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Chromatographic analysis, anti-proliferative and radical scavenging activity of Pinus wallichina essential oil growing in high altitude areas of Kashmir, India.

ARTICLE INFO

Keywords:

Pinus wollichiona

Essential oil

GC/CC--MS

Antiproliferative activity

Radical scavenging activity

ABSTRACT

Purpose: To evaluate the in vitro anti-proliferative and radical scavenging properties of the essential oil and its fractions and to determine the chemo-type of P. wallichiona essential oil.

Method: Pinus wollichiana oil was extracted by hydro-distillation and fractionated by silica gel column chromatography method. The oil and its fractions were analyzed by Gas chromatography, Gas chromatography--mass spectrometry and 13C NMR. Different concentrations of oil 12.5, 25, 50 and 100 [micro]OA and single concentration 50 of its fractions 81, B2, A2, G2, Uk r3 and 12 were evaluated for its anti-proliferative activity by in vitro {3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide) assay against human monocyte, lung carcinoma, liver adenocarcinoma, prostate and ovarian carcinoma, while as the radical scavenging activity was evaluated by different in vitro DPPH assays.

Results: The analyses indicated the presence of 17 constituents with P-pinene (46.8%) and a-pinene (25.2%) as major constituents. The oil and its fractions showed significant anti-proliferative activity. The radical scavenging activity also showed good results.

Conclusion: The oil could be used as a drug to control the diseases like cancer, cirrhosis and arteriosclerosis, caused by reactive oxygen species.

[C] 2012 Elsevier GmbH. All rights reserved.

Introduction

Oxidation and reduction or redox reactions represent the transfer of electrons from an electron donor to an electron acceptor species. The cellular redox environment is a balance between the production of reactive oxygen species (ROS), reactive nitrogen species (RNS), and their removal by antioxidant enzymes and small molecular-weight antioxidants. Oxidation is essential to many living organisms for the production of energy to fuel biological processes. Free radicals are produced in normal and pathological cell metabolism. However, the uncontrolled production of oxygen derived free radicals are involved in the onset of many diseases such as cancer, rheumatoid arthritis, cirrhosis and arteriosclerosis as well as in degenerative processes associated with ageing. Exogenous chemical and endogenous metabolic processes in the human body or in the food system might produce highly ROS especially oxygen derived radicals, which are capable of oxidizing biomolecules, resulting in cell death and tissue damage (Halliwell and Gutteridge 2003). Almost all organisms are well protected against free radical damage by oxidative enzymes such as superoxide dismutase (SOD) and catalase (CAT), or chemical compounds such as [alpha]cy-tocopherol, ascorbic acid, carotenoids, polyphenols and glutathione (Niki et al. 1994). When the mechanism of antioxidant protection becomes unbalanced by factors such as ageing, deterioration of physiological functions may occur, resulting in diseases and accelerated ageing. However, antioxidant supplement or antioxidant containing foods may be used to help the human body to reduce oxidative damage (Halliwell and Gutteridge 2003; Mau et al. 2001; Gulcin et al. 2002). Dietary antioxidant intake may be an important strategy for inhibiting or delaying the oxidation of susceptible cellular substrates, and is thus relevant to disease prevention in many paradigms. Phenolic compounds such as flavonoids, phenolic acids, triterpenes and tannins have received much attention for their high antioxidative activity (Rice-Evans et al. 1996).

Essential oils are natural extracts of aromatic plants used in many fields like agriculture, aromatherapy and nutrition. The constituents of essential oil contribute the overall activity of each essential oil against target organisms, some of them have been used in cancer treatment. However, standard cancer chemotherapy is frequently compromised by the development of drug resistance and unwanted partly life-threatening side effects. Therefore, there is an urgent need for novel treatment options with improved features. Many plant-derived compounds, e.g., Paclitaxel, ACH-1, Vinblastine, or Vincristine, and semi-synthetic derivatives of natural products, such as Etoposide and Teniposide are used as anti-cancer drugs. As pointed out recently, natural products from medicinal plants represent a fertile ground for the development of novel anticancer agents. Plants from tropical regions are considered to be one of the potential sources for the screening of anticancer agents (Efferth et al. 2007; Buttner et al. 1996, 2010).

