Ethnobotanical Leaflets 12: 772-775. 2008.

Evaluation of Brine Shrimp Lethality of Cinnamomum Species

Muthiah Maridass

Animal Health Research Unit, St.Xavier’s College (Autonomous)

Palayamkottai - 627002, Tamil Nadu, India

Email: [email protected]

Issued 01 October 2008

ABSTRACT

Cinnamomum species have long been used as spices. The preliminary bioactive constituent’s identification and brine shrimp lethality activities of ethanolic extracts of seven Cinnamomum species viz., C. travancoricum, C. walaiwarense, C. wightii, C. verum, C. sulphuratum, C. riparium, and C. perrottetii were evaluated in this study. The results of cytotoxic activity of the bark extracts of seven Cinnamomum species were more active than leaf extracts against brine shrimp lethality of Artemia salina.

Keywords: Cinnamomum species; bioactive constituents; Brine shrimp; Artemia salina; cytotoxic activity.

INTRODUCTION

In the United States in 1999, over 1500 people are expected to die of cancer each day, representing an estimated total mortality rate of about 560000. More than twice as many persons than this will be diagnosed with invasive cancer, but, overall, a slight decline in cancer incidence rates has been observed in the USA. Among many recent advances in cancer chemotherapy, phytochemicals play an important role in cancer chemotherapeutic drugs. A search for new anti-cancer drugs has taken many different approaches. The brine shrimp lethality bioassay is efficient, rapid and inexpensive tests that require only a relatively small amount samples. The technique is easily mastered, costs little, and utilizes small amount of test material. Meyer et al.(1982) has been successively employed for in-vivo lethality bioassay-guide fractionation of active cytotoxic and antitumor agents such as trilobacin from the bark of Asimina triloba (Zhao et al., 1992), cis-annonacin from Annona muricata (Rieser et al. 1996) and ent-kaur-16-en-19-oic acid from Elaeoselinum foetidum (Mongelli et al. 2002).

The genus Cinnamomum belongs to the family Lauraceae and  comprises several hundred species found in continental Asia, East and Southeast Asia, Australia, the Pacific, and a few species in Central and South America (Jantan et al.,1995). These are evergreen trees and shrubs and most of the species are aromatic. Twenty-one species of Cinnamomum have been found on the Malaysian peninsula (Kochummen, 1989). A number of these species are used in traditional medicine and their distilled essential oils or synthetic analogs are used as flavoring agents in the food and beverage industry (Burkill,1966; Jham et al., 2005). Previously, brine shrimp lethality activity has not been evaluated seven Cinnamomum species. The present study was work carried out the preliminary phytochemicals identification and cytotoxicity properties of both barks and leaf extract on seven Cinnamomum species.

MATERIALS AND METHODS

Collection of Plant materials.

Seven Cinnamomum species (C. travancoricum, C. walaiwarense, C. wightii, C. verum, C. sulphuratum, C. riparium, and C. perrottetii) were collected from Kalakad Mundanthurai Tiger Reserve Forest, Karaiyar, Tirunelveli District, South India.

Extraction.

Dried 500gms of leaves powdered were individually extracted in a Soxhlet apparatus with ethanol (1.5lrs) at 60^oC for 8hr. Collected extracts were then filtered and concentrated in vacuo at 45^oC. The ethanol free extracts were kept refrigerated (4^oC) and used for the further investigations. Done on preliminary phytochemical identification methods followed by Maridass, (2005). 

Brine shrimp lethality assay.

In vitro lethality assay of A. salina was used to detect cell toxicity (Meyer et al. 1982). Brine shrimp eggs were placed in seawater (3.8% w/v sea salt in distilled water) and incubated at 24-28^oC in front of a lamp.  Eggs were hatched within 48h providing large number of larvae (nauplii). A convenient number of nauplii were placed in vials containing 5ml of seawater and increasing concentrations of Cinnamomum species viz., C. travancoricum, C. walaiwarense, C. wightii, C. verum, C. sulphuratum, C. riparium, and C. perrottetii leaves extract (0.1-500ppm). Control was made with the same volume of 96% ethanol in seawater without addition of the Cinnamomum species leaves extract. Alive nauplii were counted after 16h and the lethal Concentration (LC₅₀) was calculated.

Statistical analysis.

Lethality assays were evaluated by Finney computer statistical program to determine the LC₅₀ values and 95% confidence intervals. All other data were expressed as mean ± SD.

