ANTIOXIDANT, CYTOTOXIC AND PHYTOCHEMICAL PROPERTIES OF THE ETHANOL EXTRACT OF PIPER BETLE LEAF

ANTIOXIDANT, CYTOTOXIC AND PHYTOCHEMICAL PROPERTIES OF THE ETHANOL EXTRACT OF PIPER BETLE LEAF

Md. Forhad Uddin ^*1, Sheikh Aftab Uddin ², Md. Delwar Hossain ¹ and Mohammed Abul Manchur ¹

Department of Microbiology ¹, Faculty of Marine Science and Fisheries ², University of Chittagong, Chittagong-4331, Bangladesh

ABSTRACT: The use of natural products for the treatment of various diseases has a long history. In the present study, the anti-oxidative, cytotoxic and phytochemical properties of ethanol extract of a local variety of Piper betle leaves cultivated in Potia, Bangladesh has been investigated. The antioxidant and cytotoxic activity of the Piper betle ethanol extract were evaluated by DPPH (1, 1-diphenyl-2-picrylhydrazyl) free radical scavenging method and brine shrimp lethality bioassay method, respectively. Preliminary phytochemical group tests were also done to investigate the phytochemical properties of the extract. Leaf extract showed significant DPPH free radical scavenging effect compared to standard antioxidant ascorbic acid. IC₅₀ value of ascorbic acid and leaf extract was found 1.81μg/ml and 151.36μg/ml respectively. In brine shrimp lethality bioassay LC₅₀ value of Piper betle ethanol extract was found 274.63862µg/ml with 95% confidence limit where the lower and upper limits were 198.39 µg/ml and 387.18µg/ml respectively, which indicates that the Piper betle leaf extract has promising cytotoxic effect. Phytochemical analysis was found to be positive for alkaloid, glycosides, terpenoids, flavonoids, tannins and saponins. The present study demonstrates that ethanol extract of Piper betle Linn. Leaf has significant antioxidant and cytotoxic effect.

Piper betle, Antioxidant, Cytotoxic, Phytochemical Screening, DPPH

INTRODUCTION: Piper betle commonly called betel leaf is a climbing shrub or small tree indigenous to tropical Asia, Australasia, and the Pacific and grown mostly in Bangladesh, India, China, Bhutan, and Malaysia ^1-3. The betel plant is an evergreen and perennial creeper, with glossy heart-shaped leaves and white catkin. The medicinal properties of betel leaf are well known since time immemorial. Traditionally the leaves are used to treat various diseases like halitosis, boils and abscesses, conjunctivitis, constipation, swelling of gums, cuts and injuries. The essential oil contained in the leaves is known to possess anti-bacterial, anti-protozoan and anti-fungal properties

Antioxidants are type of molecules that neutralize harmful free radicals, produced through a chain of reactions ⁴ that damage living cells, spoil foods; degrade materials such as rubber, gasoline, lubricating oil. Antioxidants terminate these chain reactions through the removal of free radical intermediates and inhibition of other oxidation reactions ⁵. This is why plants and animals maintain complex systems of multiple antioxidants, such as glutathione, vitamin C, and vitamin E along with some enzymes like catalase, superoxide dismutase and various peroxidases.

The use of antioxidants in pharmacology is intensively studied as oxidative stress might be an important part of many human diseases particularly stroke and neurodegenerative incidents. Antioxidants, therefore, are routinely added to meals, oils, foodstuffs, and other materials to prevent free radical damage. Recently there has been an upsurge of interest in the therapeutic potentials of medicinal plants as antioxidants in reducing such free radical induced tissue injury. A lot of new plant species have been investigated in the search for novel antioxidants ^6-9 other than well known and traditionally used natural antioxidants from tea, wine, fruits, vegetables and spices ¹⁰ but there is still a demand to find more information on the antioxidant potential of plant species. Cytotoxicity usually gives a preliminary idea on the anticancer effect of plant extract containing bioactive compounds those are toxic to living body at higher doses and pharmacologically beneficial at lower doses.

