Journal of Pharmacy And Bioallied Sciences

: 2022  |  Volume : 14  |  Issue : 2  |  Page : 93--98

Rapid preparative isolation of cleistanthin A from the leaves of Cleistanthus collinus Using reverse-phase flash chromatography

S C Santosh Kumar1, R Raveendran1, Kamsali Murali Mohan Achari2,  
1 Department of Pharmacology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Dhanvantri Nagar, Puducherry, India
2 Department of Organic Chemistry, Sri Padmavati Mahila Visvavidyalayam, Tirupati, Andhra Pradesh, India

Correspondence Address:
S C Santosh Kumar
Department of Pharmacology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Dhanvantri Nagar, Puducherry - 605006


Introduction: Cleistanthin A (CA) is an aryl naphthalene lignan, which has a potent anticancer activity by regulating the tumor microenvironment. The objective was to develop a new technique for the isolation of cleistanthin A from the acetone extract of Cleistanthus collinus utilizing reverse phase flash chromatography. Materials and Methods: Cleistanthus collinus leaves were shade dried, defatted using n-hexane and then macerated to obtain acetone extract which was further subjected to reverse phase flash chromatography for the isolation of cleistanthin A using the gradient mobile phase of 0.1% formic acid (v/v) in water and acetonitrile. Gradient elution of chromatographic run was performed for 80 min. The separated peaks that showed absorbance at λmax 254 nm were collected for the chemical characterization. Cell viability of the isolated cleistanthin A was studied on hepatocellular cancer cell line HePG2 and prostate cancer cell line PC3 using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: The chemical characteristics of the isolated compound cleistanthin A was further characterized using spectral techniques such as 1H and 13C nuclear magnetic resonance (NMR), Fourier-transform infrared spectroscopy (FT-IR), and electron spray ionization-tandem mass spectrometry (ESI-MS/MS). Cleistanthin A has decreased the cell viability of the HePG2 cell line to 52.25% at 32 μg/ml and PC3 cell line to 51.82% at 16 μg/ml in a dose-dependent manner. Conclusion: Cleistanthin A was successfully isolated from the natural source using reverse phase flash chromatography and the MTT assay has shown that cleistanthin A has decreased the cell viability in both the HePG2 and PC3 cell lines in a dose-dependent manner.

How to cite this article:
Kumar S C, Raveendran R, Achari KM. Rapid preparative isolation of cleistanthin A from the leaves of Cleistanthus collinus Using reverse-phase flash chromatography.J Pharm Bioall Sci 2022;14:93-98

How to cite this URL:
Kumar S C, Raveendran R, Achari KM. Rapid preparative isolation of cleistanthin A from the leaves of Cleistanthus collinus Using reverse-phase flash chromatography. J Pharm Bioall Sci [serial online] 2022 [cited 2022 Aug 11 ];14:93-98
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Cleistanthus collinus shrub is widely recognized for its poisonous effects where the different parts of the plant are considered toxic when consumed. In southern India, the leaves are used mainly for suicide and also for homicide. Those who ingest decoction prepared from the leaves of Cleistanthus collinus exhibit toxic symptoms such as severe metabolic acidosis, acute respiratory distress syndrome, hypokalemia, cardiac arrhythmias, renal failure, neutrophilic leukocytosis, neuromuscular weakness, and shock. Mortality has been reported in severe cases.[1],[2],[3] The toxicity profile of Cleistanthus collinus is attributed to the toxic aryl naphthalene lignan derivatives such as diphyllin, collinusin, cleistanthin A (CA), and cleistanthin B.[4],[5] Cleistanthin A, a diphyllin glycoside is the active principle moiety of Cleistanthus collinus shrub. Despite being a toxic substance, cleistanthin A possesses anticancer and alpha-adrenergic antagonistic properties.[6],[7] A study by Pradheepkumar et al.[8] suggests that the cytotoxic effect of CA in cervical carcinoma (Si Ha) and p53 deficient (K562) cell lines is due to deoxyribonucleic acid (DNA) damage, apoptosis, and inhibition of DNA synthesis. CA was found to inhibit the vacuolar-type ATPase (V-ATPase) proton pump which induced ATP hydrolysis for the transportation of proton into intracellular compartments and across the cell membrane.[9] Earlier studies have shown that different metastatic cancer types overexpress vacuolar-type H+ ATPases and acidic extracellular microenvironment created by V type H+ ATPases are correlated positively for the invasion and metastasis of tumor cells.[10] A study reported that CA was a novel V-ATPase inhibitor that decreased the expression of matrix metalloproteinases (MMP)-2 and -9 thus inhibiting the migration and invasion of melanoma A375 tumor cell lines.[11]