Pinus wallichiana (blue pine/kail), the finest pine of northwestern Himalayan region, is well known for its commercial and ecological importance. The species grows naturally along the entire length of temperate Himalaya ranging in altitude from 2000 to 3500m above mean sea level. Of the Indian pines, the wood of the blue pine is considered to be the best and stands next to deodar (Cedrus deodara) in value. Though exploited mainly for timber, the species is a good source of oleoresin also, which is used for production of turpentine oil, rosin, needle oil and camphor. In addition, it is a dominant species of forest ecosystem of the Kashmir Himalaya. The species has widespread distribution in Indian Himalaya, Afghanistan, Bhutan, Pakistan and Nepal. The extensive literature survey revealed that number of phenolic compounds have been isolated from the bark and leaves of Pinus wallichiana (Willfor et al. 2009; Naeem et al. 2010) and no earlier report on essential oil available. Therefore the objectives of the current study was to evaluate the in vitro anti-proliferative and radical scavenging properties of the essential oil and its fractions and to determine the chemical composition or chemo-type of P. wallichiana essential oil. According to our best knowledge, there is no such literature documenting chemical composition, anti-proliferative and radical scavenging activities of the P. wallichiana oil.

Material and methods

Plant material

The fresh needles of P. wallichiana were collected from Khag region of Jammu and Kashmir, India during the months of July-August 2011. The localities where the plant material was collected are usually situated between 2600 and 3500 m higher than sea level. The plant material was properly identified and further authenticated by A.H. Malik, Centre for Plant Taxonomy and Biodiversity, University of Kashmir. Voucher specimen bearing number 2070 was deposited at KASH herbarium in the Centre for Plant Taxonomy and Biodiversity, University of Kashmir, Hazratbal Srinagar.

Chemicals

DPPH (2,2-diphenyl-1-picrylhydrazyl) radical was purchased from Sigma--Aldrich, Madrid, Spain. Dimethyl sulphoxide (DMSO), anhydrous sodium sulfate, diethyl ether, methanol, ethyl acetate, toluene, n-hexane and all other reagents were of analytical grade (SISCO, Mumbai, India).

Essential oil extraction

The fresh aerial needles or leaves were subjected to hydro-distillation for 3 h, using Clevenger-type distillation apparatus. The oil was dried over anhydrous sodium sulphate (10 ml on 3g) and stored at 5 C until used.

Fraction of essential oil

Column chromatography was used for the fractionation of the P. wallichina essential oil. 100 ml of oil was fractionated using column chromatography packed with 2000g of silica gel (60-120 mesh) equilibrated with hexane. The column was sequentially eluted with 800 ml of each hexane-ethyl acetate (99:1, 98:2, 97:3, 96:4, 95:5, 94:6, 93:7, 92:8, 91:9, 90:10 and 100% (v/v) ethyl acetate) in the increasing order of polarity. The volume of each fraction was taken 100 ml and concentrated under vacuum at 40 C to obtain 33 fractions. The oil and its fractions were chromatographed on silica gel precoated TLC 60-F254 plates (Merck, India), using petroleum ether and ethyl acetate (9:1 (v/v)) as the developing solvent system and vanillin-sulphuric acid and 2, 4-dinitrophenylhydrazine as visualizing agent. In some instances they were heated for about 5-10 min at 105 *C to additional characteristic colorations. The fractions with similar TLC profile were pooled to afford only 13 fractions namely [A.sub.2], U[K.sub.2], [I.sub.2], [E.sub.2], [F.sub.2], [H.sub.2], [K.sub.2], [G.sub.2], [C.sub.2], [B.sub.1], [N.sub.2], [M.sub.2], and [B.sub.2]. The fractions were tested for anti-proliferative activity and only 13 fractions [A.sub.2], U[K.sub.13], [I.sub.2], [G.sub.2], [B.sub.1] and [B.sub.2] showed good results. The identification and purity of these fractions was carried out by Gas chromatography and Gas chromatography-mass spectrometry. The identification of some fractions was also carried out by(1) H and (13) C NMR, wherever necessary.

