"PISDER AVAL 2011 4F 6 159-8 6H Journal of Chinese Integrative Medicine, June 2011, Val. + No.6 solg+ Original Experimental Research| Identification of antiplatelet and acetylcholinesterase inhibitory constituents in betel nut Muhammad Nabeel Ghayur', Syed Faraz Kazim’, Huma Rasheed’ . Asaad Khalid’, Maliha Iqbal Jumani' » Muhammad Iqbal Choudhary" , Anwarul Hassan Gilani 1. Department of Biological and Biomedical Sciences. Aga Khan University» Karechis Pakistan Department of Medizin St, Joseph's Healtheare Hanilton, MeMaster University, Hamilton, Canada 8, De. Panjvani Center for Molecular Medicine and Drug Research. University of Karachi. Karachiy Pakistan 4A. Medicinal end Aromatic Plants Rescerch Institutes National Center for Rescerchs Khartoum» Sudan Objective: To investigate the possible mechanism and the compound(s) responsible for the antiplatelet and acetylcholinesterase CACHE) inhibitory effects of Areca catechu crude extract (ac. Cn, Methods: Aqueous-methanol (70%) was used for extraction of plant material (betel nut) Antiplatelet activity was measured in human platelet-rich plasma by using a Lumi-aggregometer while anti-AChE activity was measured spectrophotometrically in vitro. In an attempt to find the responsible compound(s) in betel nut for antiplatelet and anti-AChE activities, different com- mercially available betel nut compounds were tested. Results: Ac. Cr inhibited platelet aggregation induced by arachidonic acid (AA), adenosine diphosphate (ADP), platelet-activating factor (PAF), epinephrine and Ca°* ionophore. Ac. Cr was the most potent in inhibiting ADP- and Ca°*-lonophore-induced aggregation. In the ACHE ‘assay. Ac.Cr showed significant AChE inhibitory activity with almost complete inhibition of the: ‘enzyme. Qut of the tested compounds, none of the compounds in betel nut showed ary antipiatelet effect except for catechin that was the most potent against epinephrine-induced aggregation Catachin was significantly less potent than Ac. Cr, indicating a presence of additional compound(s) with antiplatelet activity. For the ACHE inhibitory effect, only tannic acid, gallic acid, diosgenin and isoguvacine were found to be active, whereby tannic acid was more potent than Ac. Cr. Conelusion: This study shows the possible antiplatelet and ACHE inhibitory potential of betel nut while further studies are needed to confirm and identify more compounds in betel nut for these actions. Keywords: Areca catechu; plant extracts; platelet aggregation inhibitors; cholinesterase inhibitors catechins tannic acid Rens eign ecu cis DOK, 10,3736 /jeim20110607 Mohan Maruga Raja MK. Patel RS» Mishra SH. WEE €2 1808 #2 Ieps// ww. jeimijournal. com RO HAL RO RA HR. EEN HE, ZO 9), 105-108, Ghayur MN. Kazim SF. Rasheed Hy Khalid Ay Jumani| MI. platelet and acetylcholinesterase inhibitory constituents in betel nut, J Chin Integr Med, 20115 9(6) 619-625. Ghayur MN, Kazim SP, Rasheed Hy Khalid Ay Jumani MIL. Choudhary MI. Gilani AH. 48-7? #403 ‘BAM Z. BOR HE eh. BS AH gy | “wPRTarticleID- AEM, 20115 96), 619-628, Khuds-Bukhsh AR, Bhattacharyya SS, Paul S. Boujedaini N. RHA AC AS ACREAGE HE IN A ME BIE A RAL ASTS SELRGAV HEA. PUR AK AEH. 20104 8(9), 853-862. ‘Khude-Bukhsh AR, Bhattacharyya SS, Paul S, Boujedaini N. Polymeric nanoparticle encapsulation of naturally oceurring plant scopoletin and its effects on human melanoma cell A375. J Chin Integr Med. 2010; 8(9) + 853-862. Full text available at hrtp://www. jeimjournsl. com/FullTex2, aspxarticleID= jeim20100909 Mohan Maruga Rain MK, Patel RS, Mishra SH. Identification and quantification of adrensline from the leaves of Cleradendrum uudhary MI, Gilani AH, Identification of anti Phlomidis using thirlayer chromatography. J Chin Integr Med. 20115 9C1)« 105-108, Fulltext available at heep;//www. jeimjournal. com/FullTex?, im20110117 Received December 28+ 2010; accepted March 1, 2011 published online June 15. 