Molecular docking and ex vivo study conducted by Parasuraman et al.[12] has shown that cleistanthin A has a prominent alpha-adrenergic antagonistic action on the peripheral vascular system resulting in a dose-dependent decrease in rat blood pressure. In another study, Parasuraman et al.[7] reported that cleistanthin A has inhibited the contractions induced by phenylephrine and acetylcholine on the isolated tissue of vas deferens from guinea pigs and rabbits.

Earlier studies suggest that the glycoside CA [Figure 1] was isolated by running through a neutral alumina column using benzene and ethyl acetate (1:1) as a mobile phase.[5] In recent times, the flash chromatographic technique has gained immense popularity for the separation of compounds from natural sources when compared to conventional column chromatography because the procedure is quick and inexpensive.[13] The automated flash chromatographic system allows the separation of compounds based on built-in ultraviolet-visible (UV) detection and collection of fractions. Further, it allows the use of columns of varying sizes pre-packed with different materials, which can be further selected, based on the required separation technique. The flash chromatographic technique can be employed for both normal and reverse-phase chromatographic separation and it is mainly used for the preparative separation of natural substances.[14]{Figure 1}

Herein, we report a simple, quick, and inexpensive procedure for the isolation of CA from the acetone extract of the leaves of Cleistanthus collinus using the reverse-phase flash chromatographic technique.

 Materials and Methods

Chemicals and reagents

High pressure liquid chromatography (HPLC) grade Acetonitrile was purchased from Merck Life Science Private Limited (Mumbai, India). Methanol (HPLC grade) was purchased from Loba Chemie Pvt. Ltd. (Mumbai, India). Water (HPLC grade) was procured from Milli-Q (Merck life science private limited, Vikhroli (E), Mumbai, India) an integral water purification system (India). Chromatographic separation of cleistanthin A was performed using a flash chromatography system (Combiflash Rf® 200, Teledyne Isco 4700 Superior St. Lincoln, Nebraska, U.S.A. 68504). RediSep Rf gold high-performance C18 (20–40 μ particle size) column weighing 50 g manufactured by Teledyne Isco was purchased from Septech Marketing (Mumbai, India) and used.

Preparation of extract

Cleistanthus collinus leaves were gathered from the Sedarapet village, Puducherry, India. The authenticity of the plant specimen Cleistanthus collinus (Roxb.) Benth. ex Hook. f. was confirmed by Dr. N. Ayyappan, French Institute of Pondicherry, Puducherry, India. The herbarium of the plant was deposited for future reference with accession no. HIFP 27059.

The leaves were dried completely under the shade for 15 days and the coarse powder was prepared using a mixer grinder. The coarse powder was then defatted by soaking in n-hexane for 48 h and further subjected to maceration procedure with acetone for 7 days. 4 kg coarse powder of Cleistanthus collinus leaves yielded 431 g of acetone extract. The acetone extract obtained by removing the solvent under vacuum on a rotary evaporator was subjected to a reverse-phase flash chromatography system.

Isolation of cleistanthin A

A dry sample loading technique was used. The acetone extract of 1 g was mixed with celite in a ratio of 1:9 and the dried mixture was loaded on to solid-phase empty sample loading cartridge (Teledyne Isco, USA).

