HETEROCYCLES

Review | Special issue | Vol. 79, No. 1, 2009, pp. 121-144
Received, 18th September, 2008, Accepted, 19th November, 2008, Published online, 20th November, 2008.
DOI: 10.3987/REV-08-SR(D)2

■ Constituents and Bioactivities of Clausena excavata

Ngampong Kongkathip* and Boonsong Kongkathip

Department of Chemistry, Faculty of Science, Kasetsart University, 50 Phahonyothin Road, Chatuchak, Bangkok 10903, Thailand

Abstract

Clausena excavata Burm. f. (Rutaceae) is a medicinal plant which is used in folklore medicine for treatment of cold, malaria, AIDS, dermatopathy, abdominal pain, and snake-bite. This plant is a rich source of coumarins and carbazole alkaloids. So far, fifty-three coumarins and fifty-eight carbazole alokaloids were isolated from C. excavata. Furthermore, a small group of tetranortriterpenoids, steroids, flavonoids, and essential oils were also obtained from this plant. C. excavata showed diverse therapeutic activities which are antibacterial, antifungal, antiplatelet, antiplasmodial, antitumor, antinociceptive, immunomodulatory, antimycobacterial, and anti-HIV-1 activities. The incidence of HIV-1 infection leading to AIDS has increased every year, and fungal and bacterial infections, particularly TB-causing mycobacteria are prevalent in HIV-infected patients. So Clausena excavata which showed inhibition of these diseases, is very promising to be developed for treatment of AIDS.

INTRODUCTION
Clausena excavata Burm. f. (Rutaceae) (Figure 1) is a wild shrub which is widely distributed in southern and southeastern Asia. Local Thai people usually call it by the name “Sun Soak” which is used in folklore medicine for treatment of cold, malaria, AIDS, dermatopathy, abdominal pain, snake-bite and as a detoxification agent.1 C. excavata is a rich source of coumarins and carbazole alkaloids, however a small group of tetranortriterpenoids, steroids and flavonoids has been reported. In addition, its leaves contain essential oils such as β-elemene (95.8%), β-caryophyllene (25.3%), safrole (82%), nerolidol (4.7%) and germacrene (11.8%).2 Some isolated substances from Clausena excavata as well as other Clausena genus have been reported to exhibit diverse biological activities, antibacterial,3 antiplatelet,4 antiplasmodial,5 antitumor,6 antimycobacterial and antifungal activity.7 C.excavata extracts from the leaves showed antinociceptive activity8 and that from the woods had immunomodulatory activity.9 Furthermore, the rhizomes and roots of C. excavata showed anti-HIV-1 activity.10,11

In Thailand, the incidence of HIV-1 infection leading to acquired immunodeficiency syndrome (AIDS) has dangerously increased every year, and fungal and bacterial infections, particularly TB-causing mycobacteria, often are associated with HIV-infected patients. Currently, there is no effective remedy for curing AIDS. In traditional Thai medicine, some patients with AIDS drink the extract of C.excavata obtained by soaking the roots and rhizomes in Thai whiskey that contains approximately 35% ethanol.
The crude 35% ethanol extract of the roots and rhizomes showed anti-HIV-1 activity with EC50 values of 8.67 μg/mL, IC50 values of 32.83 μg/mL and potential therapeutic index (PTI) values of 3.79. Whereas the water extract showed less activity with EC50 = 73.9 μg/mL and IC50 > 250 μg/mL and PTI = >3.38.11b

CONSTITUENTS ISOLATED FROM CLAUSENA EXCAVATA
Compounds isolated from Clausena excavata belong to different classes such as coumarins, carbazole alkaloids, and limonoids (tetranortriterpenoids) as shown in Tables 1 – 3.

2.1 Coumarins
So far, fifty-three coumarins were isolated from various parts of Clausena excavata as shown in Table 1, Figures 2 and 3. Root barks, stem barks, leaves, twigs, branch, rhizomes and roots contain quite a large amount of coumarins. Hexane, acetone, chloroform, ethyl acetate, methanol, and ethanol were used for extraction. Clausenidin (4) showed significant anti-HIV-1 activity.11
2.2 Carbazole alkaloids
Fifty-eight alkaloids were isolated from C. excavata as shown in Table 2 and Figure 4. Three carbazoles, clauzoline J (95), 3-formyl-2,7-dimethoxycarbazole (101), and O-methylmukanol (109) possessed anti-HIV-1 activity.