Analysis by Gas chromatography (GC)

Sample analyses was performed on a Perkin Elmer autosystem XL Gas Chromatograph 8500 (J & W Scientific, Rancho Cordova, CA, U.S.A.) series equipped with flame ionization detector (FID) and headspace analyzer using a fused silica capillary column (DB-5; 30 m x 0.32 mm, film thickness 0.25 [micro]m) coated with dimethyl polysiloxane (Agilent Company; Palo, Alto, CA, U.S.A.). Oven temperature was programmed from 60 to 230[degrees]C at a rate of 3[degrees]C/min, with injector temperature 230[degrees]C and detector temperature 230[degrees]C. The volume injected was 0.5 [micro]1. The identification of the components was performed by comparison of their retention times with those of pure authentic samples and by means of their linear retention indices (LRI) relative to the series of n-alkanes ([C.sub.5]-[C.sub.28]). Percentage composition of the oil components were obtained from electronic integration using FID at 250[degrees]C, dividing the area of each component by the total area of all components. Percentage values were the mean of three injections of the sample.

Analysis by Gas chromatography-mass spectrometry (GC-MS)

GC-MS analysis was performed on a Varian Gas Chromatograph series 3800 (Ohio Valley, Marietta, U.S.A.) fitted with a DB-1 MS fused silica capillary column (60 m x 0.25 mm, film thickness 0.25 [micro]m) coupled with a 4000 series mass detector under the following conditions: injection volume 0.5 [micro]l with split ratio 60, helium as carrier gas at 1 ml/min constant flow mode, injector temperature 230[degrees]C, oven temperature 40-250[degrees]C at 3[degrees]C/min. Mass spectra: electron impact (El+) mode, 70 eV and ion source temperature 250[degrees]C. Mass spectra were recorded over 50-500 amu range. Peak identification of oil and its fractions was accomplished by computer matching with those of the computer mass libraries, NIST 05, Wiley and Adams (Adams 2005) and comparison of MS with those of our library of pure standard authentic compounds. The identification of the oil components was also done by comparison of their retention indices (RI) with those from the literature (Adams 2005).

(13) C NMR analysis

(13) C NMR measurement of essential oil were performed by a Bruker Avance 300 spectrometer (Bruker, India) operating at the frequency of 75 MHz using deuterio-benzene as solvent and TMS as the internal standard. (13) C NMR spectra were recorded with spectral width of 18,000 Hz (180 ppm). The number of accumulated scans was 2000 for the sample (40 mg of the oil in 0.5 ml [C.sub.6][D.sub.6]). The identification of some constituents of oil was confirmed by (13) NMR spectroscopy, by comparing their most intense signals with those reported in the literature (Kubeczka 2002).

Cell lines and culture

The cell lines used were human monocyte (THP-1), lung carcinoma (A-549), liver adenocarcinoma (HEP-2), prostate (PC-3) and ovarian carcinoma (IGR-OV-1). The cells were cultured in (Cancer Pharmacology Division, Indian Institute of Integrative Medicines, CSIR, _Jammu, India) RPMI-1640 medium supplement with 10% heated fetal bovine serum, 1% of 2 mmol/11.-glutamine, 50 U/m1 penicillin, and 50 [micro]g/m1 streptomycin. A-549, HEP-2, PC-3 and IGR-OV-1 were cultured in 96 well plates (Bio-Grenier) at a density of [10.sup4]cells/well in Dulbecco's minimal essential medium (DMEM). Media were also supplemented with 100[micro]/m1 penicillin/streptomycin antibiotics. Cells were maintained in humidified atmosphere in 5% C[O.sub.2]at 37 C. Mitomycin-C and Paclitaxel were used as positive controls.