The flash chromatography was performed using the RediSep Rf gold high-performance 50 g C18 column. Repeated gradient flash trial runs were performed for optimizing the flow rate and selection of appropriate mobile phase and the run time [Table 1].{Table 1}

Flow rates of 30 ml/min and 25 ml/min resulted in poor separation due to low resolution whereas the 20 ml/min flow rate gave a satisfactory separation with high resolution. For the selection of appropriate mobile phase gradient composition, flash trial runs were performed using different combinations of mobile phases such as (1) water (A):methanol (B), (2) methanol (A):water (B), (3) acetonitrile (A):water (B), and (4) water (A):acetonitrile (B). Mobile phase A composed of formic acid (0.1%) in Milli-Q water and mobile phase B of acetonitrile were finally selected and used for the separation.

Gradient chromatographic run was performed starting with 10% of mobile phase B and then gradually increased to 100% B for 10 min and finally, the percentage of B was brought down to 50%. The flash run time was optimized for 80 min with a flow rate of 20 ml/min. The UV absorbance was measured at λmax× 214 nm and 254 nm to collect the separated flash fractions [Figure 2]. Cleistanthin A was separated with a retention time of 30 min as shown in [Figure 2]. The collected fractions were then subjected to lyophilization (freeze-drying) to remove solvents used as the mobile phase.{Figure 2}

Cell culture

Human hepatocellular carcinoma cell line (HepG2) and androgen-independent prostate cancer cell line PC3 were procured from National Center for Cell Science (NCCS), Pune, India. Both HepG2 and PC3 cell lines were maintained using Dulbecco's Modified Eagle Medium medium (HyClone, catalogue: SH30081.02) and supplemented with 10% fetal bovine serum (Himedia, RM;9955). The cells were cultured in a CO2 incubator in a humidified atmosphere of 5% CO2 at 37°C.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of cleistanthin A

The cytotoxic effect of cleistanthin A was determined by MTT assay. Control and experimental treatments were carried out at least three times. 70–80% confluent adherent HePG2 and PC3 cell lines were washed twice with phosphate buffered saline (PBS) to remove the media completely and trypsinized followed by centrifugation to remove the supernatant. The pellet was resuspended in fresh DMEM media and cell counting was carried out using a hemocytometer. Cells were serially diluted to yield 1 × 103 to 1 × 106 per ml and loaded onto wells in triplicates along with control which was nothing but DMEM media. The different dilutions of cells were incubated for 24 h. Post incubation cells were treated with 10 μl MTT reagent and incubated for 4 h. The purple color formazan crystals formed by the metabolic reduction of the MTT reagent were dissolved using dimethyl sulfoxide (DMSO) and absorbance was read spectroscopically at 570 nm. The cell dilution which yielded an absorbance of around 0.75 to 1.25 was considered for the study.[15]

Test drug cleistanthin A was dissolved in DMSO and the final concentration of the DMSO was < 0.1%. Hence 1 × 104 cells of both HePG2 and PC3 were seeded to each well in triplicates along with control and cleistanthin A was added at a range of 1–64 μg/ml. 24 h later the cell viability was assessed using an MTT reagent.

Statistical analysis

Data are presented as mean ± standard error of mean (SEM). Multiple comparisons were performed by one-way analysis of variance (ANOVA) followed by post hoc Tukey-Kramer. Statistical analysis was performed using GraphPad Instat (San Diego, USA) version 3.


Nuclear magnetic resonance (NMR) analysis of cleistanthin A

1H and 13C NMR were recorded at room temperature on Bruker AVANCE (Bruker) 400 spectrometer. NMR spectra for the compound CA was measured in CDCl3 using tetramethylsilane (TMS) as an internal standard. The chemical shifts were expressed as δ ppm downfield from the signal of internal TMS. 1H NMR (400 MHz, CDCl3): δH 7.77 (s, 1H); 6.92-6.81 (m, 2H), 6.72-6.58 (m, 2H), 6.00-5.96 (m, 2H), 5.47-5.44 (m, 2H), 5.21-5.17 (m, 1H), 4.96-4.85 (m, 1H), 4.57-4.55 (m, 1H), 3.99 (s, 3H), 3.70-3.67 (m, 5H), 3.59 (s, 3H), 3.51 (s, 2H), 3.35 (s, 3H); 13C NMR (100 MHz, CDCl3): δC 170.29, 170.15, 151.78, 150.23, 147.41, 143.55, 139.42, 135.49, 130.54, 128.41, 126.69, 123.57, 119.02, 114.19, 110.79, 108.11, 105.97, 104.12, 101.30, 76.18, 74.33, 69.84, 61.57, 59.92, 59.86, 57.94, 56.45, 55.82.