2011. Fulltext LinkOut at PubMed, Journal ttle in PubMed: Zhong, Xi Yi Jie He Xue Bao. ‘SEMA: This study was supported by the grants from the Pakistan Science Foundation and the Higher Eilucation Commission of Pakistan Correspondence: Anwarul Hassan Tel; rail; anwar gilani@aku. edu | aspx?artileID=jeim20110007 « PhD, Professors | More related articles at hrip,//www. jeimjouenal, com/FullText2,+ 620+ SED FH 2001 6 JR 8-6 BH Journal of Chinese Integrative Medicines June 2011, Vol 9+ No.6 Betel nut is the seed of Areca catechu Linn (Arecaceae family), a tree found abundantly in South and Southeast Asia. Betel nut is chewed by people in these areas of Asia, and other parts of the world, to give a variety of physical and psychological effects such as an enhanced ability to work, heightened alertness, euphoria, a feeling of well-being and physiological effects on the cardiovascular, gastrointestinal, and pancreatic systems’, Scientific studies conducted on the nut have shown that it possesses anti-human immunodeficiency virus"!, hypoglycaemic! antioxidant!"!, antitumor'*', antischizophrenic!"!, angiotensin-converting enzyme inhibitory'’!, gastric prokinetic'"!, vasodilator, hypotensive, cardio- suppressant!” "!, analgesic, antiinflammatory!" and hepatoprotective!" properties. Apart trom some of these beneficial effects, chewing betel nut has also been shown to induce oral submucous fibrosis"!, oral cancers"), dependency syndrome! and antiovulatory and abortifacient effects!™! Among some of the beneficial effects of betel nut, it is known to cause a drop in blood pressure and heart rate®*"!, and known to help in memory retention'"), the reason why it is used by tradi- tional healers in South Asia for hypertension! and dementia!) In light of these medicinal uses, the authors have previously reported the cardio- protective"! and memory-protective” activi- ties of betel nut. In order to continue this research, the authors report here the antiplatelet and ace~ tylcholinesterase (AChE) inhibitory effects of betel nut. The antiplatelet potential was investi- gated in human blood against platelet aggregation induced by different agonists such as arachidonic acid (AA), adenosine diphosphate (ADP), platelet- activating factor (PAF), epinephrine and Ca°*- ionophore. This study also attempts to explore the possible mechanisms of antiplatelet effects and sereen some commercially-available constituents of betel nut!"!, such as arecoline, arccaidine, catechin, tannic acid, gallic acid, diosgenin, isoguvacine and guvacine, in order to discover the compound(s) responsible for the antiplatelet and ACHE inhibitory effects of betel nut. 1 Materials and methods 1.1 Chemicals AA, ADP, arecaidine, areco- line, acetylthiocholine (ATCh), Ca°* -ionophore (A23187), C+)-catechin, diosgenin, 5, 5-dithiobis (2-nitro) benzoic acid (DTNB), electric cel AChE (type W-S). epinephi acid, guvacine, isoguvacine, PAF, and tannic acid, were all purchased from Sigma Chemical Co. (St. Louis, MO, USA). Stock solutions of all the chemicals were prepared and the dilutions were made freshly in a suitable vehicle on the day of experiment. 