a,cDepartment of Botany, Sri Parasakthi College for Women, Courtallam. India, bDepartment of Plant Sciences, Manonmaniam Sundarnar University, Tirunelveli, India
Received: 25 Dec 2017, Revised and Accepted: 05 Feb 2018
Objective: To Analyse the major phytochemical components in selected varieties of P. betle leaves using GC-MS.
Methods: P. betle leaves were shade dried and pulverized to powder in a mechanical grinder. The powder was successively extracted with ethanol (40-60 °C). The extracts were concentrated under reduced pressure in a rotary evaporator. The ethanolic extracts of the plant leaves were used for GC-MS analysis.
Results: In the present study ten chemicals were identified and compared from the P. betle varieties, the major component being Eugenol, which is responsible for the flavour and aroma of the leaves.
Conclusion: Variety Karpoori possesses the highest content of Eugenol and could be used as a promising variety in the pharmaceutical industry. The components of essential oil can also serve as a determinant to distinguish different varieties of betle vine cultivars.
Keywords: GC-MS, Eugenol, Hybrid variety 1, Hybrid variety 2, Jaipur Bangla, Karpoori, Local variety, Sirugamani
© 2018 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
P. betle is a tropical shade-loving perennial evergreen vine, with glossy heart-shaped leaves and white catkin. It may climb as high as 10-15ft. Sometimes the plant can also grow as a ground cover and it has a growth pattern similar to that of the pepper plant. Leaves are simple, alternate, ovate, cordate, acuminate or acute, entire and bright green. Flowers are dioecious. This plant has male spikes which are dense and cylindrical while female spikes are pendulous. Perianth 0. Stamens 2-4; filaments short; anthers 2 celled, the cells distinct. Ovary 1 celled; ovule solitary, style short or 0, stigma 5. Fruiting spikes up to 5 inches thick, male spikes 3-6 inches long very narrow. Roots arise from each node which aid in fixing the plant to the host tree [1, 2].
The leaf contains Water (85-90%), Proteins (3-3.5%), Carbohydrates (0.5-6.1%), Minerals (2.3-3.3%), Fat (0.4-1%), Fibre (2.3%), Essential oil (0.08-0.2%), Tannin (0.1-1.3%), Alkaloid (arakene). It also contains different Vitamins like Vitamin-C (0.005-0.01%), Nicotinic acid (0.63-0.89 mg/100 gms), Vitamin-A (1.9-2.9 mg/100 gms), Thiamine (10-70μg/100 gms), Riboflavin (1.9-30μg/100 gms). It also contains minerals such as Calcium (0.2-0.5%), Iron (0.005-0.007), Iodine (3.4μg/100 gms), Phosphorus (0.05-0.6%), Potassium (1.1-4.6%). Leaves contain bitter compounds that constitute about 0.7-2.6%. The specific strong pungent aromatic flavour in leaves is due to phenol and terpene like bodies . The total phenol content varies in the gender. The male plant contains three-fold higher total phenols content and two-fold higher thiocyanate content as compared to a female plant. The quality of the leaf depends upon the phenolic content, i.e., more the phenolic content better is the leaf quality . Recently many research works show that P. betle leaves contain starch, diastases, sugars and an essential oil composing of safrole, allyl pyrocatechol monoacetate, eugenol, terpinen-4-ol and eugenyl acetate, as the major components . Phytochemical investigation on leaves revealed the presence of Alkaloids, Carbohydrate, Amino acids, Tannins and Steroidal components . The middle part of the main vein contains the largest quantity of Tannin. The terpenoids include 1, 8-cineole, cadinene, camphene, caryophyllene, limonene, pinene, chavicol, allyl pyrocatechol, carvacrol, safrole, eugenol and chavibetol. Eugenol was identified as the antifungal principle in the oil. The fresh new leaves contain much more amount of essential oil diastase enzyme and sugar when compared to old leaves. Chavicol is four times potent as antiseptic agent as compare to carbolic acid .
