Drug discovery from Ardisia elliptica · 2015. 6. 18. · Drug discovery from Ardisia elliptica Lee...

Volume 4, Issue 1, Jan 2012 | 73

Drug discovery from Ardisia ellipticaLee Jun Feng

Ardisia elliptica is a tropical sub-shrub native to Singapore. The plant is used in traditional herbal therapy to treat parturition

complications (pain and discomfort associated with childbirth) and chest pains. This project aims to identify potent antiplatelet and anticoagulant compounds which could be used in the prevention �� ȋ��ǡ� �� stroke and heart attack) from the leaf extract of A. elliptica for possible drug development in the future. Leaves of A. elliptica were boiled in 70% methanol by volume in water in a soxhlet to obtain an extract. The extract was dried and reconstituted �� ȋ��Ȍ�and High Performance Liquid Chromatography (HPLC). An active compound !-amyrin was isolated using HPLC. !-amyrin, isolated from the leaf extract, was found to possess antiplatelet properties while the leaf extract showed both antiplatelet and anticoagulant properties in vitro in human blood and in vivo� �� ȋ��Ȍ�rats. This suggests the possibility of more potent compounds with antiplatelet and anticoagulant properties awaiting discovery in the leaves of A. elliptica. Both !-amyrin and the leaf extract ��ǡ��potent antithrombotic activity (a thrombus is �� ȌǤ�!-amyrin constitutes 0.098% by mass in the fresh leaves of A. elliptica as determined by Liquid Chromatography Mass Spectrometry (LCMS).

Cardiovascular Diseases are the number one cause of death globally (WHO 2010). Patients with CVDs related to thromboembolism are treated with antiplatelet drugs such as aspirin (Patrono et al. 2001) and anticoagulant drugs such as warfarin (Levine et al. 1995) to decrease the risk of recurrent cardiac arrest and stroke. However, there are adverse e!ects associated with current drugs such as gastrointestinal bleeding for aspirin and haemorrhage for warfarin , which prompt for a continuous search for new drugs with good e"cacy and reduced adverse e!ects. Medicinal plants, with their wealth of compounds and proven e"cacies, provide a good source of leads to begin the search.

Ardisia elliptica #unberg is a tropical evergreen sub-shrub that is native to China, Malaysia, Singapore and Sri Lanka. #e leaves and roots of this plant have been used traditionally in Southeast Asian herbal

remedies. #e leaves are boiled and the extract is drunk to treat chest pains, parturition complications, fever, diarrhoea and liver poisoning (WHO 2010). #e leaves of A. elliptica have been reported to show platelet-activating factor (PAF) receptor-binding antagonist activity (Jalil et al. 2004). #is implies that bioactive compounds could be present in the leaves to prevent the activation of platelets. Platelets which are not activated will not aggregate to form internal clots in the blood vessels. #is would help prevent CVDs.

Platelets are the main cellular components of white thrombi (clots), which tend to form in the arterial system. In contrast, red blood cells predominate in red thrombi, which form in veins. #e thrombi, or internal clots, are believed to be responsible for strokes and heart attacks due to their interruption of blood $ow downstream in the blood vessels which are narrower. #e interruption of blood $ow to the tiny vessels in the brain leads to stroke as tissues in the brain draw nutrients from the blood and the lack of nutrients leads to permanent damage of a!ected regions of the brain. Similarly, small vessels, named coronary arteries, that supply heart muscles with nutrients can be clogged by thrombi and this leads to chest pains or cardiac arrests (heart attacks). Antiplatelet drugs reduce the formation of white thrombi predominantly while anticoagulant drugs reduce the formation of red thrombi. #erefore, it is essential that potential drugs are tested for both antiplatelet and anticoagulant activities separately. #is would ensure that the most e!ective drug is prescribed for patients su!ering from atherosclerosis according to the di!erent types of thrombi formed.

Platelet aggregation is part of the sequence of events leading to the formation of a clot. It refers to the clumping together of platelets in the blood which usually precedes blood coagulation. Coagulation is a process that converts soluble %brinogen into insoluble %brin threads, turning blood into an insoluble gel in the process. #e gel plugs leaks in blood vessels and stops the loss of blood.