1.2 Plant materials and extraction procedure Betel nut (dried variety, 1 kg) was bought from a market in Karachi, Pakistan and a sample of the material was identified and deposited at the Her- barium of the Department of Biological and Bio- medical Sciences, Aga Khan University, Karachi, Pakistan (voucher No. AC-SE-05-99-18).. ‘The plant material was cleaned of adulterants, crushed, and soaked in 2 L of 70% aqueous methanol at room temperature for a total of 3 d after which the fil- trate was collected through Whatman qualitative filter papers (Grade-1) and the plant material was, resoaked twice'"!, The combined filtrate was con- centrated in a rotary evaporator under reduced pressure at 40 © to yield a viscous, dark brown Areca catechu crude extract (Ac. Cr) weighing, 144 g (yield 14.4% w/w). This extract was stored at —4 C until use and dissolved in distilled water on the day of the experiment to prepare the stock solution and different dilutions 1.3 Preparation of human platelets and measure- rent of platelet aggregation All the experiments were performed in accordance with the ethical guidelines of the Ethical Review Committee of the Aga Khan University and by standards formu- lated in the Helsinki Declaration of 1964 (revised in 2004). Experiments were carried out as previ ously described'"**"!, Blood was drawn by vein puncture from human volunteers with a mean age of 21.1 years and of both genders, 8 (58%) men and 6 (42%) women. ‘The volunteers were healthy, with no known disease or sickness and free from medication for at least 7 d. Blood sam- ples were mixed with 3.8% (w/v) sodium citrate solution (9 + 1) and centrifuged at 260 g for 15 min at 20 C to obtain platelet-rich plasma (PRP). ‘The remaining portion of the blood sample was centrifuged at 1 790g for 5 min to obtain the platelet poor-plasma (PPP). Experiments were performed within 2 h of PRP preparation Platelet aggregation was monitored by using a ‘dual-channel lumi-aggregometer (Model 400 Chronolog Corporation, Chicago, IL, USA) using 0.45 mL aliquots of PRP. The light transmission was adjusted to 0 and 100% with PRP and PPP, respectively. Aggregation was induced by the addition of aggregatory agents such as AA (1.7 mmol/L), ADP (4.3 jtmol/L), PAF (8 nmol/L), epinephrine (20 mol/L) and Ca"* -ionophore (10 #mol/L) to the PRP. The antiplatelet effect of the test substance (betel nut extract/betel nut pure compounds) was studied by pretreating the PRP with the test substance for 1 min followed by addition of the aggregatory agonist. The resulting aggregation was recorded for 5 min via change in light trans- mission as a function of time. The antiaggregatory effects of the test substances were compared to acetylsalicylic acid, a standard antiplatelet agent, which showed its antiplatelet activity against AA- induced aggregation with an inhibitory concentra- tion of 50% (ICs) value of 0.03 mg/mL (95% confidence interval (CI): 0.03—0.03, n=5).‘ise EH 2011 46 6 1859 R56 Journal of Chinese Integrative Medicines June 2011, Val. 8+ No.6: sels 1.4 Enzyme assay for cholinesterase inhibition ‘The modified spectrophotometric method of Ellman et all! was followed. Electric ecl ACHE was used, while ATCh was used as the substrate of the reaction. DTNB was used for the measure- ment of the AChE activity. In this procedure 140 uL of 0.1 mmol/L sodium phosphate buffer (pH 8.0) along with 10 L of DTNB and 20 uL of the test substance solution were mixed and incubated for 15 min at 25 C. ‘The reaction was then initiated with the addi- tion of 10 #L of ATCh. The hydrolysis of ATCh was monitored by the formation of the yellow 5- thio-2-nitrobenzoate anion formed as a result of the reaction of DTNB with acetylthiocholine iodide (ATCI), released by the enzymatic hydrolysis of ATCI at a wavelength of 412 nm. The test substance and the control were dissolved in 5% ethanol. All the reactions were in triplicate, and the initial rates were measured as the rate of change in mean optical density per minute (mOD/ min) and used in subsequent calculation. Physo- stigmine was used as a standard ACRE inhibitor and it demonstrated an inhibitory effect on AChE at an Czy value of 0.04 g/mL (95% Cl: 0.04— 0.04; n=3) 1.5 Statistical analysis All the data were expressed as ¥+ 5, and the [Cp with 95% CI concentration- response curves was analyzed by non-linear regression (GraphPad program, GraphPad, San Diego, CA, USA). 