In vitro anti-proliferative assay

Assay was carried out by MIT (3-(4,5-Dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide, a tetrazole) protocol to evaluate the anti-proliferative effect of oil and its fractions. For this purpose, a sufficient number of exponentially growing cells were used to avoid confluence of the culture during the treatment. The cell lines A-549, HEP-2, PC-3 and IGR-OV-1 were seeded at [10.sup.4]cells/well and allowed to adhere for 12 h. The THP-1 cells were seeded at 2 x [10.sup.3]cells/well and media was replaced with 200[micro]lof fresh medium before the treatment with sample. In order to evaluate the optimum concentration at which the oil inhibited the cell proliferation in all the five cell lines, cells were treated with concentration ranging from 100 to 12.5 [micro]g/ml. While as the isolated fractions were tested against the same cell lines at a concentration of 50[micro]g/ml. DMSO was used as a solvent for the dilution of oil and its fractions, which was also used as a experimental control. Mitomycin-C and Paclitaxel were used as positive controls at a concentration of 1 x 10-5 [micro]g/ml, respectively. After 48 h treatment, cell growth was evaluated by MIT assay (Alley et al. 1986, 1988). MU solution of 50 ill (5 mg/m1 of PBS) was added to each well and the plates were incubated for 3 h at 37 C in dark. The media was aspirated and 150[micro]1 of MTT solvent (4 mM HCI, 0.1% Nondet P-40, all in isopropanol) was added to each well to solubilize the formazan crystals. The absorbances of plates were measured on ELISA reader (Benchmark, BioRad) at a wavelength of 570 nm. Each sample was performed in triplicate, and the entire experiment was repeated thrice.

DPPH assay on TLC

The assay was performed as reported in the literature (Braca et al. 2002) with some modifications. The hydrogen atom-or electron-donation ability of the essential oil was measured from the bleaching of methanol solution of DPPH. In this assay, a stable DPPH (2,2'-diphenyl-1-picrylhydrazyl) radical purple in color, is reduced to yellow colored diphenylpicryl hydrazine by the action of antioxidants. Five microliters of a 1:10 dilution of the essential oil in hexane was applied as spots to the TLC plates (aluminum sheets covered with silica gel 60 F-254, Merck, India). The plate was sprayed with DPPH reagent (0.2% in methanol) and left at room temperature for 30 min. Yellowish spots formed as result of bleaching of the purple color of DPPH reagent were evaluated as positive antioxidant activity (Mimica-Dukic et al. 2003). Spectrophotometric DPPH radical scavenging assay

The capacity to scavenge the DPPH free radical was monitored according to a method previously reported (Burda and Oleszek 2001). The free radical scavenging activity of essential oil was evaluated using the stable purple colored DPPH radical spectropho-tometrically. The different concentrations of oil and [beta]-pinene 10, 20, 30, 40 and 50[micro]g/ml were added to 2 ml of methanolic solution of DPPH (10 mg/1). The degree of decoloration of methanolic solution of DPPH indicates the scavenging efficiency of the added oil. The reaction mixture was covered and left in dark at room temperature. After a time interval of 20 min, the decrease in absorbance (decoloration) was read against the control ascorbic acid at 517 nm. The reduction of DPPH radical was measured by monitoring continuously the decrease of absorption at 517 nm. Experiments were performed in triplicate. The radical scavenging capacity of oil to scavenge DPPH radical was calculated by the following equation:

scavenging effect (%) = [(A control - B sample)/A control x 100]

where A control where A control is the absorbance of blank sample and B sample is the absorbance of essential oil. The percentage of scavenging activity was plotted against the oil concentration. Radical scavenging activity was expressed as the concentration of drug scavenging DPPH radical by 50% (I[C.sub.50]). The reaction involved is:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]

Statistical analysis

All the values were expressed as mean[+ or -] S.D. The results were analyzed statistically by Analysis of Variance (ANOVA) followed by Dunnett's test. * p < 0.05, ** p <0.01.

Results

Yield and composition of oil

The yellowish hydro-distilled essential oil of P. wallichina was analyzed by Gas chromatography, Gas chromatography--coupled with mass spectrometry and (13) C NMR. The analyses and identification by these methods revealed the presence of 17 compounds, representing 94.2% of the total oil. The various compounds in order of their elution from DB-5 column are listed in Table 1. The major compounds of oil were [beta]-pinene (46.8%), [alpha]-pinene (25.2%) and myrecene (9.5%). Therefore [beta]-pinene (46.8%) and [alpha]-pinene (25.2%) determined the chemotype of the [Kashmir P. wallichina essential oil. The monoterpene (87.9%) rich essential oil of P. wallichina contained 84.6% of simple monoterpene hydrocarbons and 3.3% of oxygenated monoterpenes. While as the sesquiterpene hydrocarbons (3.6%) and oxygenated sesquiterpenes (2.7%), represented only 6.3% of the total identified compounds. The percentage yield of oil calculated was found to be 0.2% (v/w), based on their fresh weight. The fractions [Asub.2], [B.sub.2], U[K.sub.13], [I.sub.2],[G.sub.2] and [B.sub.2] represents 92.5, 84.0, 68.0, 57.2, 82.7 and 67.6%, respectively of the total compounds identified in these fractions. The main compounds identified in these fractions by analytical techniques are tabulated in the form of Table 2.

