Cytotoxic Activities of Fractions and Two Isolated Compounds from ...

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ScienceDirect Procedia Chemistry 13 (2014) 79 – 84

International Seminar on Natural Product Medicines, ISNPM 2012

Cytotoxic Activities of Fractions and Two Isolated Compounds from Sirih Merah (Indonesian red betel), Piper crocatum Ruiz& Pav. Emrizala*, Armon Fernandoa, Riska Yuliandaria, Kamal Rullaha, Nola Riska Indrayania, Adriani Susantya, Reni Yertia, Farediah Ahmadb, Hasnah M. Siratb and Dayar Arbainc a

School of Pharmacy, Sekolah Tinggi Ilmu Farmasi Riau, Pekanbaru, 28293, Riau, Indonesia b Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, Skudai, 81310, Johor Bahru, Malaysia c Faculty of Pharmacy, University of Andalas, 25163 Padang, West Sumatra, Indonesia

Abstract Cytotoxic Cytotoxic activity investigation using brine shrimp lethality test on fractions based on polarity of solvents have been done. Inhibition concentration values of three fractions of methanolic extract of sirih merah (Indonesian red betel), Piper crocatum Ruiz and Pav., showed IC50 values of 2.04; 1.34 and 2.08 μg/mL in n-hexane, ethyl acetate and butanol fractions, respectively. These results reveal that this plant is potential as antitumoral agent and also has a potential to be a candidate for the further investigation of cytotoxic compounds. Two isolated compounds, identified as β-sitosterol and 2-(5’,6’-dimethoxy-3’,4’methylenedioxyphenyl)-6-(3”,4”,5”-trimethoxyphenyl)-3,7-dioxabicyclo[3,3,0]octane have been isolated from this plant. Characterization of these compounds was carried out based on the NMR data and comparison with data from literature. © Published by Elsevier B.V. This ©2014 2014The TheAuthors. Authors. Published by Elsevier B.V.is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). Peer-review under responsibility of the School of Pharmacy, Bandung Institute of Technology. Peer-review under responsibility of the School of Pharmacy, Bandung Institute of Technology Keywords:Sirih merah, Piper crocatum, Piperaceae, Indonesian red betel, Cytotoxioc

* Corresponding author. Tel.: +62 85265070750; fax: +62 0761 58007. E-mail address: [email protected]

1876-6196 © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). Peer-review under responsibility of the School of Pharmacy, Bandung Institute of Technology doi:10.1016/j.proche.2014.12.009

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Emrizal et al. / Procedia Chemistry 13 (2014) 79 – 84