#is study uses in vitro, in vivo and ex vivo bioassays. In vitro (in-glass) bioassays are conducted with drug samples added to human blood in a glass tube; in vivo (in the living body) bioassays are conducted with drug samples fed to SD rats followed by the cutting of the tail to measure bleeding times; and ex vivo (out of the living body) bioassays are conducted with blood, drawn into a glass tube, from animals fed with the drug.

Objective#e main objective of this paper is to investigate

the potential antiplatelet and anticoagulant e!ects of ȕ-amyrin isolated from the leaves of A. elliptica. #e speci%c objectives are: to carry out preliminary

USP Undergraduate Journal | 74

identi!cation of components in the leaf extract using GCMS and HPLC; to isolate, purify and quantitate the active compound ȕ-amyrin in the leaf extract using HPLC and LCMS; to ascertain the antiplatelet and anticoagulant e"ects of ȕ-amyrin in vitro in human blood and ex vivo in SD rats; and to ascertain the e"ects of ȕ-amyrin on tail-bleeding time in vivo in SD rats.

Materials and MethodsLeaves of A. elliptica were obtained from Pasir

Ris Park with permission from the National Parks, Singapore. #e identity of the leaves was con!rmed by the Singapore Botanical Gardens and a voucher specimen is kept in the Department of Pharmacy at the National University of Singapore. #e leaf extract was prepared using soxhlet extraction with 70% methanol for six hours.

Chemical analyses of the leaf extract were done using GCMS, with a Shimadzu system consisting of a gc2010 gas chromatograph and a gp2010 mass spectrometer, and HPLC, with an Agilent 1100 series HPLC system.

An IBM workstation with GCMS solution software Version 2.10 Su2 (Shimadzu Corporation) was used for analysis of the data. A DB-5 MS column of internal diameter 0.25 mm, length 30.0 m and !lm thickness 0.25 µm was used. An auto-library search using the Wiley mass spectral database Version 7 (Wiley, USA) was made for analysing the chromatograms. #e ion source and interface temperatures were kept at 200 °C and 180 °C respectively. Samples were injected under a splitless mode with split ratio -1.0. Each injection volume was 1 µl and an injection port dwell time of 0.3 s was set. #e pressure of the Helium carrier gas was set at 100 kPa with a total $ow of 50.0 ml min-1. #e column $ow was 1.61 ml min-1 and the linear velocity was 46.3 cm s-1. Data recording was set from 5 to 81 min for 50 to 900 m/z.

An analytical HPLC system (Agilent 1100 series, CA, USA) and a Zorbax Eclipse XDB-C18 column (250 mm x 4.6 mm I.D., particle size: 5 µm; Agilent , CA , USA ) were used for the analysis of the leaf extract. #e separation of the compounds was achieved using a binary gradient elution system consisting of water and acetonitrile at the following gradients: 0–30 min, 70–95% acetonitrile; 30–40 min, 95–100% acetonitrile; 40–100 min, 100% acetonitrile. #e injection volume was 5 µl, $ow rate 1.5 ml min-1, run time was 100 min and UV (Ultraviolet) detection was set at 210 nm. #e chromatogram was compared against that obtained from another run of the same method, replacing the leaf extract with ȕ-amyrin standard (Extrasynthese, Genay, France) to ascertain the presence of ȕ-amyrin

in the leaf extract.

#is was followed by a quantitation of ȕ-amyrin in the leaf extract according to the International Conference of Harmonisation guidelines using an API 2000 triple-quadrupole mass spectrometer by Applied Biosystems. #e HPLC system consisted of an Agilent 1100 binary pump equipped with an Agilent 1100 autosampler injector with 100 ml loop (Agilent, Germany). Chromatographic separations were achieved using Zorbax Eclipse XDB-C18 column (250 mm x 4.6 mm I.D., particle size: 5 µm; Agilent , CA , USA ) with a mobile phase consisting of 100% HPLC grade methanol (Fisher Chemical, Fisher Scienti!c, USA). Flow rate was set at 1 ml min-1. #e leaf extract reconstituted in HPLC grade methanol at 500 ppm and standard solutions were auto-injected (20 µl) into the HPLC system and directed to the mass spectrometer source. LCMS experiments were performed using an API 2000 triple-quadrupole mass spectrometer (Applied Biosystems, MDS SCIEX, Ontario, Canada). #e instrument was operated in Atmospheric Pressure Chemical Ionization (APCI), Multiple Reaction Monitoring (MRM) and positive-ion detection mode, with a dwell time of 250 ms for data collection.

