1dian Journal of Experimental 13iology 'ol. 42, September 2004, pp. 893-899

Evaluation of antimitotic activity of Rotula aquatica (Lour):A traditional herb used in treatment of cancer

' l* b** c ' l S Patil ' , S Narayanan , G Eibl & C I Jolly'

' Department of Pharmacognosy and Phytochemistry. hDepartment of Pharmacology Prin . K M Kundnani College of Pharmacy, 47, R G Thadani Marg, Worli , Mumbai 400 018, India

894 I DIAN J EXP 1310L, SEPTEMBER 2004

regi on compared to that of the other tissues. This region is called the meristamatic region(meri stos : divided)9. Thi s divi sion is similar to the above mentioned cancer division in humans. Hence, these meristamatic cells can be used for preliminary screening of drugs with anticancer activity. Even though doubts can be raised about extrapolation of results from plant ti ssue to animals and finally to humans, Khilman has noted that plant cells are 1000 times more resistant to colchicine which is a potent anti-carcinogen and which acts by inhibiting the microtubule formation. Thus, it is possible that chemicals that affect plant chromosomes will also affect animal s 10.

We may thus hypothes ize that if R. aquatica extract is effective against A. cepa root cells, it will al so have antimitotic effect against animal and human cells. To evaluate thi s hypothes is, it was thought worthwhile to eva lu ate the activity of the extracts of R. aquatica on 3 established human pancreatic cell lines also.

Polyphenols have shown anticarcinogenic potential against PC-12 (human prostate cancer cells), HL-60 (human promyclocytic leukemia) by inducing apoptos is 11 . Pancreatic cancer is ex tremely resistant to the i ncluction of apoptosis by chemotherapies. Agents that regulate apoptotic sensitivity may lead to chemosensitization of pancreatic cancer 12·13. Three pancreatic cancer cell lines were used, viz. HPAF-II , BxPC-3 and CAPAN-2, which rese mble well , moderately and poorly differentiated pancreatic cancer cells respectively. The growth of pancreatic cancer is driven by multiple factors like mutations in K-ras leading to inappropriate activation of p21m', oncogenic activation of p21 ms 14. An anticancer agent may act through anyone of the mechanisms.

Phytochemical evaluation plays an important role in the standardisation of crude herbal clrugs. 15·16. The standardisation of plant material requires the estimation of active ingredients presen t in the plant material and fingerprint profil e which goes towards establishing the identity and purity of the plant part being used. HPTLC serves as an extremely reliable and precise instrument for the analysis of medicinal plants and herbal formulations prepared from them 17. In the present study, we have performed an HPTLC profile of R. aquatica that may be used as a measure for standardizing its active ingredient.

Materials and Methods Materials-The plants of R. aquatica were

collected from Umbergaon (Gujarat), India and

authenticated at Blatter Herbarium(No.:59,2683), St Xavier's College, Mumbai, India. Allium cepa bulb~ (red variety) were purchased from the local marke and stored for the entire study. Carmine stain wa~ procured from M/s Sigma Laboratories, Monsanto U.S.A. Other solvents used for extraction were of LR grade and were disti lied before use for greater purity.

Preparatio11 of plant extracts-The entire herb wa~ air-dried in the laboratory at room temperature. It wa' then powdered (24 mesh) and was ex tracted with ho1 water by boiling for 30 min to get the aqueous extract The extract obtained was concentrated and driec under controlled temperature(60"C).r,~he dri ed powcle1 was successively extracted with other solvents 18, viz a) petroleum ether (60-80"C), b) benzene, c chloroform, d) acetone, e) ethanol and f) water Extraction with each solvent was clone in a water batr for 60 min with a reflux condenser. Each time, before extracting with the next solvent, the marc was dried ir an air oven below 50°C. Each extract wa~ concentrated and evaporated to dry extract. The solvent : drug ratio for extraction was 5: I. Extracts o1 desired concentrations were prepared for further study us ing these dried extracts.

Antimitotic activity-This activity was evaluated us ing A. cepa root meristamatic cell s. A. cepa bulb~ were sprouted in tap water for 48hr at room temperature. The bulbs that developed uniform roots were used for the experiment. These roots were treated with above prepared extr were fixed in a fixing solution of acetic acid and alcohol. Squash preparations were made by stammg with acetocarmine stain .The mitotic index was calculated 1 ~ as-Mitotic Index = Number of di viding cells/Total number of cells x I 00.

