470 Leonurenone B or Leoleorin G (9,13-epoxylabd-5-en-7-on-15,16-olide) (2) C20H28O4 332.1988

Leonurenone C or Leoleorin H (15-acetoxy-9,13-epoxy-16-hydroxylabd-5en-7-one) (3) C22H34O5

Wu et al., (2013)

378.2407 Nepetaefolin 1''-methyl-11''-oxo-1'',3',3'',4',4'',7'',8'',8a''octahydro-2''h-trispiro[furan-3,2'-furan-5',5''[1,4a](methanooxymethano)naphthalene6'',2'''-oxiran]-8''-yl acetate (4) C22H28O7 404.45352 Leonurun (9,13 :15,16-Diepoxy-20-acetoxy-14-labden19,6-olide) (5) C22H30O 390.4756 Premarrubiin 9,13-epoxylabda-6(19),15(14) dioldilactone (6) C20H28O4 332.3392

45

Marrubiin

Organic extract of leaves.

(15.16-Epoxy-9a-hydroxy-13(16),14labdadien-19,6β–olide) (7) C20H28O4 332.43392

Suppression of coagulation, platelet aggregation and inflammatory markers; marrubiin significantly prolonged activated partial thromboplastin time (APTT), fibrin and D-dimer formation were significantly decreased in an ex vivo model and an obese rat model. Increase in insulin secretion, HDL-cholesterol, while total cholesterol, LDL-cholesterol, atherogenic index IL-1β and IL6 levels were normalised in an obese rat model. Cultured under hyperglycaemic conditions INS-1 cells exhibited an increased stimulatory index and insulin and glucose transporter-2 gene expressions were increased.

Rivett, (1964) McKenzie et al., (2006) Mnonopi et al., (2011)

Mnonopi et al., (2012)

Popoola et al., (2013)

Dose related antinociceptive effects in animal models. Cardioprotective effects by decreasing hypercoagulable and inflammatory states that are associated with obesity. Gastroprotective properties exhibited in mice by inhibition of gastric acid secretion (via NO synthesis, an endogenous transmitter released by endothelial cells in response to cell damaging agents). Antispasmodic effects observed in rabbit jejunum, probably mediated via a Ca2+ channel blocking mechanism. Reduction in carrageenaninduced oedema possibly via non-specific receptor actions. “Compound X”

Not reported

Not reported.

Kaplan and Rivett, (1968) Kruger and Rivett., (1988)

Methanolic extract of leaves and stems followed by further processing and purification to isolate EDD.

Dose-dependent cardiovascular effects; lower doses (0.5, 1.0 and 2.0 mg/kg) resulted in decreased systolic and diastolic pressure and mean arterial pressure, but higher doses (3.0, 4.0 and 5.0 mg/kg) increased systolic and diastolic pressure and mean arterial pressure, while all doses decreased heart rate in male Wistar rats.

Obikeze et al., (2009)

(9,13-Epoxy-15,16:19,6-labdanediolide) (8) C20H28O5 348.438 EDD (9, 13-epoxylabda-6(19),15(14)diol dilactone) (9) C20H28O5 348.2009

46

Leoleorin A or Compound Y (15,16-epoxy-9α-hydroxylabda-5,13(16), 14trien-7-one) (10)

Acetone extract of leaves by percolation at room temperature; followed by column fractionation using a gradient water:methanol solvent system.

Inhibition of radioligand binding at the 5-HT1A receptor in a battery of G-protein-coupled assays.

Kaplan and Rivett, (1968) Wu et al., (2013)

Acetone extract of leaves by percolation at room temperature; followed by column fractionation using a gradient water:methanol solvent system.

Inhibition of radio ligand binding at the H1-histamine receptor in a battery of G-protein-coupled assays.

Kaplan and Rivett, (1968) Wu et al., (2013)

Aqueous and organic extracts prepared separately suing water and methane:dichloromethane(1:1) respectively.

