Phytochemical investigations and systematic ...

Phytochemical investigations and systematic exploration of anti-cancer potential of Nyctanthes arbor-tristis Resear ch Ar ti cl e

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Phytochemical investigations and systematic explor ation of anti-cancer potential of leaves of Nyctanthes arbor-tristis Par ul Gr over 1, Shashank K umar Singh2, K .A. Sur i 3 and Gulshan Bansal 4 1School of Pharmacy, KIET Group of Instituti ons, 13th Mil e Stone, Del hi-Meerut Road, Ghaziabad, Uttar Pradesh, India 2Cancer Pharmacology Divi sion, Indian Insti tute of I ntegrative Medi cine (CSIR), Canal Road, Jammu - 180001, Jammu & Kashmi r, India 3Indian I nstitute of Integrati ve Medici ne, Jammu - 180001, Jammu & Kashmi r, India 4Department of Pharmaceutical Sciences and Drug Research, Punj abi Universi ty, Patial a - 147002, Punjab, India

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ABSTRACT Leaves of Nyctanthes arbor-tristi s have been traditi onally used as anti-cancer drug, but there is no report to support thi s tradi tional claim. The present study is undertaken to establish the anti-cancer activi ty of the N. arbortristis, and to identify the most active anticancer fraction(s) f rom its diff erent extracts. Leaves of N. arbor-tristis were extracted with solvents of varied polari ty (toluene, chl oroform, ethylacetate and aqueous ethanol (HA), and subsequently evaluated for their anti-cancer activity against HL60 cell lines usi ng MTT assay method. The toluene and HA extracts were fractionated through column chromatography and successive solvent extractions, respectively. All extracts, except ethyl acetate extract, exhibited potent anti-cancer activity. Four fractions of the toluene extract were found maxi mal ly potent whereas the petroleum ether f raction of HA was found responsible for anti-cancer activity of HA extract. I t i s concluded that the pl ant can serve as an excel lent source of potent and probabl y new anti-cancer lead(s), which can be further modif ied chemically to produce a seri es of compounds for development of potent and safe anti-cancer drugs. [M edi cinal Plants 2015; 7(3) : 227-232] K eywor ds : Nyctanthes arbor-tristi s, anti-cancer, HL60 cell line, MTT assay

I NTRODUCTION Cancer i s one of the maj or diseases responsibl e f or mortality and expenditure on health care for human bei ngs. I t i s the second l eadi ng cause of death worldwide. About 12.7 millions of cancer cases and 7.6 mi lli on deaths were recorded worl dwi de i n 2008. The f i rst li ne treatment of cancer invol ves extensive use of chemotherapeuti c agents such as anti metabol i tes,

Cor r esponding author : Gulshan Bansal e-mail : gulshanbansal@rediffmail .com [email protected] Received : 28 May 2015; Accepted : 3 July 2015 doi : 10.5958/0975-6892.2015.00031.3

Medicinal Plants, 7(3) September 2015

al kylating agents, antibioti cs and various cytotoxic agents. However, thei r use is associated with sufferings from various side effects such as bone marrow depression, al opecia and depressi on (Kaefer et al., 2008). Since times immemorial, plants have been used for treatment of cancer. Over 35,000 different plant samples from 20 countries were screened for anti-cancer activi ty (Cragg et al., 1996). Now-a-days, about 60% of the anti-cancer drugs avail able in the market are of natural origin (Boopathy et al ., 2010). Some of the plant derived anti-cancer drugs include vincristine, vinblastine, taxol, paclitaxel, camptothecin, homoharringtonine and flavopiridol. The search for more anti -cancer drugs i s sti l l to be di scovered wi th mol ecul es exhi bi ti ng superi or potency and tumor speci f ici ty. Nyctanthes arbor-tristis (f aml iy Oleaceae) is a common shrub flourishing in the Sub-Himalayan