ȕ-amyrin was isolated from the leaf extract using preparative HPLC and the ȕ-amyrin isolate was puri!ed thereafter using semi-preparative HPLC on an Agilent 1100 series HPLC system. #e leaf extract which was reconstituted in HPLC grade methanol was fractionated using a preparative HPLC system (Agilent 1100 series, CA, USA) and a preparative Zorbax Eclipse XDB-C18 column (250 mm x 21.2 mm I.D., particle size: 7 µm; Agilent, CA, USA) using methanol as the mobile phase. #e injection volume was 2 ml, $ow rate was 20 m1/min and run time was 40 min. #e fraction collected was further puri!ed using a semi-preparative Zorbax SB-C18 column (250 mm x 9.4 mm I.D., particle size: 5 µm; Agilent , CA , USA ) using methanol as the mobile phase. #e injection volume was 1 ml, $ow rate was 8 ml/min and run time was 12 min. #e UV detection for both preparative and semi-preparative HPLC was set at 210 nm.

#e isolated ȕ-amyrin was used in in vitro bioassays on human blood. #ese bioassays consisted of platelet aggregation assays with a whole blood aggregometer from Chronolog Corporation and plasma coagulation assays with an automated blood coagulation analyser CA-500 series from Sysmex.

ȕ-amyrin isolated from the leaf extract (5 mg) was dissolved in HPLC grade methanol and added to 5 ml of Bovine Serum Albumin (BSA, 0.1 g ml-1). Each addition was limited to no more than 500 µl in volume. #e mixture was homogenised with a vortex before evaporation of the methanol was done using


a rotary evaporator. ! e total volume was kept in check by keeping the suspension in a 5 ml standard " ask throughout the process of amyrin-in-methanol addition and further evaporation which was done repeatedly as necessary until a suspension of 1 mg ml-1 of ȕ-amyrin in BSA was obtained. ! e same process was repeated with the leaf extract to obtain a suspension of 12 mg ml-1 in BSA and aspirin to obtain a suspension of 5 mg ml-1 in BSA.

! e blood aggregation assays were carried out by measurement of impedance using a whole blood aggregometer (Chronolog Corporation, Havertown, PA). Whole blood (450 µl) was added to an equal volume of Phosphate Bu# ered Solution (PBS) pre-warmed to 37 °C and stirred at a constant rate of 1200 rpm. ! e blood was allowed to equilibrate for a min before adding 100 µl of the test sample (leaf extract, aspirin or ȕ-amyrin in BSA). BSA (0.1 g ml-1) was used as control in place of the test sample with the same volume of 100 µl added in a separate test. After incubating for another 2 min, 2 µl of collagen (Chronolog Corporation, Havertown, PA) of concentration 1 mg ml-1 was added. ! e test was allowed to run for 5 min before the impedance was recorded.

Plasma coagulation assays were conducted using Lyophilised human plasma (DADE, Ci-Trol, coagulation control 1) purchased from Dade Behring, Marburg, Germany. After the human plasma was reconstituted with distilled water according to the manufacturer’s instructions, 225 µl of the reconstituted plasma was added to 25 µl of each test sample (leaf extract, aspirin or ȕ-amyrin in BSA).

! e isolated ȕ-amyrin was also fed to SD rats for the in vivo tail bleeding assays and ex vivo bioassays which consisted of both platelet aggregation assays and plasma coagulation assays.

SD rats were divided into four treatment groups (six rats per group) and orally administered with either 0.5% CMC (vehicle control), aspirin (30 mg kg-1), leaf extract (300 mg kg-1) or ȕ-amyrin isolated from the leaf extract (30 mg kg-1). ! ey were anaesthetised intraperitoneally with a mixture of ketamine (75 mg kg-1) and xylazine (10 mg kg-1) at 90 min after dosing. In vivo tail-bleeding experiments were conducted following general anaesthesia, and blood (4 ml from each rat) was collected, intra-cardiacally at 110 min after dosing, directly into citrated bottles (Tapval, 4 ml blood collection tubes, 0.106 M citrate, Deltalab S.A., Barcelona, Spain) for the ex vivo assays.