Scientists have been proving that all the natural things are not good for health. Different retrospective studies done over the last 20 years indicated that the incidence of deaths occurring due to exposure to plants (as a proportion of total patients poisoned by traditional plant medicine) was about 1.5% in France, 5% in Belgium, 6.5% in Italy, 7.2% in Switzerland and 6% in Turkey ¹¹. There is, therefore, a need to have an understanding of the risks posed by herbal medicines so as to ensure that such products could be used safely. However, study on cytotoxicity of plant extract could give us an idea of the anticancer as well as toxic profile of studied plant extract.

MATERIALS AND METHODS:

Collection of plant leaves:

Fresh plant leaves of Piper betle were collected from the Potia, Chittagong, Bangladesh. The plant leaves were collected in the sterilized polythene bag. The polythene bags were sterilized with 95% ethanol. Then the bags were properly tied and labeled. After that the collected samples were brought to the laboratory.

Preparation of leaf extract:

The fresh Piper betle leaves were washed with distilled water, air dried at room temperature for about 10 days, ground into powder (500 gm) and extracted with ethanol, being stirred and macerated at room temperature (23±5⁰C) for 15 days. The ethanol was evaporated under reduced pressure below 50⁰C through rotary vacuum evaporator (Bibby RE200, Sterlin Ltd., England). The concentrated extract (52 gm blackish-green) was stored at 4⁰C for further use.

In vitro assay for antioxidant activity of leaf extract:

The antioxidant activity of Piper betle extract was assessed in comparison to standard antioxidant ascorbic acid (BDH, England) depending on the scavenging effect of 1, 1-diphenyl- 2- picrylhydrazyl (DPPH)-free radical. The whole procedure was administrated according to established procedure by Braca et al.^19-20. Ascorbic acid solution (5 ml) and different concentrations of extract (20, 40, 60, 80, 100, 200, 400 and 800μg/ml in methanol) solutions (5 ml) were mixed with 3 ml of 0.4 mM (0.004 %) DPPH solution. The mixtures were kept in dark for 30 minutes to measure the absorbance at 517 nm using UV-Visible Spectrophotometer (UV-1601 Shimadzu, Kyoto, Japan) and ascorbic acid was used as a positive control. Lower absorbance of the reaction mixture indicated higher free radical-scavenging activity. The degree of decolorization of DPPH from purple to yellow indicated the scavenging efficiency of the extract. The scavenging activity against DPPH was calculated using the following equation:

Scavenging activity (%) = [(A - B) / A] x 100

Where,

A is absorbance of control (DPPH solution without the sample),

B is the absorbance of DPPH solution in the presence of the sample (extract/ ascorbic acid).

The scavenging activity (%) or % inhibition was then plotted against log concentration and from the graph IC₅₀ (Inhibition concentration 50) value was calculated by linear regression analysis ²¹.

Assay for cytotoxicity of leaf extract:

Cytotoxic activity of plant extract was determined by Brine Shrimp lethality bioassay as described by Meyer et al. ²². Shrimp eggs were added to the artificial “sea water” (38 gm sea salt pure Sodium chloride weighed, dissolved in 1 liter of distilled water adjusted to pH 8.5 using 1N NaOH and was filtered off to get clear solution) in the larger compartment of an unequally dividend tank which was darkened by covering it with Aluminum foil ²³. The chamber was kept under illumination using a table lamp for 48 hrs for the eggs to hatch into shrimp larvae. The illuminated compartment attracts shrimp larvae (nauplii) through perforations in the dam. 10 shrimp larvae were added to 10 ml of sea water in 10 test tubes and 1000, 800, 600, 400, 200, 100, 80, 60, 40 and 20μg/ml solutions of extracts, prepared from 10 mg/ml of crude through serial dilution, were added to these test tubes. Each concentration was tested in triplicate. A control containing 10 ml of DMSO solvent was used for each solvent.