Inhibition of radioligand binding at the H1-histamine receptor in a battery of G-protein-coupled assays. Inhibited binding at the M3acetylcholine and Sigma-1 receptors. In a secondary assay of competitive binding Leoleorin C showed moderate affinity (Ki=2.9 μM) in comparison to the positive control, haloperidol, for the Sigma-1 receptor; this Is possibly the reason for the psychoactive properties of this plant. No activity against Mycobacterium tuberculosis in an in vitro assay.

Naidoo et al., (2011)

Acetone extract of leaves by percolation at room temperature; followed by silica gel column fractionation with various hexane:ethyl acetate eluent ratios.

Inhibition of radioligand binding at the 5-HT1A and the D1dopamine receptor in a battery of G-protein-coupled assays.

Wu et al., (2013)

Acetone extract of leaves by percolation at room temperature; followed by silica gel column fractionation with various hexane:ethyl acetate eluent ratios.

Inhibition of radioligand binding at the D1-dopamine and H1histamine receptors in a battery of G-protein-coupled assays

Wu et al., (2013)

Aqueous and organic extracts prepared separately suing water and methane:dichloromethane(1:1) respectively.

Inhibition of radioligand binding at the H1-histamine receptor in a battery of G-protein-coupled assays. No activity against Mycobacterium tuberculosis in an in vitro assay.

Naidoo et al., (2011)

C20H28O3 316.43452 Leoleorin B or anhydro form of Compound Y (11) (15,16-epoxylabda-5,8,13(16),14-tetraen-7one) C20H26O2 298.41924 Leoleorin C (9,13-epoxy-6-hydroxylabdan-15,16-olide) (12) C20H32O4 336.46568

Leoleorin D (9,13-epoxylabdane-6β,15,16-triol) (13) C20H36O4

Wu et al., (2013)

340.49744 Leoleorin E (9,13:15,16-diepoxylabdane-6β,15α-diol) (14) C20H34O4 338.48156 Leoleorin F (9,13:15,16-diepoxylabdane-6β,16β-diol) (15) C20H34O4 338.48156

47

Wu et al., (2013)

16-epi-Leoleorin F (9,13:15,16-diepoxylabdane-6β,16α-diol) (16) C20H34O4

Acetone extract of leaves by percolation at room temperature; followed by silica gel column fractionation with various hexane:ethyl acetate eluent ratios.

Not reported

Wu et al., (2013)

Acetone extract of leaves by percolation at room temperature; followed by column fractionation using a gradient water:methanol solvent system.

Inhibition of radioligand binding at the 5-HT3 receptor in a battery of G-protein-coupled assays.

Wu et al., (2013)

Acetone extract of aerial parts of the plant.

No oestrogen sulfotransferase inhibitory activity. No other biological effects examined.

Narukawa et al. (2015)

Acetone extract of aerial parts of the plant.

No estrogen sulfotransferase inhibitory activity. No other biological effects examined.

Narukawa et al. (2015)

338.48156 Leoleorin J (9,13-epoxylabd-5-ene-7β,15,16-triol) (17) C20H34O4 338.48156 14α-hydroxy-9α, 13α-epoxylabd-5(6)-en-7on-16, 15-olide (18) C20H28O5 348.2015

13ξ-hydroxylabd-5(6), 8(9)-dien-7-on-16, 15olide (19) C20H28O4 332.2066

Table 3. Miscellaneous compounds, other than diterpenes, from Leonotis leonurus Compound (synonym), molecular formula, molecular weight

Chemical class

Properties

Type of extraction

References

Leonurine

Guanidine alkaloid

Psychoactive, hypotensive, uterotonic, neuroprotective effects. Note: There are only anecdotal and website unsubstantiated reports that leonurine occurs in L. leonurus.

Aqueous extract of aerial parts

Chen and Kwan,( 2001) Bienvenu et al., (2002) Kuchta et al., (2012)

Flavonoid

In preclinical studies this compound has been shown to have a wide range of pharmacological activities, including antioxidant, antiinflammatory, antimicrobial and anticancer activities. Luteolin

Aqueous extract of leaves.