2 Results 2.1 Effects of Ac.Cr on human platelet aggregation Ac. Cr inhibited platelet aggregation induced by different agonists in a concentration-dependent manner (0.07 to 2.00 mg/mL) (Figure 1A). The agonists used were AA, ADP, PAF, epinephrine ‘and Ca'*-ionophore while the IC. values for the (95% Cl: 1.673—2. 163, n=5), 1.677 mg/mL (95% Cl: 1.631—1.725, m=5) and 0.987 mg/mL, (95% Cl: 0,521—1.402, n=5), respectively. 2.2. Effects of betel nut pure compounds on human platelet aggregation Different constituents of betel nut, such as arecoline, arecaidine, catechin, tannic acid, gallic acid, diosgenin, isoguvacine and guvacine were tested for their antiplatelet effect against the different aggregatory agonists. Except for catechin, none of these compounds showed any antiplatelet effect up to the tested concentration of 10 mg/mL. Catechin demonstrated an antiplatelet effect (Figure 1B) on all the aggregatory agonists such as AA, ADP. PAF, epinephrine and Ca’*-ionophore at a concentration range of 1 to 5 mg/mL and with ICavalues of 3.630 mg/mL (95% Cl: 3. 560 — 3.701, n=5), 1.876 mg/mL (95% Cl: 1.676— 2.099, n=4), 2.551 mg/mL (95% Cl: 2.501 2,603, n=4), 1.541 mg/mL (95% Cl: 1.485 1.598, n=5) and 2.640 mg/mL (95% Cl: 2.568— 2.714, n=5), respectively. 2.3 Effeets of Ae. Cr on ACHE activity The extract exhibited a strong inhibition of AChE activity in vitro (see Table 1 for IC., value) in the AChE assay. This is in accordance with the authors’ earlier findings!"!. Ac. Cr showed a max- imum of (90. 1:0.4)% inhibition of the enzyme. 2.4 Bffeets of betel nut pure compounds on ACHE activity Different pure compounds of betel nut were tested for their AChE inhibitory effect. The compounds tested were: arecoline, arecaidine, catechin, tannic acid, gallic acid, diosgenin, isoguvacine and guvacine. The study found that all of these, except for arecoline, arecaidine, cat- echin and guvacine, showed considerable AChE inhibitory activity (see Table 1 for IC:. values) The efficacy shown by these compounds, in terms of maximum inhibition of AChE activity, was (55.745. 1)% (tannic acid), (69. 8+ 0. 8)9% ibitory effect of Ac. Cr were: 1.590 mg/mL. (gallic acid), (78. 3-+4.0)% (diosgenin) and (95% Cl: 1490-1. 695, n=6), 0.628 mg/mL (85. 0)% (isoguvacine), respectively. (95% Cl: 0.2991. 318, n=4), 1.902 mg/mL B 100 100 0 fe” ge 25 35 60 a2 50- = ae ZB 40). cote 28 25 2B | oe 20 Sg eto 0 OS Sr Log fAe-Cx] maint Log [Catecin} mgimL Figure 1 Effects of betel nat crude and pare compounds on human platelet aggregation Concentration response cures show the inhibitory fet ef (A) betel not 70% amucourmethanaic extant and (8) C+ )eaecin,agenst platelet aggreesion induced by agonists such e arachidonic acd (AA, 1.7 mmol/L), adenosine dshosshate CADP, 4-3 nna L) nlc fctvating lator (PAF, 8 nmcl/L. cpincphrine (20 pnl/L) and Ca?" lonophore (10 yamal/L.) inhuman venous Boal. The symbols represent 35 from 40 8 determinations end expressed tsa percentage of agonist-induced agaregntionSED FH 2001 6 JR 8-6 BH Journal of Chinese Integrative Medicines June 2011, Vol 9+ No.6 ‘Table 1 Comparative IC values for the inhibitory fect of ete nut extract and its constituents ‘on acetylcholinesterase Tet substance = Ga vale (g/m) Te Gr 3 BaD C805. 80) Arceline 2 Coot active) Arecaidine 3 Coot active) Catechin 3 oot active) Tani 2 0,10 @.01-0.19) Callies 347.10 (88.10-58.109 Diosgenin $ 198.60 ¢97.60—109.60) Isouvecine 3 20 (18.2018. 