The test tubes were maintained under illumination. After 24 hours have elapsed, survivors were counted with the aid of a 3X magnifying glass. From the % lethality of brine shrimp, the probits were calculated for each concentration by using computer software “BioStat-2006”. Probits were then plotted against corresponding log concentration of leaf extract to get LC₅₀ (lethal concentration 50) value through regression analysis.

Phytochemical analysis:

Test for Alkaloids:

To  the  extract  added  1%  HCl  and  6  drops  of  Mayer’s  reagent  and  Dragendroff’s reagent. An organic precipitate indicated the presence of alkaloids in the sample ²⁴.

Test for Flavonoids:

5 ml of dilute ammonia solution were added to a portion of aqueous filtrate of each plant extract followed by addition of conc. H₂SO₄. A yellow coloration is observed which confirms the presence of flavonoids and it disappears on standing ²⁵.

Test of glycosides:

Dissolve small amount of an alcoholic extract of the fresh or dried material in one ml of water. Add a few drops of aqueous NaOH solution. Yellow color indicates the presence of glycoside ²⁵.

Test for Steroids:

2 ml of acetic anhydride was added to 0.5gm of ethanolic extract of each sample with 2 ml of H₂SO₄. The color change from violet to blue or green indicated the presence of steroids ²⁶.

Test for Tannins:

5 ml of extract was added to few drops of 1% lead acetate. A yellow precipitate indicated the presence of tannins ²⁶.

Test for Terpenoids:

5 ml  of  each  extract  was  added  to  2 ml  of  chloroform  and  3 ml  of  conc.  H₂SO₄ to form a monolayer of reddish brown coloration of the interface was showed to form positive result for the terpenoids ²⁵.

Test for Saponins:

The  extract  with  20 ml  of  distilled  water  was  agitated  in  a  graduated  cylinder  for  15  minutes.  The formation of 1cm layer of foam indicated the presence of saponins ²⁴.

RESULTS AND DISCUSSION:

Assay for anti-oxidative activity of Piper betle leaf extract:

The DPPH free radical scavenging activity of the Piper betle leaf extract and ascorbic acid is shown in Table 1.

TABLE 1: DPPH FREE RADICAL SCAVENGING ACTIVITY OF ASCORBIC ACID AND PIPER BETLE LEAF EXTRACT

Both ascorbic acid and Piper betle ethanol extract showed dose dependent activity. Among the eight different concentrations used in the study (20, 40, 60, 80, 100, 200, 400 and 800µg/ml) ascorbic acid showed 69.541289%, 74.12844%, 78.89908%, 82.38532%, 86.788996%, 92.11009%, 96.69724%, 98.34862% scavenging activity where highest  scavenging activity was 98.34862% at concentration 800µg/ml. On the other hand, Piper betle ethanol extract showed 11.81619%, 20.78774%, 26.69584%, 31.50984%, 37.41794%, 56.67396%, 71.99124%, and 87.52735% scavenging activity at the above mentioned eight different concentrations where highest scavenging activity of Piper betle ethanol extract was 87.52735% at concentration 800µg/ml. % of scavenging activity or % of inhibition was plotted against log concentration and from the graph IC₅₀ (Inhibition concentration 50) value was calculated by linear regression analysis. IC₅₀ value of ascorbic acid and Piper betle ethanol extract was found 1.81µg/ml and 151.36 μg/ml, respectively (Fig.1).

FIG.1: COMPARATIVE IC₅₀ VALUES OF REFERENCE ANTIOXIDANT AND STUDIED PLANT PIPER BETLE LEAF EXTRACT: (A) ASCORBIC ACID (B) PIPER BETLE.

The IC₅₀ value obtained for Piper betle extract and ascorbic acid (Fig.1) indicates that Piper betle leaf extract possess efficiency to neutralize free radicals higher than ascorbic acid (Fig. 2).