López-Lázaro, (2009) He et al., (2012)

(4{[amino(imino)methyl]amino}butyl4-hydroxy-3,5dimethyloxybenzoate) (20) C14H21N3O5 311.33364 Luteolin 2-(3,4-dihydroxyphenyl)-5,7dihydroxychromen-4-one (21) C15H10O6

48

286.2363

Luteolin-7-glucoside (22)

inhibits angiogenesis, induces apoptosis, prevents carcinogenesis in animal models, reduces tumour growth in vivo and sensitizes tumour cells to the cytotoxic effects of certain anticancer drugs. Flavonoid

Antimalarial activity against the D6 clone (2.2 μg/mL) and the W2 clone (1.8 μg/mL) of Plasmodium falciparum. No cytotoxic effects observed on mammalian kidney fibroblasts (Vero cells) up to a concentration of 4.76 μg/mL. Antioxidant activity. Anti-leishmania activity. No antimicrobial activity against Candida albicans, Escherichia coli, Pseudomonas aeruginosa, Candida neoformans, Mycobacterium intracellulare and Aspergillusfumigatus.

Ethanolic extract of flowers via percolation with 95% ethanol followed by further extraction with chloroform and ethyl acetate, and silica gel column fractionation using gradient chloroform:methanol (99:1 to 76:24). Aqueous extract of leaves.

Agnihotri et al., (2009) El-Ansari et al., (2009) He et al., (2012) (

Diterpene ester

No antimalarial or antimicrobial activity reported.

Ethanolic extract of flowers via percolation with 95% ethanol followed by further extraction with chloroform and ethyl acetate, and silica gel column fractionation using gradient chloroform:methanol (99:1 to 76:24).

Agnihotri et al., (2009)

Dicarboxylic acid

Food additive, to control acidity.

Ethanolic extract of flowers via percolation with 95% ethanol followed by further extraction with chloroform and ethyl acetate, and silica gel column fractionation using gradient chloroform:methanol (99:1 to 76:24).

Agnihotri et al., (2009) Wu et al., (2012)

Nucleic acid

Enzyme synthesis for cell function.

Ethanolic extract of flowers via percolation with 95% ethanol followed by further extraction with chloroform and ethyl acetate, and silica gel column fractionation using gradient chloroform:methanol (99:1 to 76:24)..

Agnihotri, (2009) Wu et al., 2012

Phenylethanoid

Anti-oxidant, tyrosine kinase and tumour growth inhibition, hepatoprotective.

Ethanolic extract of flowers via percolation with 95% ethanol followed by further extraction with chloroform and ethylacetate, and silica gel column fractionation using gradient chloroform:methanol (99:1 to 76:24).

Nishimura et al., (1991) Pedersen, (2000) Agnihotri et al., (2009)

Iridoid glycoside

Purgative, promotion of collagen synthesis and hypotensive effect.

Ethanolic extract of flowers via percolation with 95% ethanol followed by further extraction with chloroform and ethyl acetate, and silica gel column

Tadahiro et al., (1999) Agnihotri et al., (2009)

C21H20O11 448.37

Dihydroxyphytyl palmitate 1,2,3-Trihydroxy-3,7,11,15tetramethylhexadecan-1-ylpalmitate (23) C36H72O4 568.962 Succinic acid (24) C4H6O4 118.09

Uracil (25) C4H4N2O2 112.08676

Verbascoside (acteoside) (26) C29H36O15 624.587

Geniposidic acid (β-D-glucopyranosyloxy)-7(hydroxymethyl)-1,4a,5,7atetrahydrocyclopenta[c]pyran-4-

49

carboxylic acid) (27)

fractionation using gradient chloroform:methanol (99:1 to 76:24).