For the bleeding assays, each tail was pre-warmed for 5 min in phosphate bu# ered saline (PBS; 8 g 1–1 NaCl, 0.2 g 1–1 KCl, 1.44 g 1–1 Na2HPO4, 0.24 g 1–1 KH2PO4) at 37 °C. ! e bleeding was induced by transection of the rat tail at 2 mm from the tip.

! e distal portion (3 cm) of the tail was immersed vertically into PBS at 37 °C. ! e time between start of transaction and bleeding cessation was recorded as the bleeding time. Bleeding cessation was considered to be the time when the " ow of blood stopped for at least 30 s. If the bleeding did not cease by 15 min, the experiment was ended with pressure being applied to the wound to stop the bleeding.

Platelet aggregation assays were performed as described earlier, except that no test sample was added to the blood samples from the rats.

After exsanguination of the SD rats, the collected blood was centrifuged at 1500 g for 15 min to obtain clear plasma as the supernatant. ! e plasma was stored at -70 °C overnight and thawed for use the following day.

! e percentage inhibition of platelet aggregation, PT (Prothrombin Time), aPTT (activated Partial ! romboplastin Time), TT (! rombin Time) and BXT (Baxobin Time) are reported as means and standard deviations of experiments done in triplicates. Analyses of signi$ cance of values compared with controls, where appropriate, were done using the Student’s t-test at 5% level of signi$ cance in Microsoft Excel 2007.

ResultsChromatograms (see $ g. 1) are obtained from the

GC-MS analysis of the leaf extract and ȕ-amyrin standard. ! e chromatograms indicate that ȕ-amyrin is present in the leaf extract. ȕ-amyrin was eluted at around 67.8 min and its identity in both chromatograms was veri$ ed by mass spectrometry and an autolibrary search using the Wiley mass spectral library.

! e presence of ȕ-amyrin in the leaf extract

Figure 1: Total ion chromatograms of A) 70% (v/v)


was further veri! ed with analytical HPLC. " e chromatograms (see ! g. 2) are obtained from analytical HPLC analysis of the leaf extract and ȕ-amyrin standard. " e chromatograms indicate the presence of ȕ-amyrin in the leaf extract. ȕ-amyrin was eluted at around 63 min and its presence in the leaf extract was veri! ed by comparing the UV spectra of the peak of interest and that of the ȕ-amyrin standard.

" e composition of ȕ-amyrin in the leaf extract is about 1.2% while that in fresh leaves of A. elliptica

is 0.098% as determined by the quantitation method using LCMS.

" e results (see ! g. 3) obtained for the inhibition of collagen-induced platelet aggregation, in vitro in human blood, suggest that all the test samples (aspirin, leaf extract and the amyrins) showed antiplatelet e# ects at the indicated ! nal concentrations in the reaction mixtures. Moreover, although the composition of ȕ-amyrin in the leaf extract is about 1.2% w/w, a leaf extract ! nal concentration of 1.2 mg ml-1 and not 10 mg ml-1 was su$ cient to elicit equivalent if not more potent response than 0.1 mg ml-1 of ȕ-amyrin. " is suggests the possibility of other active compounds in the leaf extract that can also inhibit collagen-induced platelet aggregation.

" e results (see ! g. 3b) obtained for the inhibition of arachidonic acid-induced platelet aggregation suggest that only the leaf extract had demonstrated antiplatelet e# ects at the indicated ! nal concentration in the reaction mixture. Both amyrin isomers did not inhibit arachidonic acid-induced platelet aggregation. " e discrepancy between results obtained for the leaf extract and the amyrin isomers suggests the presence of other potent antiplatelet compounds in the leaf extract which remain to be identi! ed.

" e use of di# erent agonists (collagen and arachidonic acid) in the platelet aggregation assays can provide some information on the possible

Figure 2: HPLC chromatograms of A) 70% (v/v) methanol leaf extract and B) !-amyrin standard.