FIG. 2: COMPARATIVE % SCAVENGING ACTIVITIES OF PIPER BETLE LEAF EXTRACT AND ASCORBIC ACID.

Different research suggests that most of the plant extracts showing antioxidant activity are due to the presence of phenolic compounds ^27-28. Phenolic natural compounds such as flavonoids (Table 4) possess antioxidant activity due to their redox properties which allow them to act as reducing agents and singlet oxygen quencher. In addition, they have metal chelating potentials. The phenolic compounds, identified in the extract might contribute to the antioxidant activity of Piper betle leaf extract.

Assay for cytotoxicity of Piper betle leaf extract:

Percentage lethality of brine shrimp at ten different concentrations (20 to 1000µg/ml) of Piper betle ethanol extract is presented in Table 2. Plant extract showed lethality in a dose dependent manner ²⁹. More specifically 0, 0, 10, 10, 20, 40, 50, 70, 90 and 100% mortality of brine shrimp was observed at 20, 40, 60, 80 100, 200, 400, 600, 800 and 1000µg/ml concentrations respectively (Table 2).

From the % lethality of brine shrimp, the probits were calculated for each concentration by using computer program “BioStat-2006”. Response (%) or lethality (%) was then plotted against corresponding log concentration of plant extract. From this plot, LC₅₀ (lethal concentration 50) value was found by regression analysis using computer program “BioStat-2006”. LC₅₀ value of Piper betle  ethanol extract was found 274.63862µg/ml with 95% confidence limit where the lower and upper limits were 198.39 and 387.18µg/ml respectively (Table 3 and Fig.3).

TABLE 2: BRINE SHRIMP CYTOTOXICITY OF PIPER BETLE LEAF ETHANOL EXTRACT

*ProbitY were calculated using statistical software “Biostat 2006”

*Actual % = Actual formulas (n is the number of animals in a group): For the 0% dead, 100 (0.25/n), for the 100% dead, 100 (n- .25) /n.

TABLE 3: CALCULATION OF LC₅₀ VALUE, REGRESSION EQUATION, CONFIDENCE LIMIT AND CHI SQUARE BY PROBIT ANALYSIS.

FIG.3: REGRESSION LINE FOR DETERMINING THE LC₅₀ VALUE OF ETHANOL EXTRACT OF PIPER BETLE

Preliminary Phytochemical Screening:

The present study carried out on the plant samples revealed the presence of medicinally active metabolites. The phytochemical characters of both the plants investigated are summarized in the Table 4 given below.

TABLE 4: OBSERVATION ON PHYTOCHEMICAL GROUP TESTS

N.B. “++” stands for the presence and “--” indicates the absence of secondary metabolites.

Qualitative secondary metabolite tests (Table 4) showed that Piper betle possess alkaloid, terpenoids, steroids and flavonoids. Plants produce a huge variety of secondary compounds as natural protection against microbial and insect attack. Many of these compounds have been used in the form of whole plants or plant extracts for food or medical applications in man because plants are the natural reservoir of many antimicrobial, anticancer agents, analgesics, anti-diarrheal, antifungal as well as various therapeutic activities ³⁰. Presence of such alkaloid and flavonoids might have some role in showing antioxidant and cytotoxic properties of the Piper betle.

CONCLUSIONS: The results of the study demonstrate that the ethanol extract of Piper betle leaf exhibits very potential antioxidant and cytotoxic effect in experimental models which support the claims by traditional medicine practitioners. These results can be strong scientific evidence to use this plant variety as a useful source of antioxidant references. However, further studies are still necessary to elucidate a mechanistic way how the plant contributes in these pharmacologic properties. Phytochemical investigation in order to isolate the active fraction and eventually the pure compound will be a future study.

ACKNOWLEDGEMENTS: The authors thank Associate Professor Dr. Shaikh Bokhtear Uddin (Department of Botany, University of Chittagong, Bangladesh) for identification of the plant.

