Clausena (PROSEA)

Plant Resources of South-East Asia Introduction

List of species

Clausena Burm.f.

Protologue: Fl. indica: 87 (1786).

Family: Rutaceae

Chromosome number: x= 9; C. anisata: 2n= 18, 36, C. excavata: 2n= 36, C. heptaphylla: 2n= 36, C. lansium: 2n= 18

• Clausena anisata (Willd.) Hook.f. ex Benth.,
• C. excavata Burm.f.

Clausena is a relatively small genus, comprising 15 species, distributed over 4 sections. Clausena occurs primarily in continental Asia and South-East Asia, extending to north-eastern Australia (Queensland). C. anisata extends its distribution area to India and Sri Lanka and is the only representative in tropical Africa.

Many Clausena species are important medicinal plants in their original area of distribution, but some of them have been widely planted for their edible fruit as well. The juice or a decoction of the roots or leaves of several Clausena, e.g. C. anisata, C. excavata, C. lansium and C. anisum-olens (Blanco) Merr. (synonym C. sanki (Perr.) J.F. Molino), is widely used for treating colds, sinusitis, fever and stomach problems such as constipation and diarrhoea.

The leaves and fruits of C. anisata are very aromatic when crushed. The essential oil is comparable to that of C. anisum-olens. In Vietnam, a decoction of the leaves and roots is used for treating colds, rheumatism and arthritis. In Java the roots of C. excavata are used likewise. Externally, the roots of C. anisata are poulticed for sprain, contusion and fractures. In Africa the crushed leaves are considered antiseptic and analgesic and applied on wounds and burns.

In Java the juice from the leaves of C. excavata is taken for worms. Pounded leaves may be applied to the head for headache. In Java and Peninsular Malaysia, ulcerations of the nose may be treated by applying a poultice of the leaves or by fumigating with burning leaves and bark. A decoction of the flowers and leaves may also be taken for colic. In China a poultice of the leaves is applied to treat paralysis. In Indo-China the leaves are considered laxative, stomachic and expectorant and are prescribed in dyspepsia, stomach-ache and cough.

C. lansium is used in traditional medicine in Malaysia and China to treat coughs, asthma, viral hepatitis and dermatological and gastro-intestinal diseases including ulcers and dysentery. The dried unripe fruits and dried sliced roots are used as a remedy for bronchitis. Ripe fruits are said to have stomachic and cooling effects and to act as a vermifuge. A decoction of the leaves, such as those of C. lenis Drake (synonym C. kerrii Craib) from northern Indo-China and Taiwan, is used for dandruff and to preserve the hair colour. The immature fruits of C. lenis are dried and taken for bronchitis, while the roots, stem and leaves are applied on persistent furuncles.

C. anisum-olens is restricted to the Philippines and var. calciphila (Stone) Molino to Borneo. Its essential oil is a potential substitute for anise oil. Medicinally, a decoction of the roots and fruits is taken for cough with fever, that of the leaves to treat nausea during pregnancy. The leaves are also added to baths to treat rheumatism, or stuffed into pillows, as they have a soporific effect.

Clausena is of local importance only and is not traded internationally.

The leaves and fruits of Clausena contain essential oils.

For example, leaf samples of the Philippine essential-oil plant C. anisum-olens var. anisum-olens showed the existence of 3 chemovariants: "pure anethole" oil, "pure methyl chavicol" oil, and "mixed" oil (about 90% (E)-anethole and 10% methyl chavicol).

In the essential oils of the leaves of C. anisata from different sources, 3 chemovariants can also be distinguished: (E)-anethole (75-95%) containing oils (Indonesia, Ghana), methyl chavicol (80-100%) containing oils (Benin, Ghana, Nigeria) and an oil containing a large number of constituents varying in concentration from 0.2% to 20%. Examples of the latter include a leaf oil from Zimbabwe with sabinene (33%), germacrene D (17%), Z-β-ocimene (6%), germacrene B (5.5%), (E)-β-ocimene (5%) and terpinen-4-ol (4.7%) and from Cameroon, with (Z)-tagetenone (26.8%), (E)-tagetenone (19.2%), (E)-nerolidol (11.5%) and germacrene D (9.2%). Essential oils from the seeds differ in composition, without a clear distinction in chemovars: from Benin it consisted of methyl chavicol (40.8%), myrcene (22.2%), (E)-anethole (16.3%) and limonene (13.4%), and from Cameroon it contained (Z)-tagetenone (15.3%), (E)-tagetonene (14.8%), (E)-nerolidol (10.3%), myrcene (7.4%),β-caryophyllene (7.4%), 3-carene (3.9%) andα-humulene (3.5%).