Table 1

Chemical composition of Pinus wallichina essential oil from Kashmir,
India.

S. no.         Compounds (c)         % (a)  [RI.sup.Cal.]  [RI.sup.Lit]

1              [alpha]-Pinene         25.2            935           932

2              Camphene                0.9            947           946

3              [beta]-Pinene          46.8            939           974

4              Myrecene                9.5            987           988

5              [alpha]-Phellandrene    0.4           1002          1002

6              Delta-3-carene          0.8           1010          1008

7              Limonene                1.0           1026          1024

8              [alpha]-Terpineol       2.3           1188          1186

9              Trans-pinocarveol       0.4           1293          1135

10             [alpha]-Terpinyl        0.8           1340          1346
               acetate

11             Ceranyl acetate         0.1           1380          1379

12             Trans-caryophyilene     1.8           1421          1417

13             [alpha]-Humulene        0.5           1454          1452

14             [delta]-Cadinene        0.4           1509          1522

15             Caryophyllene oxide     2.1           1579          1582

16             [alpha]-Cadinol         0.9           1649          1652

17             [alpha]-Bisabolol       0.6           1656          1685

Monoterpene    84.6
hydrocarbons

Oxygenated     3.3
monoterpenes

Sesquiterpene  2.7
hydrocarbons

Oxygenated     3.6
sesquiterpene

Amount of      94.2
identified
compounds

S. no.         Compounds (c)         Identification
                                     methods (b)

1              [alpha]-Pinene        MS,RI,RT, NMR

2              Camphene              MS, RI, RT, NMR

3              [beta]-Pinene         MS. RI, RT, NMR

4              Myrecene              MS, RI, RT, NMR

5              [alpha]-Phellandrene  MS, RI, RT

6              Delta-3-carene        MS, RI, RT, NMR

7              Limonene              MS, RI, RT, NMR

8              [alpha]-Terpineol     MS, RI, RT, NMR

9              Trans-pinocarveol     MS, RI, RT

10             [alpha]-Terpinyl      MS.RI.RT, NMR
               acetate

11             Ceranyl acetate       MS, RI, RT

12             Trans-caryophyilene   MS, RI, RT, NMR

13             [alpha]-Humulene      MS, RI, RT

14             [delta]-Cadinene      MS. RI, RT

15             Caryophyllene oxide   MS, RI, RT, NMR

16             [alpha]-Cadinol       MS, RI, RT, NMR

17             [alpha]-Bisabolol     MS, RI, RT

Monoterpene    84.6
hydrocarbons

Oxygenated     3.3
monoterpenes

Sesquiterpene  2.7
hydrocarbons

Oxygenated     3.6
sesquiterpene

Amount of      94.2
identified
compounds

[RI.sup.Cal] = retention indices were experimentally measured using
homologous series of n-alkanes ([C.sub.5]-[C.sup.24]) on the DB-5
column. [RI.sup.Lit] = relative retention index taken from Adams.
RI, by comparison of RI with those reported in literature; RT, by
comparison of the retention time and mass spectrum of authentic
standard. NMR, by comparing their chemical shift values with literature
following the Kubeczka method.

(a.) Percentage obtained by FID peak area normalization without the
use of response factor.

(b.) Identification methods: MS, by comparison of their MS with those
of the computer mass libraries NIST 05 library, Wiley and Adams.

(c.) Compounds are listed in order of their elution from DB-5column.

Table 2

Chemical composition of Pinus wallichina essential oil fractions.