10) Guvacine 3 coor active) Tales represent ean along with 95% confidence icrvala parenthesis, Ip: effective concentration causing 60% inhibition Re-Crs Areca catecha crude extrot 3 Discussion ‘Ac. Cr showed concentration-dependent inhil tion of platelet aggregation induced in human platelets by different agonists such as AA, ADP, PAF, epinephrine and Ca®*-ionophore. The extract was more potent in its antiplatelet effect against aggregation induced by ADP and Ca"*- ionophore when compared with other agonists. Contrary to the findings here, Jeng et al'”! have shown that betel nut induces, rather than inhibits, platelet aggregation in rabbit blood. This discrep- ancy can be attributed to the difference in species, as has been shown in the past; the same plant com- ponents can have variable response in different species»! In human platelet membrane, AA serves as a precursor for thromboxane A: (TXA:) synthe- sis!) while PAF is also a potent stimulator of ‘TXA; production!”!, When synthesized or stimulated, ‘TXA; exerts its proaggregatory effect by binding to specific receptors, leading to aggregation by Ca®* influx". Epinephrine on the other hand interacts with a:-receptors in platelets which cor- relate with an increase in free cytosolic Ca") ADP is also known to elicit platelet aggregation via an increase in Ca°* influx'™!. The extract not only blocked ADP-mediated platelet aggregation but also inhibited aggregation induced by Ca°* -ionophore, possibly indicating interference with Ca" signal- ling. However, these are preliminary findings and more detailed studies using specific receptor and channel blockers are imperative for a defi tive conclusion detailing the mechanism of anti- platelet action of this extract To discover the compound(s) in betel nut that could be responsible for the antiplatelet effect of the parent extract, a number of commercially available betel nut compounds were tested. ‘The compounds tested were arecoline, arecaidine, catechin, tannic acid, gallic acid, diosgenin, isoguvacine and guvacine. None of these com- pounds showed any antiplatelet effects against the different aggregatory agonists except for catechin. ‘This result reiterates the earlier finding of anti platelet effect of this compound on AA-induced aggregation'"). Catechin, a flavonoid, inhibited the agonist-induced aggregation in the following order of potency: epinephrine > ADP > PAF Ca’ -ionophore> AA. It was generally less potent than the parent extract for its antiplatelet effect ‘This indicates that catechin might be only partially responsible for the antiplatelet effect of the extract. The antiplatelet role of other compounds in betel nut cannot be ruled out. An alternative explanation could be that the effect of catechin, when being presented in its natural composition, gets potentiated due to the presence of other possible chemical(s). The presence of synergistic combinations in the crude herbal products has already been documented”, The authors have earlier shown Ca®* inhibitory activity of catechin in gut, airway and vascular smooth muscle prepa- rations, This reported antiplatelet activity of betel nut, together with earlier reports of the cardio- suppressant, vasodilator and hypotensive effects of betel nut!" and vasodilator activity of its constituent catechin'” reiterates the possible presence of therapeutic constituents in this plant. However, this study is rather preliminary in scope, further pharmacological, chemical and clinical studies are required on betel nut to inves- tigate these findings in detail. This is recommended due to the fact that there are other studies done ‘on betel quid chewing (which involves chewing of betel nut along with lime and Piper betle leaf) which have shown this mixture to have detrimental effects on cardiovascular health!