REFERENCES:

1. Yusuf M, Wahab MA, Chowdhury JW, Jaripa B: Medicinal Plants of Bangladesh. BCSIR Laboratory Press, Chittagong, Bangladesh, 1994:72-73.
2. Ghani A: Medicinal Plants of Bangladesh. The Asiatic Society of Bangladesh. Dhaka, Bangladesh 2003; 181: 502-504.
3. Rahman MA: Indigenous knowledge of herbal medicines in Bangladesh. Treatment of skin diseases by tribal communities of the hill tract districts. Bangladesh Journal of Botany 2010; 39: 169-177.

4. Joseph NM, Sabharwal M, Shashi A, Mahor A, Rawal S: In vitro and in-vivo models for antioxidant activity evaluation - a review. International Journal of Pharmaceutical Sciences Review and Research 2010; 1 (1): 01-11.
5. Sies H: Oxidative stress: oxidants and antioxidants. Experimental Physiology 1997; 82 (2): 291–295.
6. Chu YH, Chang CL, Hsu HF: Flavonoid content of several vegetables and their antioxidant activity. Journal of the Science of Food and Agriculture 2000; 80: 561-566.
7. Koleva H, Van Beek TA, Linssen JPH, de Groot A, Evstatieva LN: Screening of plant extracts for antioxidant activity: a comparative study on three testing methods. Phytochemical Analysis 2002; 13 (1): 08-17.
8. Mantle D, Eddeb F, Pickering AT: Comparison of relative antioxidant activities of British medicinal plant species in vitro. Journal of Ethnopharmacology 2000; 72 (1-2): 47-51.
9. Oke JM, Hamburger MO: Screening of some Nigerian medicinal plants for antioxidant activity using 2, 2- diphenyl- picryl- hydrazyl radical. African Journal of Biomedical Research 2002; 5 (1-2): 77-79.
10. Schuler P: Natural antioxidants exploited commercially, In Food. Antioxidants, Hudson BJF (ed.), Elsevier: London 1990: 99‐
11. Gaillard Y, Pepin G: Bioactive compounds from northern plants. Journal of Chromatography 1999; 733: 181.
12. Abrahim AA, Kanthimathi MS and Aziz AA: Piper betle shows antioxidant activities, inhibits MCF-7 cell proliferation and increases activities of catalase and superoxide dismutase. BMC Complementary and Alternative Medicine 2012; 12: 220.
13. Wang S, Meckling KA, Marcone MF, Kakuda Y, Tsao R. Can phytochemical antioxidant rich foods act as anti-cancer agents? Food Research International 2011; 44: 2545-2554.
14. Mahfuzul Hoque M, Rattila S, Asaduzzaman Shishir M, Bari M L, Inatsu Y, Kawamoto S. Antibacterial Activity of Ethanol Extract of Betel Leaf (Piper betle L.) Against Some Food Borne Pathogens. Bangladesh Journal of Microbiology 2011; 28(2): 58-63.
15. Kumar N, Misra P and Dube A. Piper betle a maligned Pan-Asiatic plant with an array of pharmacological activities and prospects for drug discovery. Current Science 2010; 99: 922-932.
16. Sarkar A, Sen R, Ganguli S, Mandal G and Chatterjee M: An Ethanolic extract of leaves of Piper betle (paan) Linn. Mediates its antileishmanial activity via apoptosis. Parasitology Research 2008; 102: 1249-1255.
17. Chakraborty D and Shah B. Antimicrobial, Antioxidative and Antihaemolytic Activity of Piper betel leaf extracts. International Journal of Pharmacy and Pharmaceutical Sciences 2011; 3: 192-198.
18. Roy UB and Vijayalaxmi KK: Evaluation of Cytotoxic Activity of Piper betle Linn. Using Murine and Human Cell Lines in Vitro. International Journal of Scientific & Engineering Research 2013; 4(9): 221-233.