S. no.      Compounds         [A.sub.2]  [B.sub.1]  [B.sub.2]  [G.sub.2]

1           [alpha]- Pinene        24.5       11.0

2           Camphene                1.4

3           Myrecene               14.2

4           [beta]- Pinene         50.3       17.0       17.0

5           Limonene                2.1        9.2

6           [alpha]-                          46.8
            Phellandrene

7           Trans-                                       17.2
            caryophyllene

8           [alpha]-                                     33.4
            Humulene

9           Caryophyllene                                           82.7
            oxide

10          [alpha]- Cadinol

11          [alpha]-
            Bisabolol

Total                              92.5       84.0       67.6       82.7
identified
compounds

S. no.      Compounds         [Uk.sub.13]  [l.sub.2]

1           [alpha]- Pinene

2           Camphene

3           Myrecene

4           [beta]- Pinene

5           Limonene

6           [alpha]-
            Phellandrene

7           Trans-
            caryophyllene

8           [alpha]-
            Humulene

9           Caryophyllene
            oxide

10          [alpha]- Cadinol         68.0       21.6

11          [alpha]-                            35.6
            Bisabolol

Total                                68.0       57.2
identified
compounds


Anti-proliferative activity

Results of the anti-proliferative activity showed maximum growth inhibitions against THP-1, A-549, HEP-2, PC-3 and IGR-OV-1 cell lines, respectively, when treated with 100 [micro]g/m1 of oil and 50 [micro]g/ml of its fractions in MIT assay. The standard drug Paclitaxel at a concentration of 1 x [10.sup.-5] [micro]g/ml showed 23, 87, 34, 45 and 82% and Mitomycin-C at the same concentration showed 32, 56, 90, 72 and 29% inhibitions against THP-1, A-549, HEP-2, PC-3 and IGR-OV-1, respectively. The results are arranged and compiled in the form of Table 3. The proliferative nature of A-549 was highly inhibited at lower concentration 12.5 [micro]g/ml showing 90% growth inhibition, however with increase in concentration of oil, there was further increase in cell growth inhibition and showed maximum inhibition of 92% at 100 [micro]g/ml, with [IC.sub.50] 6.1 [micro]g/ml. The cell lines HEP-2 and IGR-OV-1 were not effectively inhibited at lower concentrations but at 100 [micro]g/ml, they were efficiently inhibited with [IC.sub.50] 9.0 and 9.9 [micro]g/ml, respectively. The results of oil also showed appreciable effects on proliferation of THP-1 cell line with [IC.sub.50] 5.6 [micro]g/ml. Like THP-1 the oil was also potent against PC-3 with [IC.sub.50] value 6.7 [micro]g/ml. Thus dose dependent inhibitory effects were recorded in all the cell lines. The fraction first of essential oil [B.sub.1] was active against PC-3 ([IC.sub.50] 21 [micro]g/m1), THP-1 ([IC.sub.50] 18 [micro]g/ml) and A-549 ([IC.sub.50] 37 [micro]g/m1), Less activity was shown by [B.sub.1] on IGR-OV-1 with [IC.sub.50] of 49 [micro]g/ml. Fraction [A.sub.2] was more active against THP-1 ([IC.sub.50] 17 [micro]g/ml) and A-549 ([IC.sub.50] 38.2 [micro]g/m1) as compared to PC-3 ([IC.sub.50] 48.2 1.4/m1) and IGR-OV-1. The oxygenated sesquiterpene fraction [Uk.sub.13] was highly effective against THP-1 ([IC.sub.50] 31[micro]g/ml) and A-549 ([IC.sub.50] 17.61.4/m1), moderately effective on PC-3 with [IC.sub.50] 39 [micro]g/m1 and least effective on IGR-OV-1 at a maximum concentration of 50 [micro]z/ml.The fraction 12 was almost equally effective against PC-3 ([IC.sub.50] 24 [micro]/ml) and IGR-OV-1 ([IC.sub.50] 25 [micro]g/m1) and moderately affected the THP-1 ([IC.sub.50] 31[micro]/m1) and A-549 ([IC.sub.50] 28.1) as shown in Table 4. Among the fractions of oil [G.sub.2] was less effective against all the cell lines used for anti-proliferative assay. Fraction [B.sub.2] showed effective results against PC-3 ([IC.sub.50] 36.5 [micro]g/m1) and A-549 ([IC.sub.50] 48.3 [micro]g/ml) and less effectiveness towards THP-1 and IGR-OV-1 cell lines. Among the fractions tested in the current study, none of them has been tested against HEP-2 cell cultures. Based on data, it could be concluded that oil and its fractions showed different inhibitory effects on different human cell lines of varied tissue origin.