C16H22O10 374.34 Luteolin (21)

Flavone aglycone

C15H10O6 286.2363 Luteolin-7-glucoside (22) C21H20O11

Monoglycoside flavone

448.37 Luteolin 7-O-β-glucoside-3'-methyl ether (34)

Chrysoeriol Luteolin 3’-methyl ether (29)

The 70% methanol and chloroform extracts exhibit strong hepatoprotective and anti-inflammatory effects. No activity observed against human tumour cell lines. Note: Biological properties reported only for methanolic and chloroform extracts and not for individual isolated and characterised compounds

Methylated monoglycoside flavone Methylated flavone

C15H10O5, 270.2369 6-Methoxyluteolin-4’-methyl ether (30) C17H14O7

Methylated flavone

330.28886 Apigenin 5,7-Dihydroxy-2-(4-hydroxyphenyl)4H-1-benzopyran-4-one (28)

Flavone aglycone

C15H10O5, 270.24 Apigenin 6-C-α-arabinoside-8-C-βglucoside (31)

Vitexin

Diglycoside flavone

Apigenin 8-C-β-glucoside (32) C21H20O10 432.38

Monoglycoside flavone

Apigetrin Apigenin 7-O-β-glucoside (33) C21H20O10

Monoglycoside flavone

432.38 Apigenin 7-O-(6''-O-p-coumaroyl)-βglucoside (35)

50

Dried ground flowering aerial parts extracted separately with 70% methanol and 70% chloroform using percolation. Solvents evaporated under reduced pressure and low temperature to dryness. Fractionation of the methanolic extract on a polyamide column with water:methanol mixtures as eluents of decreasing polarity. Isolation of pure compounds via preparative paper chromatography and further purification on a Sephadex LH-20 column with methanol as eluent.

El-Ansari et al., (2009)

Monoglycoside flavone Stachydrine

Betaine

Cardiovascular, hypotensive and tissue protective effects

1,1-dimethylpyrrolidin-1-ium-2carboxylic acid (36) C7H13NO2 143.18

51

The pulverised fresh flowering aerial parts extracted with boiling water for 60 minutes under reflux.

Kuchta et al., (2013)

Figure 1. The inflorescence of L. leonurus (a), the tip of the individual floret showing where the common name, lion’s ear, originates (b) and the plant in its natural habitat in grassland in Mpumalanga, South Africa (c). Photos A. Viljoen.

52

Figure 2. Line drawing of Leonotis leonurus showing distinct morphological features.

53

Figure 3. Geographical distribution (orange) of L. leonurus in South Africa (Iwarsson and Harvey, 2003).

54

Figure 4. General structure of labdane diterpene (Demetzos and Dimas, 2001).

55

O

OH OH

O

OH

O

O

O

O

O

O

O

O

Leonurenone A or Leoleorin I

Leonurenone B or Leoleorin G

Leonurenone C or Leoleorin H

(9,13-epoxy-15,16-dihydroxylabd-5-en-7one) (1)

(9,13-epoxylabd-5-en-7-on-15,16-olide) (2)

(15-acetoxy-9,13-epoxy-16hydroxylabd-5-en-7-one) (3)

O

O O

O

O

O

O

O

O H O

O

O

O

H O

H O

O

O

Nepetaefolin

Leonurun

Premarrubiin

1''-methyl-11''-oxo-1'',3',3'',4',4'',7'',8'',8a''octahydro-2''h-trispiro[furan-3,2'-furan5',5''[1,4a](methanooxymethano)naphthalene6'',2'''-oxiran]-8''-yl acetate (4)

20-acetoxy-9α,13α-epoxylabda-14-en6β(19)-lactone

9,13-epoxylabda-6(19),15(14) dioldilactone (6)

(9,13 :15,16-Diepoxy-20-acetoxy-14labden-19,6-olide) (5)

O

O

OH

O

O

H O

O O

O

O O

H O O

H O

Marrubiin

Compound X

EDD

(15.16-Epoxy-9a-hydroxy-13(16),14labdadien-19,6β–olide) (7)

(9,13-Epoxy-15,16:19,6-labdanediolide) (8)

(9, 13-epoxylabda6(19),15(14)dioldilactone) (9)

56

O

O

O

O

OH

O O

O H OH

Leoleorin A or Compound Y

Leoleorin B or anhydro form of Compound Y

(15,16-epoxy-9αhydroxylabda-5,13(16), 14trien-7-one) (10)

(15,16-epoxylabda-5,8,13(16),14-tetraen-7-one) (11)

O

OH OH

Leoleorin C (9,13-epoxy-6-hydroxylabdan-15,16olide) (12)