Figure 3: In vitro comparison of percentage inhibition of A) collagen-induced platelet aggregation and B) arachidonic acid-induced platelet aggregation by di# erent test samples in human blood. Error bars represent

standard deviation; all experiments were done in triplicates.


mechanisms of action of the test samples—details of which is beyond the scope of this discussion targeted at a wider audience.

! e following section presents and discusses the results of the human plasma coagulation assays in vitro. Prothrombin Time (PT) is a measure of the e" ect on the extrinsic pathway of coagulation by the di" erent test samples while activated Partial ! romboplastin Time (aPTT) is a measure of that on the intrinsic pathway. ! e di" erence between the two pathways is again beyond the scope of this discussion. ! e results (see # g. 4) of the coagulation assays for human plasma suggest that only the leaf extract has anticoagulant e" ect at the indicated concentration as demonstrated by the signi# cant prolongation of aPTT. ! e results suggest the presence of potent anticoagulant compounds in the leaf extract which remain to be identi# ed.

! e comparison of tail-bleeding times between the control and the other treatment groups shown in Table 1 suggests that both ȕ-amyrin and the leaf extract have antithrombotic e" ects as demonstrated by the prolonged bleeding times.

Test Sample (Dose) Bleeding Time/s

No. of Bleeding Times > 15 min

Control(n=6)

398 ± 54 0

Aspirin (30 mg kg-1)(n=5)

864 ± 81** 4

Leaf Extract (300 mg kg-1) (n=6)

741 ± 150** 2

ȕ-amyrin (30 mg kg-1) (n=6)

628 ± 200* 1

(* indicates p < 0.05; ** indicates p < 0.01 compared with the control)

Table 1: Tail-bleeding times after oral administration of test samples (n denotes the actual number of rats

being analysed for each test sample).

! e results from this experiment indicate that the leaf extract prolonged bleeding time more than ȕ-amyrin. ! is could be due to the presence of both antiplatelet and anticoagulant compounds in the leaf extract acting synergistically to bring about the

Figure 4: In vitro comparison of A) PT and B) aPTT due to di" erent test samples in human plasma. Error bars represent standard deviation; all experiments were done in triplicates. (* indicates p < 0.05, ** indicates p

< 0.01 compared with the control)


antithrombotic e! ect, compared with ȕ-amyrin which only has antiplatelet e! ect as discussed earlier in the in vitro bioassays.

Hence, the leaf extract " nal concentration of 300 mg kg-1 body weight which is 10 times and not 100 times that of ȕ-amyrin (30 mg kg-1 body weight) is su# cient to elicit more potent responses in vivo, suggesting that there may be synergistic e! ects of the phytoconstituents in the leaf extract. Although herbal extracts may contain only minute concentrations of bioactive compounds, such as in this case whereby ȕ-amyrin constitutes only 1.2% of the leaf extract, the overall therapeutic e! ects observed could be a result of synergy which presents itself as a high level of potency not achievable by individual isolates of the bioactive compounds.

$ e antithrombotic e! ect in vivo was further veri" ed by the ex vivo platelet aggregation assays presented in Figure 5. $ e ex vivo assays indicated the absorption of the test samples into the bloodstream via the gastrointestinal tract, suggesting that both the leaf extract and ȕ-amyrin are orally active. $ is is a highly desirable drug property as orally-active drugs would encourage greater patient compliance than those requiring intra-venous injections.

ConclusionComparing the results obtained for the leaf

extract and ȕ-amyrin in the in vitro assays, it is clear that there remains to be identi" ed compounds with anticoagulant and antiplatelet e! ects in the leaf extract which have markedly di! erent properties from ȕ-amyrin. Further isolation should be carried out to identify these bioactive compounds.

Experimental evidence has hinted at the possible e! ect of synergy occurring when the leaf extract was

tested for antiplatelet activity. $ is could be a possible explanation for the therapeutic e! ect observed in herbs despite the seemingly minute concentrations of bioactive compounds present. Sceptics of herbal remedies have often cited the low concentrations of bioactive compounds in plants as a reason for their doubts in the observed therapeutic e! ect being caused by the compounds detected. With the possibility of multiple compounds acting together to achieve therapeutic e! ects at low concentrations as demonstrated in the experiments, it is advisable that the study of medicinal plants take a turn in strategy. Isolation of bioactive compounds for individual testing may no longer be a su# ciently good analysis of the therapeutic potential of medicinal plants. It would be worthwhile examining the mechanisms of synergy occurring in the compounds present in herbs and attempt to replicate the same e! ect with a right combination of compounds in future drug development.