The aqueous extract was al ~o subjected to qualitative test which revealed the presence of tannins and steroids. Tannins were isolated from the water extract. The experiment was repeated with tannin and non-tannin fractions. Squash preparations made as above and observed. A similar experiment was undertaken to find out the probable mechanism of action through which the extract acts. Folic acid was added to the aqueous solution of methotrexate and

PATIL eta/.: EVALUATION OF ANTIMITOTIC ACTIVITY OF R.AQUATICA 895

1queous ex tract of R. aquatica. Squash preparati ons nade as above from the treated roots were observed.

Cell proliferation assay-Three cell lines( BxPC-3, ::::apan-2 and HPAF-11 ) were obtained from the Ameri can Type Culture Collecti on and grown in RPM ! 1640 med ium containing 10% fe tal bov ine 'eru m (FBS), penicillin G ( I 00 U/ml ) and strepto-mycin (100 ).lg/ml) in a humidi fied atmosphere of 5% ::::0 2 at 37°C. The effect of R. aquatica on cell viability and growth was determined using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl teti·azolium bromi de) co lorimetric assay [using a commerciall y :lYa il able kit (Boehringer Mannheim, Germany)]. The ::ompounds were disso lved in water as stock so lu tion (IOOOx) and then dilu ted with RPMI-1 640 fo r cell ::ulture ex peri ments. All so luti ons were prepared fresh Dn the day of tes ting. Cell s (Bx PC-3, Capan-2 and HPAF-Il ) were seeded at a density of lx l04 per well in 96-well pl ates in RPM I- 1640 contain ing FBS (10%). After 24 hr, fresh medi um was added ::ontaining aqueous extract of R. aquatica at concen-trati ons ofO, 10·4 , 10·3, 10·2, 10· 1, and 10 mg/ml. After 4 days of incubation, in med ium contain ing ex tract of R. aquatica cell s were further incubated fo r 4 hr with the metabolic substrate, tetrazolium salt MTT, at a final concentrati on of 0.5 mg/ml. Living cell s metaboli zed tetrazolium to fo rmazan which was detected spectrophotometri call y at 540 nm with mult iwell spectrophotometer (ELI SA Reader, Biotek Instruments Inc. , Burlington, VT).

HPTLC 111etlwd fo r f ingerprin ting-HPTLC fingerprinting studies were carried out by mi xing !Omg of aqueous extrac t in !Oml of methanol. This solu tion was spotted as 20, 40, 60 and 80).11 on a HPTLC silica GF254 plate. The plate was developed using solvent system chloroform : ammoni a: ethanol (4:5: 1). The fingerprint patterns of R. aquatica were visualised at 254 and 366nm.

Preparative TLC-A slurry of sili ca gel GF254 was made in di still ed water. This slurry was then applied to glass plates( 12.5 x 12.5cm) with the aid of a TLC spreader to obtain preparative silica gel plates having thickness of about 0.5mm. The plates were dri ed in an oven at I 05°C and acti vated 2 hr before use.

Sa 111ple preparation and application-Aqueous extract ( I mg/ml) was spotted on the pl ate as a band of length 50mm length. The plates were developed using the solvent system. The plates were visualised at 254nm, the band marked, scrapped, vortexed with

methanol and centri fuged. The supern atant was separated, evaporated and res idue dried with nitrogen gas. The dri ed res idue was subjected to spectral analys is, viz; UV, IR, Mass and elemental.

Stat istical analysis--Th e data was subjected to stati sti c analys is usi ng analys is of variance fo llowed by appropriate post-hoc tests. P

896 INDIAN J EX P 1310L, SEPTEMI3ER 2004

inhibition produced by R. aquatica ex tract was not significantly different to that of methotrexate. The cell divisions were differentiated and number of cells in each phase of cell division i.e either prophase, metaphase, anaphase, or telophase were recorded (Table 2). Thus, the number of cells entering prophase decreased with increase in concentration of the extrac ts. Since the cells do not enter prophase, further stages of cell divi sio n also decrease with increase in concentration of the aqueous ex tract.

Effect of tannin and non-tannin fractions on 111itotic activity (Table 3)-A one-way ANOV A showed th at there was a significan t decrease in mitot ic index by all 3 fractions viz., tannin, non-tannin and aqueous fraction(FJ.S = 2022.42; P

PATIL eta /.: EVALUATION OF ANTIMITOTIC ACTIVITY OF R.AQUATICA 897

acid on the antim itotic actiVIty of R. aquatica and methotrexate (F4.30 = 746.05). A con trol with only folic ac id did not show any difference in mitotic index when compared to that of plain water. The mitotic index increased when folic acid was added to the total aqueous ex tract of R aquatica and methotrexate soluti on which o therwise reduced the mitotic ac ti vity in the absence of folic acid. Thi s however, did not increase with increase in foli c acid concentration suggesting that the effect was not dose-dependent. Comparing the mitotic index of methotrexate and R. aquat ica it was observed that incorporation of folic acid increased the mitotic index sig nificantly in case of methotrexate, but not so in case of R. aquatica. Post-hoc analysis of the data showed that fo li c ac id inhbited the anti-mitotic ac tivity of methotrexate to a greate r extent as compared toR. aquatica.