O

HO OH

O

O

H

H

OH

O

H

OH

OH

Leoleorin D

Leoleorin E

Leoleorin F

(9,13-epoxylabdane-6β,15,16triol) (13)

(9,13:15,16-diepoxylabdane-6β,15α-diol) (14)

(9,13:15,16-diepoxylabdane-6β,16βdiol) (15)

OH

O

HO

O H

OH H3C

O

O

O

HO CH3

O

H

OH

H OH

H3C

O

CH3

16-epi-Leoleorin F

Leoleorin J

(9,13:15,16-diepoxylabdane6β,16α-diol) (16)

(9,13-epoxylabd-5-ene-7β,15,16-triol) (17)

57

14α-hydroxy-9α, 13α-epoxylabd-5(6)en-7-on-16, 15-olide (18)

CH3

O

HO

O N

O

HO

O

NH2

HO

OH

OH

NH2

O

O

O

OH O

CH3

O

13ξ-hydroxylabd-5(6), 8(9)-dien7-on-16, 15-olide (19)

Leonurine

Luteolin

(4-{[amino(imino)methyl]amino}butyl-4-hydroxy-3,5dimethyloxybenzoate) (20)

2-(3,4-dihydroxyphenyl)-5,7dihydroxychromen-4-one (21)

OH

OH

CH3

OH

HO O

HO HO

O

CH3

CH3

H3C

H3C

O

OH O

OH

C15 H31

O O

O

OH

OH OH

O

Luteolin-7-glucoside

Dihydroxyphytyl palmitate

Succinic acid

2-(3,4-dihydroxyphenyl)-5hydroxy-7-[(2S,3R,4S,5S,6R)3,4,5-trihydroxy-6(hydroxymethyl)oxan-2yl]oxychromen-4-one (22)

1,2,3-Trihydroxy-3,7,11,15-tetramethylhexadecan-1yl-palmitate (23)

Ethane-1,2-dicarboxylic acid (24)

OH

O

HN

HO O

N H

HO O

O

O

O

O

HO O

HO

OH

OH OH

O

HO HO

OH O OH O

HO OH

HO

O

Uracil

Verbascoside (Acteoside)

Geniposidic acid

Pyrimidine-2,4(1H,3H)-dione (25)

[(2R,3R,4R,5R,6R)-6-[2-(3,4Dihydroxyphenyl)ethoxy]-5-hydroxy-2(hydroxymethyl)-4-[(2S,3R,4R,5R,6S)-3,4,5trihydroxy-6-methyloxan-2-yl]oxyoxan-3-yl] (E)-3(3,4-dihydroxyphenyl)prop-2-enoate (26)

(β-D-glucopyranosyloxy)-7(hydroxymethyl)-1,4a,5,7atetrahydrocyclopenta[c]pyran-4carboxylic acid) (27)

58

OH

O

O

HO

O

O

H3C

5,7-Dihydroxy-2-(4hydroxyphenyl)-4H-1benzopyran-4-one (28)

Luteolin 3’-methyl ether (29)

O HO

OH OH

O

O

HO

O

HO

Chrysoeriol

OH OH OH

O

O

Apigenin

OH

O

HO

O

HO

HO

OH OH OH

6-Methoxyluteolin-4’-methyl ether (30)

OH HO

OH

O

HO HO

O

O

OH

O

O HO

OH

CH3

OH

OH HO

OH

CH3

OH O

OH O

OH O

Apigenin 6-C-α-arabinoside-8C-β-glucoside (31)

Vitexin

Apigetrin

Apigenin 8-C-β-glucoside (32)

Apigenin 7-O-β-glucoside (33)

HO

O OH

HO O

O

HO HO

O O

O

HO HO

OH

OH O

O

O

OH HO

OH

Luteolin 7-O-β-glucoside-3'-methyl ether (34)

H

O

O

O

Apigenin 7-O-(6''-O-p-coumaroyl)-β-glucoside (35)

-

+

N H3C

CH3

O

Stachydrine (36) 1,1-dimethylpyrrolidin-1-ium-2-carboxylic acid

Figure 5. Labdane diterpenoids and miscellaneous compounds isolated from Leonotis leonurus.

59