In different countries, the leaves are folded in different ways and generally, some calcium hydroxide is smeared inside. Slices of the dry Areca nut are on the upper left of the leaf and slices of the tender Areca nut on the upper right. The fold on the lower right contains tobacco—a relatively recent introduction. There is archaeological evidence that P. betle leaves have been chewed along with the areca nut since very ancient times. In most countries, the mixture of both has a ceremonial and highly symbolical value . P. betle leaves are chewed together in the wrapped package along with the Areca nut and mineral slaked lime. Catechu called “Kattha” in Hindi and other flavouring substances and spices are also added. The lime acts to keep the active ingredient in its freebase or alkaline form, thus enabling it to enter the bloodstream via sublingual absorption. The Areca nut contains the alkaloid arecoline, which promotes salivation (the saliva is stained red), and is itself a stimulant. This combination, known as a P. betle quid has been used for over thousand years .
Properties of the ingredients and their traditional uses
P. betle leaf
P. betle leaves have a strong pungent aromatic flavour and is widely used as masticators. The presence of a fairly large quantity of diastase in the P. betle leaves is likely to play an important part in starch digestion. Large quantities of saliva produced by chewing P. betle leaf act as digestive and probably the presence of diastase enhance this activity. The leaves contain a good amount of B vitamins (particularly nicotinic acid) ascorbic acid and carotene.
P. betle leaf consists of more juice which cures pharyngitis, abdominal pain and abdominal distension. Ordinary P. betle leaf cures urticaria and the effects due to the derangement in the equilibrium of the three senses of humour namely Vatha, Pitha and Kabha. It gives a pleasant odour in the mouth. P. betle leaves possess an anti-oxidant action. The anti-oxidant effect is due to the presence of phenols particularly hydroxychavicol (4-allyl pyrocatechol). The leaf produces an aromatic volatile oil containing a phenol called chavicol which has powerful antiseptic properties. The essential oil present gives rise to a sensation of warmth and well-being in the mouth and stomach. It is also known to produce a primary stimulation of the central nervous system, followed by a kind of inebriety in large doses. P. betle leaf is believed as a common household remedy for various ailments.
A paste of P. betle leaves mixed with salt and hot water can be administered for filariasis.
For treating obesity one P. betle leaf mixed with Piper nigrum is prescribed for two months.
Juice with honey or a liquid extract is useful in coughs, dyspnoea, deranged phlegm and indigestion, common in children.
The application of leaves smeared with oil is said to promote secretion of milk when applied on the breasts of lactating women.
A local application is recommended for inflammatory swellings such as orchitis, arthritis and mastitis.
In pulmonary effections of childhood and old age, leaves are soaked in mustard oil and warmed and applied to the chest in order to relieve a cough and dyspnoea.
Chewing P. betle leaf with Areca nut which has pungent and astringent taste removes derangement of humors in the body. Germs will die; it removes thirst and phlegm, prevents several diseases, reduces hunger, endears women; improves taste and cleans the abdomen. They sweeten the breath, improve voice, and remove fetor from the mouth. Also, they increase salivary secretion. P. betle chewing is considered as a good and cheap source of dietary calcium. The calcium ingested is reported to be well absorbed by the system .
Source of plant material
The present investigation was carried out in six different varieties of P. betle viz., Hybrid I (HY1), Hybrid 2 (HY2), Jaipur Bangla (JB), Karpoori (KAR), Local variety (LV) and Sirugamani (SG). Plant cuttings were collected from Sugarcane Research station, Sirugamani, Tamil Nadu.
GC MS analysis
P. betle leaves were shade dried and pulverized to powder in a mechanical grinder. The powder was successively extracted with ethanol (40-60 ° C). The extracts were concentrated under reduced pressure in a rotary evaporator. The ethanolic extracts of the plant leaves were used for GC-MS analysis.
The GC-MS analysis was carried out on a Thermo GC-TRACE ultra ver: 5.0, Thermo MSDSQ II. This instrument was employed with following conditions: Column ZB 5-MS Capillary Standard non-polar Column (30 x0.25 mm), helium was used as carrier gas at a constant flow of 1 ml/min and an injection volume of 1.0 ml was employed in an injector with a temperature of 260 °C. The oven temperature was programmed from 70 °C raised gradually to 260 °C at the rate of 6 °C/min .