Dried plants of C. anisata are used in Africa and elsewhere to repel mosquitoes. In tests, the leaf essential oil proved toxic to the 3rd nymphal instar of the grasshopper Zonocerus variegata. Its major component was identified as estragol, which proved 1.5 times more toxic than the oil itself. Results of other bioassays showed that the essential oil was also very effective against stored-product insect pests, Sitophilus zeamais and other species, including Tenebrio molitor and Rhizopertha dominica. It caused 99.3% mortality and completely inhibited the reproduction of Tribolium castaneum when used for fumigation at a dosage of 20 mg/litre, or mixed with wheat flour at a concentration of 0.2% by weight.

Furthermore, using the hole-plate diffusion method for antibacterial testing, the essential oil from the leaves originating from Zimbabwe exhibited significant activity against Alcaligenes faecalis, Bacillus subtilis, Enterococcus faecalis (synonym Streptococcus faecalis), Flavobacterium suaveolens, Leuconostoc cremoris and Serratia marcescens. The mycelium growth inhibition method was used to test for antifungal activity. The oil exhibited significant activity against Alternaria alternata, Aspergillus parasiticus, Candida albicans, Geotrichum candidum and Penicillium citrinum.

The fruits, aerial parts and stem bark of C. anisata contain series of coumarins belonging to the furanocoumarin type (e.g. imperatorin, isoimperatorin, oxypeucedanine, bergaptene, xanthotoxin, xanthotoxol and chalepin), geranylcoumarin (e.g. anisocoumarin A–I), or furanocoumarin-lactone type (indicolactone, anisolactone). Several of these constituents show biological activities. Imperatorin is mentioned as an anticonvulsant agent. In addition, of imperatorin, oxypeucedanine and chalepin, only chalepin is found to have anticoagulant activity when administered to rats in a single dose. Aniline hydroxylase activity was appreciably depressed by each of the substances. Ethylmorphine demethylase, hepatic DNA, reduced glutathione and glucose-6-phosphatase were unaffected by these compounds when administered at a dose of 50 mg/kg for 3 days prior to slaughter. Under similar conditions only chalepin treatment resulted in α-1-globulin increase and a decrease in β-globulin content of the serum. Only intraperitoneal treatment with chalepin (100 mg/kg) for 2 days resulted in the death of 40% of the rats used within 48h of treatment. Livers of dead rats showed generalized necrosis of hepatocytes. Rats surviving after 8 weeks showed no changes in hepatic enzyme activity, reduced glutathione and DNA concentrations. However, chalepin and imperatorin induced alterations in the serum protein pattern within this period. Liver lesions were observed in chalepin-treated animals and were characterized by very mild necrosis of hepatocytes. No lesions were observed in the livers of rats treated with imperatorin or oxypeucedanine.

Additionally, methanolic root extracts showed molluscicidal activity in a bioassay with Bulinus globosus, the intermediate snail host in schistosomiasis. The LC₅₀ and LC₉₀ were 60 and 80 ppm, respectively. The powdered crude drug resulted in 60% mortality at 100 ppm. Two coumarin derivatives, heliettin and imperatorin, isolated from the methanolic extract, were more toxic to the test snail than other simple coumarins used in the bioassay, both giving 100% kill at 8 ppm.

Furthermore, C. anisata stem bark contains several quinolone/carbazole type alkaloids, including clausenol, clausenine, clausamines A-–G, 1-methyl-3,4-dimethoxy-2-quinolone and 3-formyl-1-hydroxycarbazole. The latter two can also be isolated from the roots. Clausamines D-G act as inhibitors of Epstein-Barr virus early antigen activation induced by 12-O-tetradecanoylphorbol-13-acetate in Raji cells. In addition clausenol was found to be active against Gram-positive and Gram-negative bacteria and fungi.

The essential oil of C. excavata from Vietnam contains more than 50 components, of which the main ones are β-caryophyllene (25.3%), germacrene B (11.8%) and β-phellandrene (9.2%). In addition, coumarins and alkaloids are also mentioned. For example, the binary carbazole alkaloid clausenamine-A, and a carbazole-pyranocoumarin dimer, carbazomarin-A, were isolated from the stem- and root bark. Other (series of) carbazole alkaloids include clausemine A, clausenaquinone-A, clauszoline A-L, clausines A-V and clausevatine D-G. Furthermore, typical furano-coumarins are the clauslactones A-J, N-Q, together with claucavatin-A, -B, clausarin, umbelliferone, scopoletin and xanthoxyletin.