2.3 Limonoids (Tetranortriterpenoids)
Six limonoids were isolated from the ethanol extracts of aerial parts and rhizomes as shown in Table 3 and Figure 5. Clausenolide-1-ethyl ether (117) isolated from the rhizome of C.excavata showed anti HIV-1 activity.10

BIOACTIVITIES
Clausena excavata is used in diverse aspects of traditional medicine in Asian countries. Several bioassays were done on this plant and many properties were found which support the folk medicine. The therapeutic activities of this plant are:

3.1 Antinociceptive activity
3.2 Antiplatelet activity
3.3 Antimicrobial activity
3.3.1 Antifungal activity
3.3.2 Antibacterial activity
3.4 Immunomodulatory activity
3.5 Antimycobacterial activity
3.6 Anti HIV-1 activity

3.1 Antinociceptive activity
In 2002, Rahman and coworkers8 have found the ethanol extract of C.excavata leaves showed significant antinociceptive activity on acetic acid-induced writhing in mice by oral at doses of 125.25 and 500 mg/kg body weight. The ethanol extract was obtained by maceration of dried leaves in ethanol (4.08% yield). The report has not mentioned about the active constituents responsible for this activity. It was just stated that phytochemical screening indicated the presence of coumarins, flavonoides and glycosides. This plant is used in Bangladesh traditional medicine; for example, the sap of the leaves reduced the muscular pain; the root is used to reduce malarial fever; stems and roots are used for treatment of stomach problem; leaves and stem barks are used as tonic, diuretic and astringent.

3.2 Antiplatelet activity
Safrole, a volatile oil, has been isolated from the leaves of C.excavata4 and inhibited rabbit platelet aggregation in 70% at the dose of 20 μg/mL, which were induced by arachidonic acid (100 μM). When collagen (10 μg/mL) was used instead, safrole only inhibited in 48% at a dose of 50 μg/mL. A carbazole, clausine-D (65) isolated from the leaves of C. excavata also exhibited antiplatelet activity in vitro. It inhibited platelet aggregation and released reaction of washed rabbit platelets. The inhibitory effect varied depending on the types of aggregation inducers. Clausine-D (65) inhibited most strongly when induced by arachidonic acid (AA) and inhibited less when induced by collagen whereas there was no effect when induced by U46619, PAF, and thrombin.4
The IC50 values of clausine-D (65) on arachidonic acid (AA) and collagen-induced platelet aggregation were 9.0 ± 1.1 and 58.9 ± 0.9 μM, respectively. Clausine D (65) also inhibited increased intracellular concentration of calcium in platelet aggregation caused by AA and collagen. In human citrated platelets-rich plasma, cluasine-D (65) inhibited the secondary phase, but not primary phase of aggregation induced by epinephrine and ADP. The results showed that the antiplatelet effect of clausine D (65) was due to inhibition of thromboxane A2 formation.

3.3 Antimicrobial activity

3.3.1 Antibacterial activity
In 1982, Wu and Furukawa3 have investigated that a coumarin, nordentatin possessed inhibitory affect against the bacteria Bordetella brochiseptica 4614, Bacillus subtilis 6633, Pneumococcus, Staphylococcus aureus 6538-P and Pseudomonas aeruginoso NCTC 10490 at more than 10 ppm concentration.

3.3.2 Antifungal activity
In 2002, we reported7 that four carbazoles isolated from the rhizome and roots of C.excavata; 3- formylcarbazole (100), 2-hydroxy-3-formyl-7-methoxylcarbazole (105), 3-methoxycarbonylcarbazole (107) and mukonal (108) showed antifungal activity against Candida albicans with IC50 values of 13.6, 2.8, 9.5 and 29.3 μg/mL, respectively. In addition, these four carbazoles did not exhibit cytotoxicity against KB and BC-1 cell lines. This indicates a good sign for modification of these compounds by derivatization and evaluation for their antifungal activity.