Gas chromatography-mass spectroscopy analysis (GC-MS)
Gas chromatography Mass Spectroscopy, a hyphenated system which is very a compatible technique and the most commonly used technique for the identification and quantification of secondary compounds. The unknown organic compounds in a complex mixture can be determined by interpretation and also by matching the spectra with reference spectra .
GC-MS analysis was carried out in the powdered leaf of P. betle varieties viz., HY1, HY2, JB, KP, LV and SG and the results are given in the table 1 and Figs (1 ‒ 12). In general P. betle leaf contains the phytocomponents viz., Hydroxychavicol, Eugenol, Safrole, Caryophyllene, Caryophyllene oxide, Silicone oil, Campesterol, Stigmasterol, Vitamin E and Sitosterol . HY 1 variety contains Hydroxychavicol, Eugenol, Safrole, Caryophyllene, Caryophyllene oxide, Silicone oil, Campesterol and Stigmasterol. Campesterol and Stigmasterol were absent in HY2. JB contains all the major compounds. Safrole, Silicone oil and Vitamin E were absent in Karpoori and Local Variety. Hydroxychavicol, Vitamin E and Sitosterol were absent in SG. Eugenol content is more in all the varieties and was highest in Karpoori (53%). Hydroxychavicol was highest in LV and absent in SG.
The leaves of P. betle possess a strong spicy aromatic flavour and widely consumed as a mouth freshener . The leaves are credited with wound healing, digestive and pancreatic lipase-stimulant activities in traditional medicines. The most important factor determining the aromatic value of the leaf is the amount and particularly the nature of essential oil present. P. betle leaves from different regions vary in aroma and taste. The oil content of different Indian types also varies. Table 4 gives the profile often important chemical constituents.
Table 1: Phytochemicals present Piper betle varieties analysed by GC-MS P
|S. No.||Compounds||Molecular formula||Molecular weight||Area %|
In the present study, ten chemicals were identified and compared from the P. betle varieties, the major component being Eugenol, which is responsible for the flavour and aroma of the leaves [15-17]. Among the selected varieties Karpoori possesses the highest content of Eugenol and could be used as a promising variety and form a valuable source of eugenol, which is widely used in pharmaceutical industry. Eugenol is a Phenolic compound and its content also varies with gender.  Pradhan et al. (2013) reported that male plants contain three-fold higher content of this phenolic compound as compared to the female. In the present study also higher Eugenol content was present in HY2 (female) compared to HY1 (male). The components of essential oil can also serve as a determinant to distinguish the gender and also different varieties of P. betle vine .
Chandra vikash et al. (2012)  reported the main constituents as Hydroxychavicol (HC)/Hydroxychavicol acetate (HCA), Allylpyrocatechol (APC), Chavibetol (CHV) and Piperbetol. Other constituents are arecoline, carvacrol, caryophyllene, piperitol, piperbetol, eugenol, isoeugenol, allylpyrocatechol, chavicol, safrole, anethole, chavibetol, cadinene-hydroxychavicol, β-sitosterol, β-sitosteryl palmitate, dotriacontanoic acid, tritriacontane, stearic acid, cepharadione, piperine, piperlonguminine, chavibetol acetate, allylpyrocatechol monoacetate, allyldiacetoxy benzene, benzene, estragole, methyl eugenol and hydroxycatechol, methylpiperbetol, piperol A, piperol B, cavacrol, eugenol acetate and allyl pyrocatechol diacetate. Leaves posses pharmacological properties like antibiotic, antiulcer and platelets aggregation antifertility, cardiotonic, antitumor, antimutagenic, respiratory depressant antihelminthetics which may be attributed to the presence of these chemicals.
Fig. 1-2: GC-MS chromatogram profile of hybrid 1(HY1)
Fig. 3-4: GC-MS chromatogram profile of hybrid 2 (HY2)
Fig. 5-6: GC-MS chromatogram profile of Jaipur Bangla (JB)
Fig. 7-8: GC-MS chromatogram profile of karpoori (KP)
Fig. 9-10: GC-MS chromatogram profile of local variety (LV) 9
Fig. 11-12: GC-MS Chromatogram profile of sirugumani (SG)
In the present study, ten major chemicals were identified and compared. Among the chemical compounds, the major component is Eugenol, which is responsible for flavour and aroma of the leaves. Variety Karpoori possesses the highest content of Eugenol and could be used as a promising variety in the pharmaceutical industry. The components of essential oil can also serve as a determinant to distinguish different varieties of betle vine cultivars.
