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Saro (Cinnamosma fragrans Baillon) essential oil: Application in Health and Medicine Olivier Behra1, Pascal Danthu2, Samira Sarter2, Rina Radaniela3, Cesar Fourcade4, Roger Randrianarivelo, Boris Ranaivosoa5 and Bérengère Arnal-Schnebelen6 1

NGO “Man And The Environment”, Antananarivo, Madagascar CIRAD (International Center for Agronomic Research for Development) Antananarivo, Madagascar and Montpellier, France 3 Label CBD, Antananarivo, Madagascar 4 Faculty of Medicine, Paris, France 5 FOFIFA (National Agronomic Research Institute), Antananarivo, Madagascar 6 DUMENAT (University Department of Natural Medicines) Paris, France

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The richness of medicinal plants of the island Madagascar is unique in the world and has largely been understudied. Saro, Cinnamosma fragrans, is produced from a Madagascan plant which is locally well-known for its antimicrobial activities and also for its powerful anti-poisoning effects. While searching for local strategies to enhance the value of this renewable natural resources for the benefit of local populations and biodiversity conservation this plant appeared as a very promising candidate with an essential oil that could be used in many new ways. This paper presents research on the plants essential oil composition including the variation found across environments where the plant is endemic as well as the results of associated biological tests conducted on the essential oils’ toxicity, skin and eye tolerance and on mutagenesis potential. The properties of the Saro essential oil have been tested and show very interesting results as an antibacterial and antiviral agent and as a health protection enhancer.

© 2009 American Chemical Society

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486 Madagascar, an island as large as the state of Texas, is located in the southern Indian Ocean. The island was separated from Africa about 65 million years ago. This isolation has created an amazing diversity of plants and animals. The island hosts up to 13,000 plant species of which 85% are endemic (1), while endemism (96%) is even higher in the case of trees and large shrubs (2), the endemic richness of species in Madagascar is thus unparalleled in the world (3). Saro, or Cinnamosma fragrans (Canellaceae), is an aromatic shrub or small tree, up to five meters high, evergreen and very aromatic. This small family includes only nine species in four genus, Winterana and Cinnamodendron in America, Warburgia in western Africa and Cinnamosma endemic to Madagascar. The genus includes, C. madagascariensis, in the eastern forests of the island between 900 to 1500 meters of altitude, with a variety C. madagascariensis namorensis, in the littoral forest at low altitude. C. macrocarpa, in the eastern forest between 600 to 900 meters altitude but much rarer; and C. fragrans, in the western forest (4). Leaves are full alternate transparent when young becoming hard when adult. Flowers are white small (5 to 6 mm) and hermaphrodites. The fruit is an ovoid berry dark brown when mature, having 15 to 25 seeds. Saro is often found in tropophyle forests, close to the water or in rocky shady areas, and growing in chalky or silicitic soils. Flowering occurs between September and November and fruits develop during the rainy season which occurs December and May (5). Some initial research was conducted on Saro (6) though research came to a halt due to the sociopolitical isolation of the island from 1972 until recently. Initial work also had been conducted on the vanilloides present in this plant which had originally suggested potential applications as memory enhancers, appetite regulation and emotions, among others (7). These new applications may be surprising to scientists but the plant has been well-known by the local populations and its uses have been described by different authors since 1910 (810). Although, it has incredible natural wealth, Madagascar is still one of the poorest nations of the world. The United Nations has declared Madagascar a world major priority for conservation because of the threats on its biodiversity (11). Thus, the discovery of new products with new potential applications can drive the commercialization process and add value to the natural resources and by generating income for local populations which in turn can contribute to sustainable development. The sustainable production of essential oils, through sustainable harvesting of the leaves is an alternative to other destructive agricultural practices, like slashing and burning (12). Thus, the ability to be able to continually harvest leaves of this plant for its essential oils would be a key strategy to contribute to sustainable development. The first humans arrived to the island 2000 years ago. The newcomers had to discover the properties of plants to treat themselves and since then the local population (80%) still use medicinal plants (13). Saro is one of these plants and its’ local name reflects the fact that it has many therapeutic uses including anti poisoning. Saro’s local name is “Mandravasarotra” which literally translates to “which keeps bad things away” or “annihilates diseases”. The plant C. fragrans has different other names following tribes and in the west it is also called