19. Braca A, De Tommasi N, Di Bari L, Pizza C, Politi M, Morelli I: Antioxidant principles from Bauhinia tarapotensis. Journal of Natural Products 2000; 64 (7): 892‐
20. Brand-William W, Cuvelier ME, Berset C: Use of a free radical method to evaluate antioxidant activity. LWT Food Science and Technology 1995; 28 (1): 25‐
21. Emran TB, Rahman MA, Hosen SMZ, Khanam UH, Saha D: Antioxidant, cytotoxic and phytochemical properties of the ethanol extract of Leea indica Journal of Pharmacy Research 2012; 5 (5): 2938-2941.
22. Meyer BN, Ferrigni NR, Putnam JE, Jacobson LB, Nichols DE, McLaughlin JL: Brine shrimp: a convenient general bioassay for active plant constituents. Planta Medica 1982; 45 (5): 31-34.
23. Persoone G, Kumar A, Ilavarasn R, Jayachandran T, Decaraman M, Aravindhan P, Padmanaban N, Krishnan M, Artemia Salina 3:428 (Universa Press, Witteren, Belgium,       1980).
24. Rice-Evans CA, Miller NJ, Paganga G: Antioxidant properties of phenolic compounds. Trends in Plant Science 1997; 4: 152-159.
25. Ayoola GA, Coker HAB, Adesegun SA, Adepoju-Bello AA, Obaweya K, Ezennia EC, Atangbayila TO: Phytochemical Screening  and  Antioxidant  Activities  of  Some            Selected  Medicinal  Plants Used  for Malaria Therapy in Southwestern Nigeria. Tropical Journal of Pharmaceutical Research 2008; 7 (3): 1019-1024.
26. Edeoga HA, Okwu DE, Mbaebie BO: Phytochemical constituent of some Nigerian Medicinal Plants. African Journal of Biotechnology 2005; 4: 685-688.
27. Ramarathnam N, Ochi H, Takeuchi M: Antioxidant Defense System in Vegetable Extracts. In: Shahidi F. (ed.) Natural Antioxidants: Chemistry, Health Effects and Applications, Aocs Press, Champaign, IL, USA (1997).
28. Vertuani S, Angusti A, Manfredini S: The antioxidants and pro‐antioxidants network: An overview. Current Pharmaceutical Design 2004; 10 (14): 1677‐
29. Zhao G, Hui Y, Rupprecht JK, McLaughlin JL, Wood KV: Additional bioactive compounds and trilobacin, a novel highly cytotoxic acetogenin from the bark of Asimina triloba. Journal of Natural Products 1992; 55 (3): 347-356.
30. Nakanishi K: Recent studies on bioactive compounds from plants. Journal of Natural Products 1982; 45 (1): 15-26.
31. Gupta S, Kumar N and Gupta SM: Antibacterial and antifungal activity in the extract and oil of Piper betle (Linn.) landrace Bangla Mahoba. Advanced Zoology 2009; 31:16-20.
32. Abdulelah HA, Zurainee MN, Hesham MA, Adel AA, Rohela Mahmud. Antimalarial activity of methanolic leaf extract of piper betle L. Molecules 2011; 16: 107-18.
33. Agarwal T, Singh R, Shukla AD, Waris I, Gujrati A. Comparative analysis of antibacterial activity of four Piper betle varieties. Advances in Applied Science Research 2012; 3(2): 698-705.
34. Satish AB, Deepa RV, Rohan VG, Nikhil CT, Yatin YR, Vinodkumar SD and Ashwin T. Phytochemistry, pharmacological profile and therapeutic uses of piper betle linn–an   overview Research Revision. Journal of pharmacognosy & phytochemistry 2013; 1(2): 10-19
    

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How to cite this article:

Uddin F, Uddin SA, Md. Hossain D and Md. Manchur A: Antioxidant, Cytotoxic and Phytochemical Properties of the Ethanol Extract of Piper Betle Leaf. Int J Pharm Sci Res 2015; 6(10): 4252-58.doi: 10.13040/IJPSR.0975-8232.6(10).4252-58.

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