Of the isolated constituents, the alkaloid clausenaquinone-A shows potent cytotoxicity in HCT-8, RPMI-7951, and TE671 tumour cells, as did clausemine-A against a variety of human cancer cell lines in vitro.

In addition, the extract from the stem bark showed significant inhibition of rabbit platelet aggregation and caused vasocontraction. The crude methanol extract, partitioned layers and chromatographic fractions revealed the presence of promotive and inhibitive constituents simultaneously. Other research revealed some more information on active constituents: clausine-D inhibited arachidonic acid- and collagen-induced aggregation of rabbit platelets in a dose-dependent manner (IC₅₀ values of 9 and 59μM, respectively). In human citrated platelet-rich plasma, clausine-D inhibited the secondary phase, but not the primary phase, of aggregation induced by epinephrine and ADP. Therefore, it was concluded that the antiplatelet effect of clausine-D is due to the inhibition of thromboxane A2 formation. Also safrole, isolated from the leaves, showed significant anti-platelet aggregative activity: 70% at 20μg/ml, after rabbit platelet aggregation was induced by 100μM arachidonic acid, and 48% at 50μg/ml, after aggregation was induced by 10μg/ml collagen. Clausenaquinone is active in the same assay as well.

The volatile oil obtained from the fruits of C. lansium contained some 40 compounds. Monoterpene hydrocarbons represented 60% of the total volatiles, whileβ-phellandrene (40.5%) was dominant. Among the sesquiterpenoids found, α-santalal was the most abundant. Other compounds are carbazole type alkaloids (e.g. murrayanine, glycozoline, indizoline, 3-formyl-6-methoxycarbazole, methyl 6-methoxycarbazole-3-carboxylate and 3-formyl-1,6-dimethoxycarbazole), and (furo-)coumarins, of which chalepensin, chalepin, lansiumarin A-C, wampetin, dehydroindicolactone and clausenacoumarin are examples. The latter coumarin can lower blood glucose levels in normal- and alloxan diabetic mice at a dose of 200 mg/kg for three days when given orally. It also antagonized the elevation of blood glucose caused by injecting adrenaline in normal mice. However, no effect on blood lactic acid content was observed.

Further characteristic constituents from C. lansium include a series of amides: clausenamide, neoclausenamide, cycloclausenamide, clausamide-I, -II, lansamide 1-4, and lansiumamide A-C. As isolated compounds, several showed distinct biological activities. Lansamide 2-4 were found to have a marked spasmolytic activity on the isolated guinea-pig ileum in vitro. Furthermore, the hepatoprotective activity of clausenamide, neo-clausenamide, cylcoclausenamide and some related derivatives was studied. Except for demethyl-secoclausenamide, at a dose of 250 mg/kg, they significantly depressed the elevated serum transaminase levels in mice intoxicated with CCl₄. Further studies on seco-clausenamide and clausenamide demonstrated that they decreased the hepatotoxicity of thioacetamide and acetaminophen in mice as well. Clausenamide was additionally shown to significantly inhibit CCl₄-induced lipid peroxidation of liver microsomes and ¹⁴C-CCl₄ covalent binding to microsomal lipids. In another experiment clausenamide inhibited ferrous-cysteine induced lipid peroxidation (malondialdehyde formation) of microsomes from the rat brain, heart, liver and testes. Electron spin resonance (ESR) indicated that the compound scavenges oxygen radicals produced by human polymorphonuclear leukocytes (PML, previously stimulated with phorbol myristate acetate (PMA). However, no inhibitory effect on oxygen consumption of PMLs during respiratory burst was measured by spin label oximetry. It may be concluded that clausenamide exhibits its antioxidant activity via scavenging oxygen free radicals. In addition, multiple doses of clausenamide were shown to inhibit the liver lipid peroxidation caused by 50% alcohol and to increase the GSH-peroxidase activity significantly in rat liver and brain cytosols.