487 “Motrobeatinainy” which means “large fire in the belly” as all its aerial parts have a hot and spicy taste (14). Roots are used against coughing, asthma and dysenteries. Liquid expressed from leaves are known for their powerful treatment of wounds and abscesses. The most characteristic use of this plant is as a tonic and antitoxic against poisoning. Leaf decoctions are also used for weak overweight children and for underweight children, for difficult and exhausting labor or for weak persons to protect them from diseases. The Tanala tribe uses Saro during traditional sportive fights between men (Tolona) and traditional rodeos (Tolon’omby). Cinnamosma fragrans leaves are boiled with leaves of “voafotsy” (Aphloïa theiformis) a traditional tea which has been found to contain high levels of interesting antioxidants (15). Fighters drink the decoction before battle. The plant is also known to treat nervous and sensorial system diseases (16). Isolated and unpublished work has been conducted in Saro, though the composition of the oil and their pharmacological properties are less known. In view, of the ethnobotanical uses of this plant by the Malagasy people, the aim of this work was to study the chemical diversity of Saro essential oil from different regions of Madagascar and to evaluate its safety and pharmacological activities.

Material and Methods Leaves of Saro were collected manually during the rainy season and processed. The leaves were steam distilled over a 4 h period and captured using a Clevenger-type apparatus. Distillations were performed less than 24 h after harvesting. The essential oils were dried over anhydrous sodium sulphate until the last traces of water were removed and then stored in dark glass bottles at 4°C. The botanical identity of C. fragrans was confirmed and a herbarium voucher was deposited at the Madagascar Academia. Study sites ranged from the extreme north of the island around Antsiranana to Manja at the southern extreme of the western domain. A special emphasize was placed on this latest site to compare different specimens with some at around 500 km northern on the site of Antsalova between 18°32’10’’ to 18°41’21’’ south and 44°11’16’’ to 44°15’30’’ east. The Saro essential oils (EO) were analyzed by GC/MS using a HewlettPackard GC/MS System. Rtx-WAX column (30 m x 0.25 mm x 0.25 film thickness) was used with hydrogen (H2) as the carrier gas. GC oven temperature was kept at 50°C for 10 min and programmed to 250°C at a rate of 5°C/min, an then kept constant at 250°C for 10 min. The FID and injector temperature was at 255°C. The results were expressed as relative (area) percentage. Different toxicity tests were conducted to evaluate safety of Saro essential oil. These include primary eye tolerance tests, cytotoxicity and mutagenity potential. Each test was conducted in compliance with the Principle of Good Laboratory Practice regulation by internationally recognized laboratories. Primary eye tolerance tests were conducted by Eurotox Registered Toxicologists at the “Institute d’Expertise Clinique” (Clinical Expertise Institute), Lyon, France following the Good Laboratory Practises published in the J.O.R.F. (French Republic Official Texts) of December 1996. The method