Infrared spectra of cleistanthin A

Fourier-transform infrared spectroscopy (FT-IR) spectra were recorded on Perkin Elmer spectrum one FT-IR instrument with a scan range of middle infrared (MIR) 450-4000 cm−1 and are reported in the frequency of absorption (cm−1). IR (KBr, cm−1): 3425, 2920, 2850, 1754, 1621, 1570, 1510, 1456, 1347, 1263, 1192, 1168, 1075, 810, 790.

UV and mass spectra of cleistanthin A

UV absorbance showed at λmax 258 nm and 291 nm. Mass spectra were determined using tandem electron spray ionization-tandem mass spectrometry (ESI-MS/MS) (m/z): [M + H]+ found 541 and calculated 541 for C28H29O11.

Physical properties and percentage of yield of cleistanthin A

After final clean up, cleistanthin A was separated using reverse-phase flash chromatography is amorphous and appears yellow to white in color. It is soluble in acetonitrile and methanol.

4 kg of powdered leaves of Cleistanthus collinus yielded 6.4 g of cleistanthin A. Hence, the percentage of yield of cleistanthin A is 0.16.

Antiproliferative effects of cleistanthin A on liver cancer line HePG2 and prostate cancer cell line PC3

To assess the cell viability of the isolated cleistanthin A on liver cancer cell line HePG2 and prostate cancer cell line PC3, an MTT assay was performed. As shown in [Figure 3] and [Figure 4], treatment using cleistanthin A in the dose range from 1 μg to 64 μg has significantly decreased the cell viability of the HePG2 and PC3 cell lines in a dose-dependent manner. The percentage of hepatocellular carcinoma cell line HePG2 cell viability with cleistanthin A at different doses of 1 μg, 2 μg, 4 μg, 8 μg, 16 μg, 32 μg, and 64 μg was 98.61%, 96.05%, 80.88, 71.51%, 60.02%, 52.25%, and 35.55, respectively. The percentage of prostate cancer cell line PC3 cell viability with cleistanthin A at different doses of 1 μg, 2 μg, 4 μg, 8 μg, and 16 μg per ml was 97.5%, 94.71%, 86.35%, 71.25%, and 51.82%, respectively. With respect to the PC3 cell line, there was a statistically significant difference in the cell viability between 4 μg, 8 μg, and 16 μg per ml of cleistanthin A. After 24 h, cleistanthin A at 32 μg has decreased the HePG2 cell viability to 52.25%. Treatment of 16 μg of cleistanthin A to the PC3 cell line resulted in 51.82% of cell viability.{Figure 3}{Figure 4}


The use of conventional methods for the isolation of natural compounds is encountered with many problems like the open column chromatography is tedious, tends to be associated with manual errors, requires a substantial volume of organic solvents as a mobile phase, and gives less yield. Short column life and low sensitivity and efficiency of the open liquid columns may result in improper separation of the mixture of compounds from natural sources.[16] Contemporary flash chromatographic technique for the preparative separation of compounds from natural sources offers some advantages such as the availability of automated integrated pumps which enable gradient mixing and in-built UV detection coupled with the collection of fractions. Hence the procedure is quick and inexpensive.[17]

Govindachari et al.[5] isolated CA by neutral alumina column using benzene and ethyl acetate as mobile phase. Anjaneyulu et al.[18] isolated CA from the heartwood of Cleistanthus collinus by running through a silica column using the same mobile phase as mentioned earlier. Parasuraman et al.[12] also used a similar combination of mobile phase for the isolation of CA. Nowadays benzene is not preferred as a mobile phase for chromatographic techniques due to its confirmed carcinogenic and possible mutagenic effects in humans. Target organ damage is expected after repeated or long-term exposure to benzene.[19],[20] In comparison with the older methods, the currently developed method requires a lesser volume of mobile phase and also avoids the use of such carcinogenic solvents. The procedure was repeated for at least five times a day for 1 week to assess the reproducibility of the technique.