1. Introduction The utilization of plants as medicinal materials has become a heritage. In Indonesia, the raw medicine materials are found in abundance but unfortunately, only a small number of these plants have scientifically have been scientifically studied. It has been estimated that only 5-15 % of the estimated half million plant species in the world have been investigated and screened for its chemical and biological activities. It is clearly shown that traditional medicines offer a rich and largely unexplored source of therapeutic leads for pharmaceutical preparations1,2. Approximately, 80 % of populations in the world still depend on herbal and other naturally based medicines as sources for the treatment of various diseases and ailments. However, very little effort has been made to establish the scientific basis of traditional medicine and to develop the availability of the plants into useful and valuable pharmaceutical products3. The utilization of these plants initially took the form of crude drugs, such as tinctures, teas, poultices, powders, and other herbal formulations. The specific plants to be used and the methods of application for a particular ailment were passed down through oral history. In more recent history the use of plants as medicines has involved the isolation of the active compounds, beginning with the isolation of morphine from opium in the early 19 th century. Isolation and characterization of pharmacologically active compounds from medicinal plants continue until today4. A well-known objective for studying medicinal plants is the discovery of new bioactive components, in the search for promising drugs. The genus Piper has a great greatest diversity in geographical distribution with about 700 species in American tropic and nearly 340 species in Asian tropic including Indonesian tropical rainforests5. The chemistry of members of this genus is of great interest owing to the variety of biological properties. A structural diversity and bioactivity survey reveals that this genus specializes in the production of amides, phenylpropanoids, lignans and neolignans, benzoic acids and chromenes, alkaloids, polyketides, and a plethora of compounds of mixed biosynthetic origin6. In the family of Piperaceae, approximately, 677 different compounds have been isolated from 112 species of this family. They were found to be 190 alkaloids/amides, 49 lignans, 70 neolignans, 97 terpenes, 39 phenylpropanoid, 15 steroids, 18 kavapyrones, 17 chalcones/ dihydrochalcones, 16 flavones, 6 flavanones, 4 piperolides (cinnamylidone butenolides) and 146 miscellaneous compounds that do not fit into the common major groups of secondary metabolites7. In this paper, we report the extraction and fractionation of sirih merah (Indonesian red betel), Piper crocatum Ruiz & Pav., from West Sumatra, Indonesia, based on the polarity of solvents and also cytotoxic activities of the fractions using brine shrimp lethality test (BSLT). In Indonesia this plant is traditionally used as herbal medicine for the treatment of diabetic people and for prevention of coronary heart diseases, especially in Batusangkar, West Sumatra, where this plant is collected. Peoples consumed this plant by macerating a piece of leave in a glass of hot water and consume it twice a day. Based on our knowledge, there are no report on the isolation of pure compounds from this plant so far. 2. Experiments 2.1. Material Aerial parts of Piper crocatum Ruiz and Pav., (EM-06/12-2011) were collected from Batu Sangkar village, Kabupaten Tanah Datar, West Sumatra, Indonesia, about 100 km from Padang in March 2012. This plant was identified by Nurainas and a sample specimen was deposited at the Andalas Herbarium (ANDA), University of Andalas, Padang, Indonesia. Artemia salina encysted eggs (10 mg) were incubated in 100 ml seawater under artificial light at 28 °C, pH 7–8. After incubation for 24 h, nauplii were collected with a Pasteur pipette and kept for an additional 24 h under the same conditions to reach the metanauplii stage.


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2.2. Methods 2.2.1. Extraction method and fractionation based on polarity The dried and powdered whole plant of Piper crocatum Ruiz and Pav., (0.841 kg) were extracted at room temperature with MeOH to get the crude methanolic extract 253.27 g (30.1153%) after drying. The fractionation of this crude extract was based on the polarity of the solvent by using non polar (n-hexane), semi polar (ethyl acetate) and polar (butanol) solvents. Each of these fractions were concentrated under reduced pressure using a rotary evaporator to afford a green gum of n-hexane 24.22 g (9.56 %), a green gum of ethyl acetate 53.17 g (20.99 %) and a brown gum of butanol 61.23 g (24.18 %) fractions. 2.2.2. Phytochemical investigations All fractions were submitted to secondary metabolite analysis8, 9. The hexane and ethyl acetate fractions showed positive test for terpenoids and steroids, respectively, while the methanolic and butanolic fractions gave positive test for phenolic, flavonoid, terpenoid and steroid compounds. 2.2.3. Purification using multiple column chromatographic methods Fractions giving a good separation by the thin layer chromatography (TLC) were purified using multiple column chromatographic methods with silica gel 60 as stationary phase and n-hexane; a combination of n-hexane : ethyl acetate; ethyl acetate: methanol solvents as mobile phase, based on the step gradient polarity. These chromatographies yielded fractions which were monitored by TLC to evaluate the separation profiles. 2.2.4. Purification of n-hexane fraction Purification of the n-hexane fraction with SiO2 column chromatography using a polarity step gradient yielded 650 fractions which were monitored on a TLC plate to evaluate the separation profiles of compounds. These TLC profiles gave 14 combined fractions which were labelled as PC1 to PC14. From these fractions, there were two fractions that showed good separation of compounds. These two fractions were further purified by recrystallization to yield two pure compounds. The first compound was a steroid and the second compound was a lignan. These compounds were characterized and identified based on the physical properties and spectroscopic analyses. 2.2.5. Evaluation of cytotoxicity activity A general bioassay that appears capable of detecting a broad spectrum of bioactivities present in crude extracts is the brine shrimp lethality bioassay (BSLT). The technique is easily mastered, costs little, and utilizes small amounts of the test material10. The samples (triplicate) to be assayed were dissolved in DMSO (dimethylsulfoxide) (2 mg/400 μl or 2 mg/1000 μl) and diluted serially (10, 20, 30 and 50 μl/5 ml) in seawater. About 10–20 nauplii were added to each set of tubes containing the samples. Controls containing 50 μl of DMSO in seawater were included in each experiment. Twenty-four hours later, the number of survivors was counted, recorded and the lethal concentration 50% (LC50 value) was calculated by Probit analysis11, 12. 2.2.6. Statistical analysis Statistical analyses were performed using Sigma Plot 8.0. Data were presented as means of standard error of triplicate samples.