All the author have contributed equally
Gamble JB. Flora of the presidency of madras II. Botanical Survey of India, Howrah, India; 1957.
Satish AB, DR Verma, RV Gavankar, C Nikhil, V Didwana, A Trikannad. Phytochemistry, pharmacological profile and therapeutic uses of Piper betle linn.-an overview. Res Rev: J Pharmacogn Phytochem 2013;1:10-9.
Bajpai V, D Sharma, B Kumar, KP Madhusudanan. Profiling of Piper betle Linn. cultivars by direct analysis in real time mass spectrometric technique. Biomed Chromatogr 2010;24:1283-6.
Bissa S, D Songara, A Bohra. Traditions in oral hygiene: chewing of betle (Piper betle L.) leaves. Curr Sci 2007;92:26-8.
Kanjwani DG, TP Marathe, SV Chiplunkar, SS Sathaye. Evaluation of immunomodulatory activity of methanolic extract of Piper betle. Scandinavian J Immunol 2008;67:589-93.
Sugumaran M, M Poornima, S Venkatraman, M Lakshmi, S. Sethuvani. Chemical composition and antimicrobial activity of sirugamani variety of Piper betle linn leaf oil. J Pharm Res 2011;4:3424-6.
Kumar N, P Mishra, A Dube, S Battacharya, K Dikshit, S Ranade. Piper betle Linn.-A maligned pan asiatic plant with an array of pharmacological activities and prospects for drug discovery. Curr Sci 2010;99:922-32.
Palaniappan G, A Sengottiyan, T Saravanan. Betle leaf: the green gold of India: facts for you. EFY enterprises PVT Ltd., New Delhi. India; 2012. p. 21–4.
Gupta PC, CS Ray. Epidemiology of betle quid usage. Ann Acad Med Singapore 2004;33:31-6.
Williams SA. Betle quid chewing: a community perspective. In: Betle-Quid Chewing Among Bangladeshi Community in the United Kingdom, London. R Bedi, P Jones. Eds. Centre for Transcultural Oral Health; 1995. p. 11-25.
Gopalakrishnan S, E Vadivel. GC-MS analysis of some bioactive constituents of Mussaenda frondosa Linn. Int J Pharma Bosci 2011;2:313-20.
Elizabeth Thomas A, T Aneesh, Della Grace Thomas,
R Anandan. GC-MS analysis of phytochemical compounds present in the rhizomes of Nervilia aragoana gaud. Asian J Pharm Clin Res 2013;6:68-74.
Chandra Vikash, Tripathi Shalini, NK Verma, DP Singh, SK Chaudhary, Roshan Asha. Piper Betle: phytochemistry traditional use and pharmacological activity-a review. IJPRD 2012;4:216-23.
Santhanam G, S Nagarajan. Wound healing activity of Curcuma aromatica and Piper betle. Fitoterapia 1990;61:458-9.
Rathee JS, BS Patro, S Mula, S Gamre, S Chattopadhyay. Antioxidant activity of Piper betle leaf extract and its constituents. J Agric Food Chem 2006;54:9046-54.
Mazura MP, N Hashim, RM Ali, Kiong, L Sui. Evaluation of Piper betle on platelet activating factor (PAF) receptor binding activities. Malaysian J Sci 2007;26:79-83.
Nalina T, ZHA Rahim. The crude aqueous extract of Piper betle L. and its antibacterial effect towards Streptococcus mutans. Am J Biotechnol Biochem 2007;3:10-5.
Pradhan D, KA Suri, DK Pradhan, P Biswasroy. Golden heart of nature: Piper betle L. Ixoreal biomed. Pvt. Ltd., APIIC, Green Industrial Park, Jadcherla, Mahaboobnagar, Hyderabad, Andhra Pradesh. India. J Pharmacogn Phytochem 2013;1:6.
Rawat AKS, RD Tripathi, AJ Khan, VR Balasubrahmanyam. Essential oil components as markers for identification of Piper betle L. cultivars. Biochem Systematic Ecol 1989;17:35-8.