$ e study of prescriptions involving multiple herbs have often proved to be challenging to the novice. Prescriptions with instructions such as brewing the medicine in a claypot and consuming it warm may also seem unimportant details to the scientist employing modern scienti" c methods in the study of such herbal remedies. Such instructions are often explained with unfathomable terms such as drinking the warm medicine will better promote the % ow of “qi” in the body for quick recovery compared with drinking it cold. While the concept of “qi” is hardly explicable in scienti" c terms, it is important that the scientist relates these instructions to a sound knowledge of science wherever possible.

For instance, there have been instances in our laboratory in which boiling prescriptions in water in pyrex glass or metal pots have failed to produce the bioactive compound responsible for the therapeutic

Figure 5: Ex vivo comparison of percentage inhibition of collagen-induced platelet aggregation after treatment with di! erent test samples in SD rats. Error bars represent standard deviation and experiments on each

animal were done in triplicates. Doses of test samples are indicated in brackets, while the actual number of rats being analysed for each test sample is denoted by n.


e!ect of the medicine. "e compound could only be obtained when brewed in a claypot on a charcoal stove according to traditions. "ere is a possibility that claypot brewing on a charcoal stove may allow slightly higher boiling temperatures and pressures to develop in the claypot which aids the extraction process of bioactive compounds from the herbs or even establishes the right conditions for complex reactions to occur between the organic compounds to form the desired bioactive ones.

As for drinking the medicine warm, it is important to relate the simple concept of the increasing solubility of sparingly soluble compounds in water with higher temperatures. Prescriptions of medicine to be drunk warm generally have doses adjusted such that the right concentration of the bioactive compound will be present in the water extract when it is warm. When analyses are done cold or at room temperature, the concentration of the sparingly soluble bioactive compound will naturally be much lower or even undetectable in the water extract. In such instances, analyses of the sediments will generally show toxic concentrations of the bioactive compound. "us, we see that even though Chinese physicians use perplexingly abstract concepts like “qi” in their explanation of drinking certain prescriptions warm, modern science has its own theories to explain the observations made. It is important to appreciate the di!erences in the approach of experimentation and application of theories in the two disparate methods of studying medicine. Only scholars and scientists who can embrace the beauty of an open mind will have what it takes to acquire the imaginative capacity of applying modern scienti#c theories to the study of herbal remedies.


ReferencesChing, J., Chua, T. K., Chin, L. C., Lau, A. J., Pang,

Y. K., Johannes, M. J., Tan, C. H., and Koh, H. L. ȕ-amyrin from Ardisia elliptica is more potent than aspirin in inhibiting collagen-induced platelet aggregation. Indian Journal of Experimental Biology 48, 275–279 (2010).

Jalil, J., Jantan, I., Shaari, K., and Ra!, I. A. A. Bioassay-guided isolation of a potent platelet-activating factor antagonist alkenylresorcinol from Ardisia elliptica. Pharmarceutical Biology 42, 457–461 (2004).

WHO, “Cardiovascular diseases,” http://www.who.int/mediacentre/factsheets/fs317/en/index.html (accessed 17 Jan, 2010).

Patrono, C., Coller, B., Dalen, J. E., FitzGerald, G. A., Fuster, V., Gent, M., Hirsh, J., and Roth, G. Platelet-active drugs: the relationships among dose, e"ectiveness, and side e"ects. Chest 119, 39S-63S (2001).

Levine, M. N., Raskob, G., Landefeld, S., and Hirsh, J. Hemorrhagic complications of anticoagulant treatment. Chest 108, 276S-290S (1995).

Lee Jun Feng is an alumnus of NUS and USP. He graduated in July 2010 with honours in Life Sciences and is currently a teacher employed by the Ministry of Education, Singapore. This article is a revised version of his honours thesis which was presented at the Universitas 21 Undergraduate Research Conference 2010, held at the University of Melbourne, representing NUS.

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