Furthermore, clausenamide at a concentration of 10^-5M inhibited the contraction of basilar artery preparations caused by serotonin, PGF2α and arachidonic acid, indicating that clausenamide can also act as a cerebral protective agent. Several other tests with clausenamide in neurological assays indicated that only (-)-clausenamide was active; (+)-clausenamide was inactive. The effect of (-)-clausenamine as a new cognition enhancer, on memory deficit and on regional acetylcholine (ACh) levels and anisodine-induced ACh decrease were examined in mice. The protective action of the compound against anisodine-induced amnesia was shown to be due to its ability to reverse ACh decrease. Furthermore, the neurotrophic effects of on rat frontal cortex neurons in culture was also studied.

(-)-Clausenamine increased choline acetyltransferase activity and protein content, stimulated proliferation of neuronal cells, and supported survival and neurite outgrowth of neurons. The neurotrophic action was similar to that of nerve growth factor. In addition, the effects of (-)-clausenamine on synaptic transmission of the hippocampal dentate gyrus in freely-moving rats was studied using an extracellular recording technique. The results obtained suggest that orally-administered clausenamide can enter the brain tissue to affect synaptic transmission.

The root bark of C. harmandiana yields the alkaloids heptaphylline, 7-methoxyheptaphylline and 2-hydroxy-3-formyl-7-methoxycarbazole. The latter constituents showed a slight activity against certain carcinoma and leukaemia cell lines in bioassays. Furthermore, the coumarins clausarin, dentatin, osthol, xanthoxyletin, nordentatin and clausine K were isolated. These compounds, except for clausine K, exhibited activity against Plasmodium falciparum in vitro.

The essential oil from the leaves of possibly C. heptaphylla from Thailand contains (E)-anethole (97%), anisaldehyde (1.5%), estragole (0.6%), anisyl ketone (0.4%) andα-elemene (0.1%) as the main components. Samples collected from other regions (e.g. eastern India) showed that methyl chavicol was the main oil component (57.5-75.6%), followed by anethole (21.7-40.3%). This species probably also shows the existence of chemovariants. Similar results were obtained for the fruits.

Furthermore, in addition to coumarins such as clausmarin A, the carbazole alkaloids clausenapin, clausenal (from the leaves) and clausenalene (from the stem bark) were isolated. Clausenal was found to be active against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Salmonella typhi, and Pseudomonas aeruginosa, and the fungi Candida albicans and Trychophyton rubrum in vitro. The minimum inhibitory concentrations of clausenalene against Bacillus subtilis, Proteus vulgaris, Escherichia coli and Staphylococcus aureus were 15, 20, 25 and 33μg/ml, respectively.

• Trees or erect shrubs, unarmed, usually evergreen, young branches patently short-hairy.
• Leaves spirally arranged, at the apex of branches often densely crowded, imparipinnate, surface pellucid punctate, strong smelling when crushed; petiolate; stipules absent; leaflets alternate to subopposite, ovate-oblong to ovate, base often oblique; petiolules short.
• Inflorescence a terminal, or sometimes axillary, panicle, hairy.
• Flowers bisexual, 4-5-merous, globose in bud, fragrant, pedicellate; calyx 4-5-dentate, lobed or more deeply incised, persistent; petals 4-5, free, ovate-oblong, glandular, imbricate, soon caducous; stamens (7-)8 or 10, free, unequal, in 2 whorls, often yellow or orange, towards the base usually conspicuously broadened, curving outward at anthesis, anthers dorsifixed; gynophore short, ovary entire or faintly lobed, glabrous, pubescent or scaly, cells with 2 superposed ovules, style short, thick, caducous or persistent.
• Fruit a globose or ellipsoid, glandular berry, 0.8-2 cm in diameter, seeds 1-3, embedded in mucilaginous pulp, usually sweet-tasting, bright coloured.
• Seed ellipsoid to ovoid, greenish.
• Seedling with epigeal germination.

Clausena is usually evergreen, but species with a wide distribution area, subject to seasonality either through temperature or rainfall patterns, do show ecotypes which are deciduous. C. harmandiana and C. excavata can be found flowering and fruiting throughout the year.

In South-East Asia the fruits of C. lansium mature from June to October, in Queensland in November-December. The fruits of Clausena are mainly dispersed by birds, but probably also by small mammals.

Clausena species often vary in their vegetative characteristics. This is particularly the case with C. anisata in Africa, where it has probably been introduced relatively recently. Identification of Clausena species is mainly based on floral characters, and in particular the pistil, just before or after anthesis.

An orthographic variant of Clausena is Claucena.