The current isolation method has very good reproducibility compared to older open column methods since the flash chromatography has automated pumps where mobile phase ratios are finely adjusted and the flow rate is maintained accordingly. The availability of pre-packed column cartridges for flash chromatography with different materials is handy and also increases the reproducibility of the method.[15] The use of reverse-phase flash chromatography for the isolation of cleistanthin A has immensely decreased the time taken for the compound isolation when compared to older open liquid chromatographic methods.[5] Cytotoxic aryl naphthalene diphyllin glycoside cleistanthin A is the investigational anticancer molecule. Cell viability assay was carried out to assess the role of cleistanthin A on the hepatocellular cancer cell line HePG2 and androgen-independent prostate cancer cell line PC3. V-ATPase inhibitor cleistanthin A has induced cytotoxicity to the HePG2 and PC3 cell lines and these results demonstrate the antiproliferative effect of cleistanthin A on the cancer cell lines. Pradheepkumar et al.[8] suggested cleistanthin A had induced cell death and decreased cell viability in Chinese hamster ovary (CHO) cells in a dose-dependent manner at 10, 15, and 20 μg/ml. The effects of cleistanthin A are mediated by apoptotic induction, blockage of DNA synthesis, and DNA damaging properties. Zhao et al. chemically synthesized cleistanthin A and its derivatives to study the antiproliferative and V ATPase activity on solid tumor cell lines such as HepG2, Hela, A549, and HCT 116.[6] Cleistanthin A and its derivatives have shown cytotoxic effects on the above mentioned four solid tumor cell lines at concentrations of submicromolar levels, but cleistanthin A was found to be more potent than its derivatives.[9] Pan et al.[11] studied the cytotoxic effects of cleistanthin A and its role in invasion and metastasis of melanoma cells. The results of the MTT assay of the study suggest that cleistanthin A inhibited the growth of A375 cells in a dose-dependent manner and at submicromolar concentration levels.

The cell viability of the PC3 cell line with cleistanthin A at 16 μg/ml was 51.8% and for HePG2 it was 53.8% at 32 μg/ml. Hence, the present study results show that cleistanthin A is more potent in inhibiting PC3 proliferation. The growth of PC3 cells does not depend on androgen and does not express androgen receptors.[21]

The present technique can be exploited for preparative scale production. It also offers the easiest way of isolation of CA from Cleistanthus collinus. This technique will be of immense help to researchers to obtain CA from natural sources for pharmacological and toxicity studies.


In this study, we have developed a universal technique for the isolation of bioactive dyphyllin glycoside CA from the acetone extract of the leaves of Cleistanthus collinus by reverse phase flash chromatographic technique. Cleistanthin A has decreased the cell viability of the HePG2 and PC3 solid tumor cell lines in a dose-dependent manner.

Financial support and sponsorship

This study was funded by JIPMER intramural research grant.

Conflicts of interest

There are no conflicts of interest.