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tract, in the states of Uttar Pradesh, Assam, Bengal, Madhya Pradesh and in Godavari bel ts in South India (Suresh et al., 2010). It has long been used in Ayurvedic system of medici ne for the cure of snake bi te, bi tes of wild animals, cachexyia, cancer, sores, ulcers, dysentery menorrhagia and obstinate sci atica (Satyavati et al ., 1987; Thomas et al ., 2013; Khanapur et al., 2014). It has been reported to be effective against lei shmanial, vi ral and amoebi c i nfections (Tandon et al ., 1991; Chitravanshi et al ., 1992; Rathee et al., 2007). The juice of its leaves is used i n chronic and bilious f evers, rheumati sm, i nf l ammati on, and as a l axati ve, di aphoretic and di ureti c (Pattanayak et al ., 2013; Aggarwal et al ., 2013). The l eaves are reported to possess a number of constituents rangi ng from nonpolar molecules such as  -amyrin,  -sitosterol and friedelin to pol ar ones such as l oganin, arborsides and nyctanthoside (Wagh et al., 2012, Woyengo et al., 2009, Fidelis et al ., 2012; Jain et al., 2011). The present study has been designed to evaluate anti-cancer activity of leaf extract of N. arbor-tristis using sol vents of varied polarity. M ATERI AL AND M ETHODS Pl ant mat er i al Leaves of Nyctanthes arbor-tristis were coll ected from botanical garden of Punjabi University, Patiala i n the month of February, 2011. The sample was submitted to Herbarium, Punj abi University, Pati ala (authenticati on no. 55155, dated 4/3/2011). Extr action and f r actionati on Contaminant analysis of plant mat er i al The leaves were got analysed for contaminati on due to heavy metals, microbes, pathogen and pestici des from Oscar Analytical Pvt. Ltd., Baddi, Sol an, Himachal Pradesh (India). The l eaves were washed under runni ng water and shade dri ed. The dri ed l eaves were crushed and subjected to successive soxhlet extraction (36-40 h f or each sol vent) using solvents i n order of increasi ng polarity starti ng with toluene f oll owed by chloroform, ethyl acetate and f i nal ly 30% aqueous methanol (hydroal cohol). The toluene extract was fractionated by column chromatography through gradient el uti on with ethyl acetate and n-hexane. The elution was started

wi th n-hexane (100%) and pol ari ty was i ncreased wi th ethyl acetate i n steps using an increment of 0.5% up to 20% ethyl acetate fol lowed by increment of 1% up to 25% ethyl acetate and f inally 100% ethyl acetate. Solvent of each polarity was run till no component was detected in the el uent as revealed by TLC. However, a mi nimum of 200 mL of solvent of each polarity was run on the column. The f ractions were collected and pool ed on the basis of their si milar TLC prof iles. Each pooled fraction was condensed under vacuum and the dried fracti ons were stored at 0oC ti ll use. Phyt ochemical scr eening Each extract was screened f or the phytoconstituents l i ke al kal oi ds, tanni ns, f l avonoi ds, carbohydrates, gl ycosides, saponins, steroids, free amino acids, fats and f i xed oi l s and starch usi ng standard methods (Farnsworth, 1966; Kokate, 2006). Ant icancer activity Each extract and f ractions of toluene and HA extract were eval uated on human l eukemia cancer cell lines (HL-60) using MTT assay (Mossman, 1983; Verma et al., 2008). Brief ly, the cell s (2000-3000 in 100 L of foetal bovine serum) were pl aced in each wel l of a 96 well plate and incubated for 24 h so that the cel ls adhered to the surface. One L of each of the four test soluti ons (of di fferent concentrations i.e., 20, 2, 0.2 and 0.02 mg/mL) of each extract as well as fracti on prepared in DMSO (10%) were added separately into the wel l s contai ni ng adhered cel l s/100  L of the medium so that the f inal concentrati on of the test sol uti ons become 200, 20, 2 and 0.2  g/mL , respectively. One L of DMSOO O was added i n the control well. Control and each test solution was treated in triplicate (n=3). The plate was incubated at 37 °C for 48 h. Subsequently, i n each of the control and test treated well, 25 L of (3-(4,5-dimethylthiazol-2-yl)-2,5di phenyl tetrazoli um bromide (MTT) (2.5 mg/mL of phosphate buffer solution of pH 7.4) was added and incubated f or 4 h. The medi um as wel l as the excess dye was removed by gentl e pressing on soaking paper. The formazan produced by the viable cells in each well was solubilized by addi tion of 100 L of DMSO (1% v/v) and shaken gently for f ive mi nutes. Absorbance of the solution in each wel l was recorded at 570 nm by ELISA reader. The per cent cell viability was calculated and the IC50 value of each test solution was determined from the plot of % cell viabili ty and concentration. Medicinal Plants, 7(3) September 2015

Phytochemical investigations and systematic exploration of anti-cancer potential of Nyctanthes arbor-tristis

RESULTS

Table 2. Yi el d and ant i-cancer acti vi ty of ext r act s and fr act ions

Contaminant analysis and phytochemical scr eeni ng The leaves were found free from any contamination of heavy metals, pathogens and pesticides. Total microbial count as well as total yeast and mould count were al so found to be wel l withi n the limits. The phytochemical composition and yields of various extracts and fractions are given in Tables 1 and 2, respectively.




