STUDIES ON PIPER BETLE OF SRI LANKA

More t h a n twe l ve cult ivars o f Piper betle are reported i n Sri Lanka bu t no chemical studies have been carried o u t o n t h e m . T h e present s t u d y describes t h e morphological, physic0 chemical, chemical and antimicrobial activities o f six main cultivars o f P. betle Linn namely Galdalu, Malzamaneru, K u d a m a n e r u , R a t a d a l u , Nagawa l l i and Malabulath. T h e chemical consti tuents identified i n t h e essential oil o f Malabulath by Gas Chromatography-Mass Spectrometry (GC-MS) were dif ferent t o those from t h e other c u l t i v a r s . T h e m a j o r compound i n M a l a b u l a t h w a s allylpyrocatechol diacetate while t ha t i n all other cultivars categor ized a s c o m m o n b e t e l w a s sa f ro l e . C h e m i c a l compositions o f t h e essential oil o f t h e lea f , s talk, s tem, fruit and root were dif ferent. T h e major compounds i n t h e lea f , Gampaha, Kalutara , Kurunegala, Kegalle, Ratnapura, Matale and Galle are the main betel cultivating districts in the country. In Sri Lanka, more than twelve cultivars of betel have been recognized by the villagers but no systematic nomenclature and scientific classification of these cultivars are recorded. The objectives of the present study are to identify the different cultivars of betel found in Sri Lanka using morphological characters and chemical properties of the oil.(to assist a scientific classification of the different P. betle Linn. cultivars found in Sri Lanka). In most s tem, stalk and root oil was safrole bu t i n t h e fruit i t was POf the the constituents depend on the phellandrene. T h e composition o f t h e oil also varied w i th maturity and the part analysed. Hence in the matur i ty . T h e essential oil from common betel was active agains t Escherichia coli ( N C T C 101481, Pseudomonas present study, the chemical constituents of the aeruginosa (NCTC 106621, Staphylococcus eprdermidis oils from different parts of the plant (NCTC 4276), Staphylococcus aureus ( NCTC 8532 ), and a t different maturi ty levels were also Streptococcus pyrogens. T h e essential oil and t h e ethanol investigated. extract were also active against Cladosporium sp.

Gampaha, Kalutara, Kurunegala, Kegalle, Ratnapura, Matale and Galle are the main betel cultivating districts in the country. In Sri Lanka, more than twelve cultivars of betel have been recognized by the villagers but no systematic nomenclature and scientific classification of these cultivars are recorded. The objectives of the present study are to identify the different cultivars of betel found in Sri Lanka using morphological characters and chemical properties of the oil.(to assist a scientific classification of the different P. betle Linn. cultivars found in Sri Lanka). In most stem, stalk and root oil was safrole b u t i n t h e fruit it w a s P- Of

METHODS AND MATERIALS
Plant material : P. betle plant materials were collected from Narammala, Panduwasnuwara, Thissawa, Thabbomulla, Walpitagama, Udagama (Kurunegala district), Wadinapaha, Divulapitiya, Dewalapola (Gampaha district), Karagoda, Bibullawella, Uyangoda (Matara district) and Hiyara east, Akmeemana, Yakkalamulla, Paradaradeniya (Galle district). Six cultivars of P. betle namely G a l d a l u , M a h a m a n e r u , K u d a m a n e r u , R a t a d a l u , N a g a w a l l i a n d Malabulath were used in the present study. From 5-6 plants of each type 6-8 wk old leaves (250 g) from the sixth to eight position from the apex of the vine were collected along with some plagiotrophic branches. The roots and the fruits from the same plant were also collected. The samples were identified by the third author.
Morphological studies: Morphological observations regarding colour and size were recorded for each of the leaf samples collected. Internodal distances of the plagiotrophic branches of each sample were measured.
Study of the anatomy: Cross sections of the stem and leaf of each of the samples were obtained and stained. These cross sections were mounted and observed under the microscope. The microscopic photographs were taken and used for comparative study. Similarly microscopic cross sections of the leaves were photographed and studied.
Five betel leaves were selected from each sample. From each leaf the lower epidermal peels were trimmed from the leaf lamina between the tip and the base and from half-way between the margin and the mid rib. The lamina peels of each were examined under the microscope using grid eye piece. The stomata1 indices were calculated using the Salisburgs formula.
Determination of the moisture content: The leaves (5.11 g ) were dried in an oven at 105 O C for 2 h, cooled and weighed. The procedure was repeated till a constant weight was obtained. The determination was done in triplicate. Similarly the moisture contents in the stem, the stalk, the fruit and t h e root of common betel were determined.
Determination of the essential oil content : Fresh leaves (251.9 g ), roots (160.7 g ), stems (175.8 g ), stalks (245.9 g), and fruits (176.7 g ) obtained from each cultivar except from the cultivar identified as Malabulath were cut in to small pieces and the essential oil extracted separately as follows. In the case of Malabulath only the leaves were used for the extraction. The cut plant materials were separately water distilled for 6 h using a Clevenger oil arm fitted with condensers through which cooled water was circulated to prevent low volatiles from escaping. The volatile oil was collected in hexane-pentane mixture. The yield was calculated on a dry weight basis. Determinations were carried out in triplicate.
Determination of the physical properties of the essential oil: Refractive index of the oil was measured using an Abbe Atogo IT refractometer. The temperature was maintained at 23 O C using a thermostat controlled water circulator. Specific gravity of the oil was measured using a specific gravity bottle.
Preparation o f the ethanol extract: Fresh leaf samples ( 20.8 g) were cut into small pieces and refluxed with ethanol (400 ml) using a Soxhlet extractor for 8 h. The extract was evaporated under reduced pressure and a dark brown mass ( 0.96 g ) was obtained.
Determination of the chemical constituents of the essential oil : The volatile oil extracted from each of the betel cultivars was subjected to Gas Liquid Chromatography (GLC) analysis using Shimadzu GC -14 B equipped with F I detector and Supelcowax TM-10 fused silica capillary column. Retention d a t a a n d peak enhancement techniques were used for the identification of compounds. The GLCs of cultivars Galdalu, M a h a m a n e r u , K u d a m a n e r u , R a t a d a l u and Nagawalli were similar and they were referred to as common betel. The volatile oil from the leaves, stalks, stems, fruits and roots of common betel and from the leaf of Malabulath were subjected to the GC-MS using Hewlett-Packard 5890 Series I1 equipped with FI detector and DB-5 MS capillary column. The volatile oils of young and mature P. betle leaves of common betel were also analysed .
Antibacterial screening: In the antibacterial studies cultivars G a l d a l u , M a h a m a n e r u , Kudamaneru, R a t a d a l u and Nagawalli were classified as common betel. The leaf oil and the ethanol extract obtained from the common cultivars of betel were used for the antibacterial studies. The antibacterial activity was studied by the 'Disk diffusion method'" using Escherichia coli       Antifungal study: The antifungal activity was studied by the Bioautographic TLC Assay7 using Cladosporium sp. The experiment was conducted in triplicate.