Clausena is closely related to Murraya, but differs from the latter by its short, thick style, which is less than half the length of the pistil, ovary and gynophore often differentiated, and stamens towards the base conspicuously broadened.

Clausena is generally found in subtropical to tropical climates, and occurs as understorey in primary and secondary forests, also on forest edges or savanna. They are often planted around villages, and prefer richer, well-drained soils, but are found on a wide variety of soils.

Clausena can be propagated by seed, but several species are propagated by cuttings, air layers or grafts. The seeds do not have to pass the digestive tract of animals, and they often germinate readily within a few days, at the base of the tree. They are relatively short-lived, and desiccate or rot quickly when not in favourable conditions.

In China seeds of C. lansium were collected at weekly intervals from mid-maturation to the fully ripened stage. As seed development progressed, desiccation tolerance increased. Seeds remain viable for several weeks if stored cool and if not excessively dried.

In vitro propagation has been successfully tried with e.g. C. excavata.

C. excavata is generally used as a rootstock for C. anisum-olens. In experiments in Sumatra C. excavata has been used as rootstock for C. anisata. Pruning of Clausena is recommended to avoid overcrowding of the branches.

Few important diseases and pests on Clausena are recorded, although some can occasionally be a nuisance. A new leaf disease, sooty leaf blotch, was found on C. excavata in Java, and the causal fungus was described as Mycovellosiella clausenae. Fusarium sp. causes leaf spot on C. anisata. The causal fungus of wampee shoot rot on C. lansium is Gibberella baccata.

In Malaysia C. excavata was found to be an important natural host of the vector Trioza erytreae (Hemiptera), which causes citrus greening disease.

Furthermore, C. lansium is a host for the immature citrus blackfly (Aleurocanthus woglumi (Homoptera)). In China, one of the major insect pests of C. lansium is Phyllocnistis wampella.

C. anisata is a host for the nematode Tylenchulus semipenetrans.

All plant parts of Clausena that are used can be harvested when needed, especially of the species that are cultivated near villages.

The parts of Clausena that are harvested are used fresh or dried and sometimes powdered for future use.

The Clausena species treated here have a large area of distribution, either naturally or as a result of cultivation, and do not seem to be at risk of genetic erosion.

Small germplasm collections of C. anisata, C. excavata and C. lansium are present at the Instituto Valenciano de Investigaciones Agrarias in Valencia, Spain and of C. anisata, C. excavata, C. harmandiana and C. lansium in USDA-ARS National Germplasm Repository for Citrus & Dates, in Riverside, California, United States. The other germplasm collections are of C. lansium, and can be found in Taiwan and Bangkok, Thailand.

Clausena contains a variety of compounds, for instance clausenamine, which exhibits interesting biological activities. This compound, as well as several others, merits further research to fully evaluate their future potential. Furthermore, the anethole-containing essential oils may be of interest as a local substitute for anise oil, or as an industrial starting material in the production of this component. Seen in this light, the potential for cultivation of Clausena species in South-East Asian countries may need further investigation.

• Chakraborty, A., Saha, C., Podder, G., Chowdhury, B.K. & Bhattacharyya, P., 1995. Carbazole alkaloid with antimicrobial activity from Clausena heptaphylla. Phytochemistry 38(3): 787-789.
• Emerole, G., Thabrew, M.I., Anosa, V. & Okorie, D.A., 1981. Structure-activity relationship in the toxicity of some naturally occurring coumarins - chalepin, imperatorin and oxypeucedanine. Toxicology 20(1): 71-80.
• Molino, J.-F., 1994. Révision du genre Clausena Burm.f. (Rutaceae) [Revision of the genus Clausena Burm.f. (Rutaceae)]. Bulletin du Muséum National d’Histoire Naturelle, Section B, Adansonia 16: 105-–153.
• Quisumbing, E., 1978. Medicinal plants of the Philippines. Katha Publishing Co., Quezon City, the Philippines. pp. 457-458.
• Xu, H.H., Zhao, S.H., Zhu, L.F. & Lu, B.Y., 1994. Studies on insecticidal activity of the essential oil from Clausena dunniana and its toxic component. Journal of South China Agricultural University 15(2): 56-60.
• Zhang, A. & Lin, G., 2000. The first synthesis of clausenamine-A and cytotoxic activities of three biscarbazole analogues against cancer cells. Bioorganic and Medicinal Chemistry Letters 10(10): 1021-1023.

• G.H. Schmelzer