*""! ‘Ac. Cr was then evaluated for possible AChE tory effects. The extract showed a robust ACHE inhibitory effect. This AChE inhibitory effect, along with the known presence of cholin- ergic constituents in betel nut!””"!, possibly in cates its usefulness in memory disorders. AChE inhibitors and muscarinic agonists are the drugs of choice in such conditions'!. However. these are conclusions drawn upon enzymatic assays, which demand further investigations in detail In order to determine the probable compound(s) in betel nut for this AChE inhibitory effect, the effects of different commercially-available compounds of betel nut on ACHE were tested, namely: arecoline, arecaidine, catechin, tannic acid, gallic acid, diosgenin, isoguvacine and guvacine. Tannic acid was found to be more potent compared with the parent extract and all the other compounds (see Table 1 for comparison of ICs) values), indicating that it may be the principal compound with this, activity in betel nut. The four compounds were found active on the ACHE inhibition assay here (tannic acid, gallic acid, diosgenin and isoguvacine) and are being reported for their anti-AChE activity for the first time in the literature. Although arec-"PISDER AVAL 2011 4F 6 159-8 6H Journal of Chinese Integrative Medicine, June 2011, Val. + No.6 oline was found inactive in this ACHE assay, it has been shown in the past to improve memory in patients with Alzheimer disease'‘! and increase cognitive performance in primates‘! Recently in another study, arecoline was shown to have neurotoxic effects in rats through enhancement of oxidative stress!!, These contradictory findings again show the need for more comprehensive studies in this area In conclusion, this study shows the antiplatelet and anti-AChE effect of betel nut and some of its known compounds. The extract showed inhibition of platelet aggregation induced by all of the aggregatory agonist used in this study. More detailed studies are needed to ascertain the mech- anism of this antiplatelet effect. Catechin was found to be, at least, partially responsible for the antiplatelet effect while tannic acid, gallic acid, diosgenin and isoguvacine were possibly responsible for the anti-AChE activity. The results indicate the presence of therapeutic compounds in betel nut while further studies are needed to investigate the new pharmacological and therapeutic ques- tions that have arisen from this study. 4 Acknowledgements ‘This study was supported by grants made available to Professor Anwarul Hassan Gilani from the Pakistan Science Foundation and the Higher Edu- cation Commission of Pakistan. Dr. Asaad Khalid is grateful to Sudan Academy of Sciences (Khar- toum, Sudan) for the financial support. 5 Competing interests ‘The authors declare that they have no competing, interests REFERENCES 1 Chu NS. Elfects of betel chewing on the central and autonomic nervous systems. 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Aga Khan University, Karachie Pakistan 2) Department of Medicine, Se. Joseph's Hesltheare Hamilton, MeMaster University. Hamilton, Canada 5. De, Panjwani Center for Molecular Medicine and Drug Research, University of Karachi, Karachi. Pakistan 4A. Medicinal and Aromatic Plants Research Institute, National Center for Research+ Khartourns Sudan FLAY ESE BEEF (Areca catech uc) BARC AYP HH ts AR BZ HHA AR SE ALBUM aH ARI 70.96 BAC ATA AD a AT AL AL. GL A Ay a ARE AE AN A A PA FLA Ay A A Lb a A A A Ah A UAE AL. ALAR oF a A A Oy DML ADT ot HM HZ RF Sg LAE te 6 AP HE A AE a AE Sa A SL AS ART AR» Seat — eR WR AE BA BEE | Ry a ls RL FA HERERO NE, ERM Se ALR RA LG A RRA SHOE HAAN AES AT ROT MRR RRS PRR TT AA RRA TM TAH I RE NZ. A HE Oh I ET TR SAB I oh A ah HMR IL Zw EE AF PE SE hI PES. SORIA SHR AEs HA REMC Ah yA AR A PH A LIE Rs