RESULTS AND DISCUSSION
According to morphological a n d anatomical studies the parameters such as stomata1 index and leaf length to width ratio were similar in Kudamaneru, Mahamaneru, Galdalu R a t a d a l u and Nagawalli b u t different i n Malabulath. (Table 1&2). The physical parameters of the essential oil ( Table 3) and constituents of the esse~tial oils of Kudamaneru, Mahamaneru, Galdalu, RatadaEu and Nagawalli too were similar but were different to those of Malabulath. Safrole was detected as the major compound in the leaf oil of common betel but Allyl pyrocatechol diacetate was the major compound in the leaf oil of Malabulath. Hence the results show that the cultivars of betel analysed in the present study fall into two chemically and morphologically different groups namely Malabulath and common betel. Common betel includes Kudamaneru, Mahamaneru, Galdalu, Ratadalu and Nagawalli. The appearance of Nagawalli leaves differed from the other cultivars. Therefore it can be concluded that chemotaxanomically there are two different groups of betel in Sri Lanka namely Malabulath and the other referred to as common betel.
Over 100 cultivars of betel have been identified by farmers and traders in India. The chemical studies on P. betle of India have revealed that composition of the volatile oils in the leaves can be used as markers for the identification of different cultivarss. The chemical composition of common betel oil of Sri Lanka appears to be closer to that of cultivar, Deshawari in India.g GC-MS analysis of oil of Malabulath and common betel leaves indicated that the major constituents of common betel oil are safrole (48.69 %) a n d chavibitol acetate (12.55%) while Malabulath does not contain these two compounds ( Table 5). The major compound in Malabulath oil is allylpyrocatechol diacetate (34.01%) which is the third major compound in common betel oil (11.34%). Furtherp-cymene ,4-terpineol, safrole, eugenol, P-caryophellene and chavibitol acetate detected in common betel leaves were not detected in Malabulath.
Hence in the present study GC-MS and microbiological studies were carried out considering the cultivars Kudamaneru, Mahamaneru, Ratadalu, Galdalu and Nagawalli as one group and referred to as common betel.
The GC-MS analysis of the essential oil of different parts of common betel (Table 5) indicated that the composition of the stalk was different to that of the other parts. The stalk did not contain detectable amounts of allylpyrocatechol diacetate. The major compound detected in the oil from the leaf, the stem, the stalk a.nd the root was safrole but in the fruit, it was 0phellandrene. Table 6 indicates that the composition of the oil also differs according to the stage of maturity of the leaf. It was observed that the major compounds safrole (48.69%) and chavibitol acetate (12.55%) content in the leafwas maximum at harvesting stage. Further it is seen from table 6 that eugenol and Pphellandrene content decreased with maturity. It was observed that 0phellandrene content remained constant after maturity. Allyl pyrocatechol diacetate content increased up to harvesting stage and remained constant thereafter.
The change in the composition of the oil with matui-ity and the part of the plant explains why the ayurvedic physicians specify the maturity of the plant and part of the plant in drug preparations.
The antimicrobial activity of local P. betel leaf oil and the ethanol extract has not

5.
a, 3 2 cd U1 been studied previously. In the antibacterial study the essential oil from the common betel leaves showed activity against Escherichia coli, Streptococcus pyogenes and Staphylococcus aureus. The MIC values were 3.12 x lo2, 2.50 x lo3, and 5.00 x lo3 respectively. The ethanol extracts showed high activity against Streptococcus pyogenes, Escherichia coli and Staphylococcus aureus. The MIC values were 1.25x103, 5.00x103 and 5.00x103re~pectively (Table  8 ). This antibiotic activity can be related to the Antifungal activity of P. betle on C l a d o s p o r i u m sp. has not been previously reported. The antifungal activity against Cladosporium sp. indicates that the essential oil of P. betle possess a t least three fungicidal compounds having R, 0.54,0.40,0.11. The ethanol extract contained a t least one fungicidal compound having R, 0.61 on silica gel TLC plates. These studies will be useful in the development of value added products from betel.