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3. Results and Discussion The investigation of cytotoxic activity using the brine shrimp lethality test to detect cytotoxic fractions from a Piperaceae plant, Piper crocatum Ruiz and Pav showed variation of their cytotoxic activity. Inhibition concentration values of three fractions of the crude methanolic extract of sirih merah (Indonesian red betel), showed IC50 values of 2.04; 1.34, 2.08 and 27.40 μg/mL in n-hexane, ethyl acetate, butanol fractions, and methanolic extract, respectively. These results showed a good brine shrimp larvicidal activity according to literature10, who classified crude extracts and pure substances into toxic (LC50 value < 1000 μg/ml) and non-toxic (LC50 value > 1000 μg/ml). Phytochemical investigation of n-hexane and EtOAc fractionsshowed positive test results for terpenoid and steroid, while butanolic fractions showed positive test results for phenolic, flavonoid, terpenoid and steroid compounds. LC50 values for the extract and all fractions proved to be the most active larvicidal plant. These results reveal that this plant has a potential as antitumoral agent and also has a potential to be a candidate for the further investigation of cytotoxic compounds. Table 1. LC50 values of the crude extracts and fractions from the leaves of Piper crocatum Ruiz and Pav., by The brine shrimp lethality test Sampel

LC50 ± SD (μg/mL)

methanolic extract

27.40 ± 2.6

n-hexane fraction

2.04 ± 1.1

ethyl acetate fraction

1.34 ± 3.2

butanolic fraction

2.08 ± 0.2

Lapachol (positive control)

12.30 ± 0.3

Chromatographic fractionation of the n-hexane fraction of this plant gave two pure isolated compounds. The first compound was characterized and identified as β-sitosterol,thiscompound yielded 89.12 mg as white crystalline needles with m.p. 66-67 oC; Rf value is 0.46 in n-hexane-EtOAc (4:1); IR (KBr) νmax cm-1: 3435 (O-H), 2966 (C-H sp3), 1052 (C-O); 1H NMR (CDCl3, 300 MHz): δ 0.68 (3H, s, H-18); 0.77 (3H, d, J = 6.6 Hz, H-27); 0.81 (3H, d, J = 6.6 Hz, H-26); 0.86 (3H, t, J = 3.9 Hz, H-29); 0.92 (3H, d, J = 6.3 Hz, H-21); 1.00 (3H, s, H-19); 1.06-2.34 (22H, m, 11 x CH2); 3.53 (1H, m, H-3); dan 5.35 (1H, d, J = 4.8 Hz, H-6); 13C NMR (CDCl3, 75 MHz): δ 11.61 (C-29); 12.20 (C-18); 18.62 (C-21); 18.83 (C-27); 19.22 (C-19); 19.81 (C-26); 21.05 (C-11); 22.91 (C-28); 24.03 (C-15); 25.82 (C-23); 28.01 (C-16); 28.91 (C-25); 31.43 (C-2); 31.70 (C-8); 32.10 (C-7); 33.87 (C-22); 36.01 (C-20); 36.44 (C10); 37.06 (C-1); 39.51 (C-12); 42.22 (C-4); 42.60 (C-13); 45.65 (C-24); 50.01 (C-9); 55.92 (C-17); 56.53 (C-14); 71.72 (C-3); 121.70 (C-6); dan 138.23 (C-5); EIMS (% rel int): m/z 414 (78), [M+] (C29H50O), 396 (52), 382 (29), 329 (26), 303 (39), 273 (34), 255 (68), 231 (27), 213 (46), 199 (22), 159 (73), 145 (81), 81 (100). The identification of this compound was based on its physical properties and comparison with NMR spectral data from literature13. 29 28 21 18

22 20

25 26

19

HO

27

24

3 6

Fig. 1. β-Sitosterol.