3.4 Immunomodulatory activity
In 1998, Manosroi’s group9 found that the Thai folklore extract from wood of C.excavata increased CD4 and CD8 level as well as the CD4/CD8 ratio in cancer patients. They have proved the immunomodulatory activity of this plant using aqueous extract, acetone extract and Thai folklore extract (35% aqueous ethanol solution) to test in vitro mouse macrophage phagocytosis and splenocyte proliferation assay. The results showed as follow; all extracts showed phagocytic modulation, but no dose-response relationships. The aqueous extract (62.5 μg/ml) gave the most effective nitroblue tetrazolium (NBT) dye reduction9a which was approximately 30% and 20% more than that from the acetone and the folklore extracts, respectively. The aqueous extract (162.5 μg/ml) also exhibited potential lysosomal enzyme activity approximately 80% and 40% higher than those from the acetone and the folklore extracts, respectively. All extracts showed cell proliferation simulation with no dose-response relationship. The 35% aqueous ethanol extract gave the maximum proliferation enhancement both with and without mitogens by the MTT assay. All extracts did not cause cell death, with the percentage of viability of macrophages and splenocytes of more than 90% and 80%, respectively. All extracts stimulated phagocytic activity on lysosomal enzyme activity higher than NBT dye reduction. Although this work did not mention about the active constituents which had immunomodulatory activity, it is still useful to support the folklore medicine.
In addition, the same group9b has reported in 2004 that the hot aqueous extract and the acetone extract were more splenocyte-proliferation active than the folklore extract (35% aqueous ethanol extract). The following year, this group9c showed the effect in vivo of the crude extracts of C.excavata on the production of haemagglutinating antibodies (HA) in mice by intraperitoneal administration and oral route. They found that both aqueous extract and 35% aqueous ethanol extract gave the maximum antibody titer of 1/800 which was two times of the control. Oral administration appeared to reach the maximum heamagglutinating antibody (HA) titer9c faster than the intraperitoneal administration. Antibodies produced by orally administration of both extracts decreased with times without maintaining similar to cimetidine. Phenolic compounds in the aqueous extract and 35% aqueous ethanol extract were preliminary observed by TLC spraying with FeCl3 solution. Therefore the immunomodulating activity in both extracts might be due to the presence of phenolic groups. The aqueous extract and 35% aqueous ethanol extract (folklore) from wood of C. excavata showed potent in vitro and in vivo immunomodulating activity in mice. The folklore extract exhibited effective antibody production stimulation whereas the aqueous extract demonstrated superior cell-mediated immune (CMI) response.9c

3.5 Antimycobacterial activity
We reported7 the antimycobacterial activity in 2003. The crude chloroform extract from the rhizomes of C.excavata exhibited antimycobacterial activity with a minimum inhibitory concentration (MIC) of 25 μg/ml. Further isolation by vacuum liquid chromatography (VLC) and then column chromatography (CC) gave dentatin (25), nordentatin (26), 3-formylcarbazole (100), methylcarbazole-3-carboxylate (107), and mukonal (108). The hexane extract of C.excavata rhizomes gave clausenidin. The crude chloroform extract of C.excavata roots were further isolated by VLC and CC to give clauszoline-J (95) and 2-hydroxy-3-formyl-7-methoxycarbazole (105). O-Methylated clausenidin (118) was obtained from methylation reaction of clausenidin (4) using methyl iodide in the presence of potassium carbonate. The antimycobacterial activity of courmarins and carbazoles were shown in Table 4.

Among these, dentatin (25), methylcarbazole-3-carboxylate (107) and O-methylated clausenidin (118) showed significant antimycobacterial activity (MIC = 50 μg/mL).

3.6 Anti-HIV-1 Activity
Some thai AIDS patients consume the extract of C.excavata obtained by soaking the rhizomes and roots in thai whisky (35% ethanol). Therefore we10,11 have searched for anti HIV-1 coustituents from the extract of C.excavata rhizomes and roots since 2000. It was found that one limonoid,10 clausenolide-1-ethyl ether (117); one pyranocoumarin,11 clausenidin (4) and three carbazole alkaloids,11 clauszoline-J (95), 3-formyl-2,7-dimethoxycarbazole (101), O-methylmukonal (109) inhibited HIV-1 viruses. The IC50 values of the inhibition and PTI are shown in Table 5.

The above promising results spurred us to investigate the synthesis and modification of the structure of these compounds in order to increase the anti-HIV-1 activity.
In addition, determination of these compounds in the extracts from different regional sources in Thailand was investigated. We have found that the extracts from various sources of Thailand showed different amounts of the anti-HIV-1 compounds and anti-HIV-1 activity.32
We have found optimum conditions for quantitative determination of the anti-HIV-1 compounds in the extract (without purification) by HPLC which would be very useful for AIDS patients.

CONCLUSION
In conclusion, Clausena excavata is a magic plant which showed several therapeutic activities especially anti-HIV-1, antimycobacterial, immunomodulatory and antimicrobial activities. The activities of this plant is so promising that it could be developed for anti-HIV drug. Moreover, this plant showed no toxicity to normal Vero cells. Finally, this plant is very worthy to develop for the treatment of AIDS.

ACKNOWLEDGEMENTS
This review is for Professor John W. Daly who passed away in March, 2008. Professor John W. Daly was very kind, generous and helpful to all of us. Four of our students, Dr. Uthai Sakee, Dr. Nisachon Chaosuancharoen, Dr. Naratitt Noimai and Dr. Arunrat Sunthitikawinsakul have been working for 6-8 months at Professor John Daly’s Laboratory and gained a lot of results, knowledge and experiences from him and his colleague co-workers especially Dr. Thomas F. Spande. Everytime we visited NIH, he accorded us very wonderful hospitality even picking us at the airport. This review also includes some papers which have been suggested by him. Finally, we pray for him to be in the heaven.
Some work mentioned in this review was supported by the Kasetsart University Research and Development Institute (KURDI).

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