488 used was that described by Luepke (1985) which allows checking of irritation potential of product through application on hen’s egg chorioallantoic membrane at day 10 of incubation when the embryo has no cerebral activity and when the membrane does not have any sensorial receptors (17, 18). For mutagenic activity, the risk of carcinogens and mutagens compounds which might be presented in the oil was also evaluated according to the international guidelines (OECD 471 and commission directive N° B13/14). Tests have been conducted at the CIT (International Center of Toxicity) Safety and Health Research Laboratories, 27005 Evreux, France. This test evaluates the potential of the Saro essential oil to induce reverse mutation in Salmonella typhimurium, knowing that the bacterial reverse test is able to identify substances that cause point mutations, by affections of DNA base-pairs (19, 20). Five strains of S. typhimurium: TA 1535, TA 1537, TA 98; TA 100 and TA 102 were supplied for the study by B.N. Ames Laboratory (University of California, Berkeley or Oakland Research Institute, USA). Cytotoxicity tests have been conducted at the “Institut d’Expertise Clinique” (Clinical Expertise Institute),Lyon,France, on isolated cultivated rabbit cells registered at the American Tissues Culture Company (ATCC, CCL60) following the Good Laboratory Practices for cosmetic products as published by the French legislation (J.O.R.F., 2004) which prohibits animal testing. The test was based on the evaluation of the cytotoxicity of the product by the determination of the concentration inducing 50% of cell mortality (C.I. 50) through direct application on rabbit eye cell fibroblast monolayer and neutral red release as described in Official Journal of the French Republic (J.O.R.F., 1999). Antimicrobial activity was evaluated using Mueller-Hinton agar plates by the disc diffusion technique. Solution of the essential oils were prepared with Tween 80 (1 Vol. Saro + 1 Vol. Tween) and kept at 4° Celsius, then diluted in sterile distilled water at 10 mg/ml. 0.1 µl of the bacterial suspension was applied to Mueller-Hinton agar plates for sensitivity testing. Sterile antibiogram discs were moistened with 10 μl of pure Saro oils and its decimal dilutions and then applied to the surface of the agar plates. After incubation of the plates for 24h the diameters of the inhibition zones were measured with a caliper and expressed in mm. The results were expressed as +++ high activity, ++ medium activity, + low activity, - no activity. Tests were conducted at the National Centre for Research on the Environment (CNRE, Antananarivo, Madagascar). Additional antimicrobial tests on microbes of gynecological origin (Neisseria gonorrhoe, Gardenerella vaginalis, and Candida albicans) were conducted by Dr. Berengère Arnal-Schnebelen (22).

489

Results and Discussion


Physicochemical Properties of the Essential Oil The essential oil of Saro is a yellow and mobile liquid with a slight and characteristic fresh and eucalyptus aroma. The oil is less dense than water (0.9452), and the refractive index was 1.4636 (Table I). The essential oil is characterized by high levels of 1,8-cineole (40-55%), with minor amounts of αpinene (4-7%), β-pinene (5-8%) and linalool (4-9%). The Saro EO also contained at lower levels terpenyl acetate (1-4%), α-terpineol (2-5%) and terpinen-4-ol (2-6%) (Table II). Prior studies showed the presence of at least two EO chemotypes of C. fragans, the first one rich in linalool (49.2 to 95.8% (22) with only traces of 1,8cineole. The second EO chemotype was the opposite, being rich in 1,8-cineole with a range from 37.1% to 71.6% (22, 23). Saro essential oil, produced in the Northwestern region of Madagascar is of the second type and regular analyses of the essential oil chemical composition for over a year has shown it to be stable with a level of 1,8-cineole between 40 to 55 % (Table II). Table I. Physicochemical properties of Saro essential oil Character Aroma Color Density Refractive index

Value Slight and characteristic fresh and eucalyptus aroma Light yellow 0.9452 1.4636

Table II. Essential oil composition of Saro essential oil Essential oil components Sabinene α Pinene β Pinene 1,8–Cineole Linalool Terpinen-4-ol α Terpineol Terpenyl acetate β-Caryophyllene

Percent (as % of total EO) 1 -3 4-7 5-8 40-55 4-9 2-6 2-5 1-4 2-5

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Biological Activities of the Essential Oil The primary eye tolerance tests showed that the Saro essential oil has eye irritation levels of 2.8 on a scale of 9 and thus, can be considered as a low irritant oil, since its value is within 1 and 5 of the scale. The study appeared to be valid according to the criteria specified in the international guidelines and the Saro essential oil did not show any mutagenic activity in the bacterial reverse mutation test with S. typhimurium. Values of the test reference quality control satisfied the standards and following the published J.O.R.F. classification Saro essential oil can now be stated as having a “negligible cytotoxicity”. Saro essential oil appears also interesting in terms of tolerance. However, high doses 1,8-cineole is epileptogen and is also an enzyme inductor of hepatic microsomes presenting when taken internally unknown interactions with other drugs (24, 25). Although some pharmacists recommend the use of similar essential oils (with 1,8-cineole like ravintsara) for young children and pregnant woman (26), we disagree. We do not recommend the use of Saro essential oil for children without medical advice, and this EO should not be given to pregnant or lactating women.