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The second compound was identified as 2-(5’,6’-dimethoxy-3’,4’-methylenedioxyphenyl)-6-(3”,4”,5”trimethoxyphenyl)-3,7-dioxa-bicyclo[3,3,0]octane as a white crystalline solid as 18.9 mg with R f value of 0.32 in nhexane-EtOAc (1:1); IR (KBr) νmax cm-1; 3030 (CH sp2), 2920 (C-H sp3), dan 1560 (C=C); 1H NMR (CDCl3, 300 MHz): δ 6.28 (1H, s), 6.19 (1H, s), 5.85 (1H, q), 5.37 (1H, s), 5.23 (2H, dd), 3.80 (9H, s, 3-OCH3), 3.71 (3H, s, OCH3), 3.30 (3H, s, -OCH3), 3.35 (1H, d, J = 8 Hz), 2.49 (2H, m), 2.35 (2H, m), 2.28 (3H, s, CH 3), 1.61 (12H, s, 4CH3), dan 1.30 (3H, d, J =3.3 Hz); 13C NMR (CDCl3, 75 MHz): δ 169.35; 152.85; 152.84; 132.98; 133.72; 124.42; 106.04; 106.04; 94.71; 78.71; 60.68; 59.64; 55.86; 55.86; 55.39; 55.02; 48.75; 48.02; 35.06; 20.99; and 17.23. This compound was identified and characterised based on its physical properties and also comparison of its NMR spectral data with literature14. These pure isolated compounds were reported in this paper for the first time from Indonesian red betel leaves, Piper crocatum Ruiz and Pav., Piperaceae.

Fig. 2. 2-(5’,6’-dimethoxy-3’,4’-methylenedioxyphenyl)-6-(3”,4”,5”-trimethoxyphenyl)-3,7-dioxabicyclo[3,3,0]octane

4. Conclusion Inhibition concentration values of three fractions of the crude methanolic extract of sirih merah (Indonesian red betel), showed IC50 values of 2.04; 1.34, 2.08 and 27.40 μg/mL in n-hexane, ethyl acetate, butanol fractions, and methanolic extract, respectively. These results showed a good brine shrimp larvicidal activity according to literature10, LC50 values for extract and all fractions proved to be the most active larvicidal plant. These results reveal that this plant has a potential as antitumoral agent. Two compounds have been isolated from this plant and indetified based on literature data and spectroscopic analyses, as β-sitosterol and 2-(5’,6’-dimethoxy-3’,4’methylenedioxyphenyl)-6-(3”,4”,5”-trimethoxyphenyl)-dioxabicyclo[3,3,0]octane. Acknowledgement Authors are grateful acknowledges to The Directorate General of Higher Education, Ministry of Education and Culture, Republic of Indonesia for financial supported (Fundamental Scheme 2012-2013, No. Scheme: 160/010/KU/2012 and 34/Kontrak/010/KM/2013). Thanks are also due to Nurainas from Andalas Herbarium (ANDA), University of Andalas, Padang, Indonesia, for plant identification. References 1. Darise, M. and Yamasaki, K. Chemical Constituents of Some Indonesian Medicine Plants. Proceeding of the Eighth Asian Symposium on Medicinal Plants, Spices and Other Natural Products (ASOMPS VIII). 1994 June 13-16; Melaka (Malaysia). Malaysia: The Malaysian Natural Products Society; 1996.p. 219-30. 2. Heinrich, M. Ethnopharmacy and Natural Products Research-Multidisciplinary Opportunities for Research in the Metabolomic Age. Phytochem Letts 2008, 1: 1-5.

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