RESULTS AND DISCUSSION

The cytotoxic activity of both barks and leaves of seven Cinnnamomum species were investigated in vitro tested against the brine shrimp (Artemia salina). The results are given in Table 1. All the crude extracts of Cinnamomum species resulting in LC₅₀ values of less than 250µg/ml were considered for active against brine shrimp.  This bioassay has a good correlation with cytotoxic activity  in several plant extracts of Ocimum sanctum, Lagerstroemia reginae, Cissampelos pareira, Acacia conccina, Punica granatum, Aconitum species, Rosa damascene, Cinchona species, Bacopa monnieri, Symplocos racemosa were showed significant lethality to brine shrimp( Krishnaraju et al. (2006).  In less active against brine shrimp lethality effect of ehtanolic extract of Annona crassiflora leaves are earlier reported (Pimenta et al. 2003). In the present reports of both plant parts of bark and leaf of all the Cinnamomum species showed good brine shrimp lethality. In bioactive constituents identification of seven Cinnamomum species with cytotoxic properties, activity due to the presence of triterpenes, fixed oils, sugars, saponins and tannins were observed (see Table 2). Alkaloids were absent in both leaves and barks of Cinnamomum species but all the plants samples gave positive reaction of both Liberman-Burchard and Salkowski tests indicating the presence of triterpenoids. The ferric chloride test gave positive results indicating the presence of catechol tannins in both parts of leaves and barks of all Cinnamomum species. Previously, strong reactions froth test were observed saponins compound present in the leaf of Gomphandra lysipetala, Maesa ramentacea, Alphitonia excelsa, and Kleinhovia hospital (Lailay, 2002). The present results agree with the presence of terpenoids, essential oils, fixed oils, saponins and tannin may be acte constituents useful for the utilization of cytotoxic principles of these extracts of Cinnamomum species. Further studies should be going on fractionation and identification of bioactive constituent to human cell line culture of cytotoxic effect.

Acknowledgement

            Author wants to thank Department of Science and Technology, SERC- Fast Track Scheme, New Delhi, India for the financial supports.

References

Burkill, I.H. 1966. A Dictionary of the Economic Products of the Malay Peninsula. Vol. 1, 2nd edition 2444pp, Ministry of Agriculture and Cooperatives, Kuala Lumpur.

Jantan, I., S.I. Wiselius, S.C. Lim and M.S.M. Sosef.1995. Cinnamomum Schaeffer. In: Plant Resources of South-East Asia No. 5 (2) Timber trees: Minor commercial timbers. Edits., R.H.M.J. Lemmens, I. Soerianegara and W.C. Wong, pp 130-140, Backhuys Publishers, Leiden.

Jham, G.N., O.D., Dhingra, C.M. Jardin, and M.M. Valente, 2005. Identification of the major fungitoxic component of cinnamon bark oil. Fitopatol. Bras. 30: 404-408.

Kochummen, K.M.1989.  Family: Lauraceae, Tree Flora of Malaya. Vol.4, pp 124-132, Longmans, Kuala Lumpur.

Krishnaraju, A. V.,  V. N.Tayi Rao, D. Sundararaju, M. Vanisree,H.S. Tsay, and G. V. Subbaraju. 2006.  Biological Screening of Medicinal Plants Collected from Eastern Ghats of India Using Artemia salina (Brine Shrimp Test). International Journal of Applied Science and Engineering, 2:115-25.

Lailay, Din, N. I. Yusoff, M W. Samsudin, U. Suki, K. M. Salleh, A. Z. Ibrahim, A. Latiff and I. M. Said. 2002. A preliminary phytochemical survey of plants in crocker range, Sabah, Malaysia. ASEAN Review of Biodiversity and Environmental Conservation (ARBEC), pp.1-7, updated, http://www.arbec.com.my/pdf/art7julysep02.pdf.

Maridass, 2005. Studies on phytochemical and biological activites of Eulophia epidendraea (Retz.) Fisher (Orchidaceae), Ph.D.Thesis, Manonmaniam Sundaranar University, Tirunelveli,Tamil Nadu,South India.

Meyer, B.N., N.R. Ferrigni, J.E.Putnam, L.B.Jacobsen,D.E. Nichols, and J.L. McLaughlin, 1982.  A convenient general bioassay for active plant constituents. Planta Medica, 45: 31-34.

Mongelli, E., Pomilio, A. B., Sanchez, J. B., Guerra, F. M., & Massanet, G. M. (2002). ent-Kaur-16-en-19-oic acid, a KB cells cytotoxic diterpenoid from Elaeoselinum foetidum. Phytotherapy Research, 16:387-388.

Rieser, M. J.,  Z.M.Gu, X.-P. Fang, L.Zeng, K. V.Wood, and J. L. McLaughlin.1996. Five novel mono-tetrahydrofuran ring acetogenins from the seeds of Annona muricata. Journal of Natural Products, 59:100-108.

Santos Pimenta, L.P., G.PB.Pinto, J.A.Takahashi, L.G.F.e.Silva and M.A.D.Boaventura, 2003. Biological screening of Annonceous Brazilian Medicinal plants using Artemia salina (Brine Shrimp Test). Phytomedicine, 10:209-212.

Zhao, G., Y. Hui, J. K., Rupprecht, J. L., McLaughlin, and K. V.Wood.1992. Additional bioactive compounds and trilobacin, a novel highly cytotoxic acetogenin, from the bark of Asimina triloba. Journal of Natural Products, 55, 347-356.

Table 1. Brine shrimp bioassay results of leaves extract of Cinnamomum species.

LC₅₀’s were estimated using logit transformation. Therefore confidence intervals are not provided.

An average of three replicates.

Table 2. Preliminary phytochemicals identification of Cinnamomum species.

“+” Presence of bioactive compound “-” Absence of bioactive compound