Anti-cancer activity Off the four extracts, the ethyl acetate extract was found to be i nacti ve whil e tol uene, chl orof orm and HA extracts showed potent cytotoxi city on the HeLa cell lines (Table 2). Each fraction of toluene and HA extracts was evaluated f or the activity i n order to isolate the active fraction(s). The petroleum ether and n-butanol fractions of HA extract were found to have cytotoxicity equivalent to the HA extract itself (IC50 10 g/ml) while the other fracti ons were f ound to be si gnif icantly less active (IC50 >20 g/ml). I n total, 23 fracti ons were i sol ated f rom tol uene extract. Amongst these, the fracti ons 2, 4, 5 and 12 were found to be signif i cantly active with IC50 100

Hydroalcoholic (HA)

20.6

10 ± 0.49

Petroleum ether f racti on of HA

3.24

10 ± 0.50

Chl oroform fracti on of HA

5.78

25 ± 0.99

Ethyl acetate fraction of HA

4.08

82 ± 1.12

n-butanol fraction of HA

3.39

10 ± 1.43

Aqueous fracti on of HA

18.3

>100

Residual HA

0.571

ND

Tabl e 3. Y iel d and anti -cancer act ivi t y of f r act ions of tol uene extr act Fr act ions

Yield (% )

I C 50 (g/mL )

1.

3.4

>100

2.

4

10 ± 0.21

3.

6

50 ± 0.35

4.

4

5 ± 0.05

5.

5.2

5 ± 0.17

6.

6

76 ± 0.83

7.

2.6

100 ± 0.77

8.

3.2

100 ± 0.69

9.

3.4

70 ± 0.42

10.

3.6

40 ± 0.32

11.

2.6

60 ± 0.54

12.

3.8

15 ± 0.22

13.

3.6

45 ± 0.07

14.

3.4

25 ± 0.23

15.

3.2

50 ± 0.60

16.

3.6

20 ± 0.09

17.

4

30 ± 0.19

18.

4

>100

19.

3.6

65 ± 0.34

20.

3.4

>100

21.

4.8

>100

22.

4

>100

23.

5.6

>100

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H

H

H

H

O

H

OH

HO

Oleic acid (Olea europrae) Log P 6.32

H

Lupeol (Calotropis gigantia) Log P 7.37

O




O

O

O

O

O N H

OH

OH

O

O

O

O

H

HO

OH O

OH

O O

O

Pomiferin (Maclura pomifera) Log P 4.15 Taxol (Taxus brevifolia) Log P 3.96 H 3C OH H3COOC

Et

N

OH

OCH 3

OHC

H 3COCO Et

O

N

N

H 3C

O H3COOC

OH

N

OCHO3

H N

O

H 3CO HN

OCH 3

Et N Et

Vinblastine (Catharanthus roseus) Log P 3.70

N OH

Vincristine (Catharanthus roseus) Log P 2.82

Fig. 1. Log P values of some anti-cancer plant derivati ves Medicinal Plants, 7(3) September 2015




Phytochemical investigations and systematic exploration of anti-cancer potential of Nyctanthes arbor-tristis

arbor-stristis. Chlorof orm and HA extracts were found more cytotoxic than the tol uene extract. A study of ClogP values of various anti-cancer compounds isolated from diff erent plants has revealed that most of these compounds such as lupeol, ol eic acid, pomiferin, taxol, vinblastine and vincristine are lipophilic in nature (Fig. 1). In the present study, the plant material was extracted sequentially starti ng with toluene (log P 2.7) followed by chl oroform (l og P 2.2), ethyl acetate (0.7), and aqueous ethanol . Toluene and chlorof orm have almost si mil ar l ipophi licity and hence these are expected to extract si mi l ar types of consti tuents. Theref ore, consi dering the facts that : (i) a vast maj ori ty of anticancer phytoconstituents are non-polar; (ii) toluene and chlorof orm are almost equi li pophi lic; (ii i) yiel d of toluene extract was maximum, it was hypothesized that probabili ty of f inding constituent(s) responsibl e f or anti-cancer activity of the plant is greater from toulene extract than from the extracts of other solvents used in the study. This hypothesi s of exploiting toluene (nonpolar) extract was further supported by the observation that petrol eum ether f racti on of HA extract was equicytotoxi c to the HA extract itself. Hence, toluene extract was sel ected f or f urther f racti onati on. Cytotoxi city of the toluene extract was found to be chief ly due to its four fracti ons i.e., fracti ons 2, 4, 5 and 12. Further, phytochemical screening of the toluene extract suggested that i ts acti vi ty may be due to al kal oids and tannins in the leaves. CONCLUSI ON The toluene, chlorof orm and aqueous methanol extract of leaves of N. arbor-tri stis were found to have anticancer properties against HL60 cell lines. Four fractions of the toluene extract were found to be the best one. The study supports the traditi onal use of leaves of N. arbor-tri sti s as anti -cancer herb and suggests that i ts acti vi ty may be due to the non-pol ar constituents. Hence this shrub can serve as an excellent source of potent and probably new anti-cancer lead(s) which can be further anal yzed to produce a seri es of compounds for devel opment of potent and safe anti-cancer drugs. ACK NOWL EDGEM ENT The authors are thankful to Counci l f or Scienti f ic & I ndustrial Research (CSIR), New Delhi (I ndia) for provi ding f inancial assi stance to conduct the study (Reference No. 02(0132)/13/EMR-II , April 12, 2013). The authors are also thankful to Lord Shiva College of Medicinal Plants, 7(3) September 2015

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