Effect of R. aquatica on cell viability of luti/Wll pancreatic concer cell lines (Fig. 1 )-A o ne-way analysis of the data of each of the cell lines showed a significant effec t of the extract on each of the cell lines (F5.42 = 58.53 , 58.53, 131.67) respective ly. Post-

HPAF - 11

120

D ~ o=~~ -- -10 5

Capan • 2 120

100 01

898 INDI AN J EXP BIOL, SEPTEMBER 2004

Plate viewed under 254nm Plate viewed under 366nm

Fig. 2--HPTLC fin gerprinting of R. aqumica at - (a) 254 nm; and (b) 366 nm

glycos ide gets hyd ro lysed in the process of extraction . The melting point of compound for spot I and spot

2 was found to be 170° and 190°C respec tively. Both the compounds showed positive response to Liebermann - Burchard's test, thus confirm ing the identity of a stero ida l alcohol.

Discussion The results from our s tudy showed that the aqueous

ex trac t of R. aquatica had excellent anti-mito tic activity, that was comparable to the activity of methotrexate. The fractionation of the aqueous ex tract into tannin and non-tannin fractions showed that each of these individual fractions possessed good anti -mitotic activity but were not as potent as the total aqueous ex tract. The addition of folic ac id inhibited the anti-mito ti c acti vity of R. aquatica significantly, but not completely. It completely inhibited the activity of methotrexate.

Aqueous extrac t of R. aquatica was also effective in reducing the ce ll viability of 3 human pancreatic cancer cell lines that may be acting following the same mechanisms as those in the o nio n cells. By vi rtue o f thi s, if the extract is admini stered in humans it may prevent cell proliferation by direct ly combining with DNA leading to DNA fragmentation, by inhibiting the folic acid pathway or by combining with cell receptors/enzymes and eliciting sig nals for

ce ll apoptosis. The phytochemical analys is of R. aquatica revealed

that the plant was ri ch in polyphenols (tannins) and steroids. The polyphenols have been show n to be potent anti-mitotic agents in numerous assays. Thus, it was not surpri sing that the aqueous extrac t of R. aquatica showed good anti-mitotic act ivity. The tannin and non- tannin f racti ons showed good anti-

mito tic activity, but less than that of the total aqueous extract. It may be, therefo re, concluded that the non-tannin fracti on contains ste roids along with other pol ar constituents. Though, the probability of steroids extracted in a po lar solvent was low, these s teroids occurred as ag lycones of the saponin g lycos ides after the glycosi des was hydrolyzed . There might be other po lar compounds in the to ta l extrac ts that might be acting sy nergist ically with the tannins and steroids of R. aquatica. Steroids have been shown to be useful in treatment of cancer. Hence, the steroids from R. aquatica must be contributing to the anticancer potenti al of the he rb.

The aqueous extract of R. aquctica seems to prevent prophase stage in ce ll di v ision where the DN A duplication occurs. Methotrexate is a known anticancer· drug that inhibits DNA synthesis. When folic acid was supplemented to the cells with methotrexate and the total aqueous extract it was seen that mitotic index increased. Thus, it may be concluded that the ex tract may be acting through this pathway inhibiting tetrahydrofo lic ac id and hence fo lic acid required for DNA synthes is thereby arres ting cel l di vision. Methotrexate is known to compete with folic acid for the enzyme THF reductase, the total aqueous extract of R. aquatica may also be competing with fo lic ac id thus inhibiting the DNA sy nthes is. Hence, increase in folic ac id concentration increases the mito ti c index due to more availability of folic acid. However, the mitotic index does not increase significantl y in case of R. aquatica, as compared to that of methotrexate. This may be because the extract may be mediat ing its effect through other mechanisms a lso . The extrac t may be binding with different cell proteins responsible for cell division. The antioxidant potential of the extract

PATIL eta/.: EVALUATION OF ANTIMITOTIC ACTIVITY OF R.AQUATICA 899

established in another experiment in our laboratory. he extract shows good antioxidant activity which so may be one of the contributing factors to it's 1ticancer potential. The spectral analysis of the isolated compounds

tows that the compounds may be steroidal glycosidic kaloid and a steroidal alcohol. The study throws ght on potential use of R. aquatica in treatment of mcer.

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