1Shankar V, Jose VM, Bangdiwala SI, Thomas K. Epidemiology of Cleistanthus collinus (oduvan) poisoning: Clinical features and risk factors for mortality. Int J Inj Contr Saf Promot 2009;16:223-30.
2Eswarappa S, Chakraborty AR, Palatty BU, Vasnaik M. Cleistanthus collinus poisoning: Case reports and review of the literature. J Toxicol Clin Toxicol 2003;41:369-72.
3Mohan A, Naik GS, Harikrishna J, Kumar DP, Rao MH, Sarma K, et al. Cleistanthus collinus poisoning: Experience at a medical intensive care unit in a tertiary care hospital in South India. Indian J Med Res 2016;143:793-7.
4Ramesh C, Ravindranath N, Ram TS, Das B. Arylnaphthalide lignans from Cleistanthus collinus. Chem Pharm Bull 2003;51:1299-300.
5Govindachari TR, Sathe SS, Viswanathan N, Pai BR, Srinivasan M. Chemical constituents of Cleistanthus collinus (Roxb.). Tetrahedron 1969;25:2815-21.
6Zhao Y, Zhang R, Lu Y, Ma J, Zhu L. Synthesis and bioevaluation of heterocyclic derivatives of Cleistanthin-A. Bioorg Med Chem 2015;23:4884-90.
7Parasuraman S, Raveendran R. Effect of cleistanthin A and B on adrenergic and cholinergic receptors. Pharmacogn Mag 2011;7:243-7.
8Pradheepkumar CP, Panneerselvam N, Shanmugam G. Cleistanthin A causes DNA strand breaks and induces apoptosis in cultured cells. Mutat Res Toxicol Environ Mutagen 2000;464:185-93.
9Zhao Y, Lu Y, Ma J, Zhu L. Synthesis and evaluation of Cleistanthin A derivatives as potent vacuolar H(+) -ATPase inhibitors. Chem Biol Drug Des 2015;86:691-6.
10Stransky L, Cotter K, Forgac M. The function of V-ATPases in cancer. Physiol Rev 2016;96:1071-91.
11Pan S, Cai H, Gu L, Cao S. Cleistanthin A inhibits the invasion and metastasis of human melanoma cells by inhibiting the expression of matrix metallopeptidase-2 and -9. Oncol Letter 2017;14:6217-23.
12Parasuraman S, Raveendran R, Vijayakumar B, Velmurugan D, Balamurugan S. Molecular docking and ex vivo pharmacological evaluation of constituents of the leaves of Cleistanthus collinus (Roxb.) (Euphorbiaceae). Indian J Pharmacol 2012;44:197-203.
13Moosmann B, Kneisel S, Wohlfarth A, Brecht V, Auwärter V. A fast and inexpensive procedure for the isolation of synthetic cannabinoids from 'Spice' products using a flash chromatography system. Anal Bioanal Chem 2013;405:3929-35.
14Wohlfarth A, Mahler H, Auwärter V. Rapid isolation procedure for △ 9-tetrahydrocannabinolic acid A (THCA) from Cannabis sativa using two flash chromatography systems. J Chromatogr B 2011;879:3059-64.
15Kumar P, Nagarajan A, Uchil PD. Analysis of cell viability by the MTT assay. Cold Spring Harb Protoc 2018;2018:469-71.
16Muhammad S, Han S, Xie X, Wang S, Aziz MM. Overview of online two-dimensional liquid chromatography based on cell membrane chromatography for screening target components from traditional Chinese medicines. J Sep Sci 2017;40:299-313.
17Jubie S, Dhanabal SP, Chaitanya MVNL. Isolation of methyl gamma linolenate from Spirulina platensis using flash chromatography and its apoptosis inducing effect. BMC Complement Altern Med 2015;15:263.
18Anjaneyulu ASR, Ramaiah PA, Row LR, Venkateswarlu R, Pelter A, Ward RS. New lignans from the heartwood of cleistanthus collinus. Tetrahedron 1981;37:3641-52.
19Salemi R, Marconi A, Di Salvatore V, Franco S, Rapisarda V, Libra M. Epigenetic alterations and occupational exposure to benzene, fibers, and heavy metals associated with tumor development (Review). Mol Med Rep 2017;15:3366-71.
20Falzone L, Marconi A, Loreto C, Franco S, Spandidos DA, Libra M. Occupational exposure to carcinogens: Benzene, pesticides and fibers (Review). Mol Med Rep 2016;14:4467-74.
21Mitchell S, Abel P, Ware M, Stamp G, Lalani EN. Phenotypic and genotypic characterization of commonly used human prostatic cell lines. BJU Int 2000;85:932-44.