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Table III. Microbial activity of essential oil against bacteria and the application of Saro essential oil against potential human diseases. Bacterial strains


Alcalescens dispar Bacillus cereus Candida albicans

Saro activity ++ +++ ++

Cryptococcus neoformans Enterobacter cloacae Escherichia coli

+++

Gardenerella vaginalis Klebsiella pneumoniae

++ +++

Listeria ivanovii Listeria seeligeri Listeria welshimeri Neisseria gonorrhoe Pasteurella multicida Salmonella typhi Shigella sonnei Staphylococcus aureus (Cocci Gram+)

++ + +++ +++ ++ +++ +++

Streptococcus pneumoniae

+++

Trycophyton rubrum Trycosporon mucoides

+++ +++

+++ ++

Suggested uses for potential human diseases. Dysenteric syndrome Food borne illnesses Vaginal mycosis, perionyxis, skin infection. Skin and nails wounds, Gastro intestinal toxicity Urinary tract infections, meningitis, peritonitis, mastitis, septicemia, gastrointestinal toxicity and Gramnegative pneumonia. Vaginal infections Nosocomial infections, opportunistic pathogen : chronic pulmonary disease, enteric pathogenicity, nasal mucosa atrophy and rhinoscleroma. Gastrointestinal toxicity Gastrointestinal toxicity Gastrointestinal toxicity ORL infections, genitals infections. Gastro intestinal toxicity Typhoid Gastro intestinal toxicity Skin infections, abscesses, pneumonia, meningitis, endocarditis, toxic shock, syndrome, septicaemia. Skin infections, pneumonia, endocarditis, pericarditis, gastrointestinal toxicity, osteo-articulary infections Nail mycoses Intertrigos, onyxis

NOTE: Listed in alphabetical order by microbe. Results shown as =+++: high activity; ++: medium ; +: low ; -: without activity.

For gynecologic infection prevention or treatment, when it is not only used as an external pubian massage and taken internally, caution should be taken that it might affect the Doderlein flora, and natural beer yeast should be taken simultaneously to help regenerate the Doderlein flora (21). Tests conduced on Saro essential oil demonstrated powerful properties even when compared to the most interesting essential oil known in Aromatherapy.

492 The antimicrobial results suggest that the oil could have activities against vaginal infections, candidiasis, listeriasis, diarrhea, common infections and even animals diseases like pork pledge and chicken cholera.


Suggested Applications Saro essential oil showed high activity against a broad range of bacteria (Table III). The oil was particularly active against both gram positive (e.g. Staphylococcus aureus, Bacillus cereus, and Streptococcus pneumoniae) and negative bacteria (e.g. Shigella sonnei). The oil showed moderate activity against Escherichi coli, Salmonella typhi, Gardenerella vaginalis, Alcalescens dispar and Candida albicans. The oil showed low or no activity for different members of the genus Listeria (Table III). These results showed that the oil could have potential application as antimicrobial. Possible applications of Saro essential oil are vast considering its properties: antiviral, antibacterial, antimycosis, antiparasitic, immune modulant, analgesic, antispasmodic, among other uses. The antimicrobial properties might be related to the high content of 1,8-cineole in the Saro essential oil coupled with α-pinene as these compounds have expectorant and mucolytic properties (27). Saro’s use as a general tonic (immunomodulation) should increase the attractiveness for this use as well our finding showing that the Saro essential oil can be considered non-toxic, with the possibility of becoming an organic certified product. Application in gynecology might be relevant especially as art of a larger holistic approach to serious infections and in the prevention of recurrences. This essential oil could also be a good complement for preventive and curative treatments of HPV (Human papilloma virus) and HSV (herpes simplex virus). Saro essential oil uses could be effective for minor infections and especially to prevent them in topic application (27). The antalgic properties of Saro essential oil might also be useful in relaxing massage oils as they will be associated with its anti inflammatory properties (21, 27).

Conclusions Madagascan Saro essential oil was found to have an attractive odor with a fresh eucalyptus type aroma, being the oil dominated by 1,8-cineole. The oil was found to be safe for human use, with low irritation levels, no mutagenicity and cytotoxicity. This EO also showed antimicrobial activity against several mold and bacteria. Due to its favorable organoleptic and chemical profiles, its safety and effective microbial activity, this oil may have application in the cosmetic, perfumery and pharmaceutical industries. The identification of new uses and application of essential oils, can ultimately assist rural communities in Madagascar by generating interest and market access to their products only if these new applications find a sustainable market.

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