Table 3

Anti-proliferative activity of essential oil and its fractions on
human monocyte (THP-1), lung carcinoma (A-549), liver adenocarcinoma
(HEP-1), prostate (PC-3) and ovarian carcinoma (IGR-OV-1).

Materials                  Percentage of growth inhibition (%)
Concentration of          Leukemia         Lung     Liver (HEP-2)
oil ([mu]-g/ml)            (THP-1)      (A-549)

DMSO               4 [+ or -] 0.31   3 [+ or -]   3 [+ or -] 0.02
                                         0.02 *

Pw-12.5           83 [+ or -] 1.04  90 [+ or -]  56 [+ or -] 1.42
                                 *       3.04 *                 *

Pw-25             86 [+ or -] 2.43  91 [+ or -]  56 [+ or -] 1.56
                                 *         02 *                 *

Pw-50             86 [+ or -] 1.78  91 [+ or -]  58 [+ or -] 2.04
                                 *       1.78 *                 *

Pw-100            96 [+ or -] 2.08  92 [+ or -]  62 [+ or -] 3.05
                                 *       1.04 *                 *

[B.sub.1]-50      82 [+ or -] 0.82  61 [+ or -]                NT
                                 *       0.14 *

[UK.sub.13]-50    90 [+ or -] 1.90  81 [+ or -]                NT
                                 *       1.86 *

[I.sub.2]-50      60 [+ or -] 2.84  65 [+ or -]                NT
                                 *       4.19 *

[G.sub.2]-50      45 [+ or -] 0.64  42 [+ or -]                NT
                                 *       0.84 *

[A.sub.2]-50      89 [+ or -] 1.25  65 [+ or -]                NT
                                 *       3.81 *

[B.sub.2]-50      30 [+ or -] 3.20  55 [+ or -]                NT
                                 *       1.53 *

Pacilitaxel-1 x   23 [+ or -] 0.32  87 [+ or -]   34[+ or -] 1.03
[10.sup.-5]                      *       1.43 *                 *

Mitomycin-C 1 x   32 [+ or -] 0.42  56 [+ or -]  90 [+ or -] 2.88
[10.sup.-5]                      *       2.81 *                 *

Materials
Concentration of             Ovary     Prostate
oil ([mu]-g/ml)         (IGR-OV-1)       (PC-3)

DMSO               1 [+ or -] 0.04   1 [+ or -]
                                           0.04

Pw-12.5           51 [+ or -] 2.04  68 [+ or -]
                                 *       1.78 *

Pw-25             52 [+ or -] 3.01  73 [+ or -]
                                 *       1.04 *

Pw-50             58 [+ or -] 2.86  80 [+ or -]
                                 *        0.2 *

Pw-100             59 [+ or -]3.89  83 [+ or -]
                                 *       2.04 *

[B.sub.1]-50      56 [+ or -] 1.34  73 [+ or -]
                                 *       0.31 *

[UK.sub.13]-50    43 [+ or -] 0.64  63 [+ or -]
                                 *       1.74 *

[I.sub.2]-50      75 [+ or -] 3.14  61 [+ or -]
                                 *       3.17 *

[G.sub.2]-50      18 [+ or -] 1.71  10 [+ or -]
                                 *       2.86 *

[A.sub.2]-50      32 [+ or -] 4.00  59 [+ or -]
                                 *       0.61 *

[B.sub.2]-50      26 [+ or -] 0.60  66 [+ or -]
                                 *       2.63 *

Pacilitaxel-1 x   82 [+ or -] 4.56  45 [+ or -]
[10.sup.-5]                      *       2.81 *

Mitomycin-C 1 x   29 [+ or -] 1.99  79 [+ or -]
[10.sup.-5]                      *       0.32 *

Where n = 3, values are expressed as Mean[+ or -]S.E,M. DMSO =
dimethylsalphoxide control sample, Pw-12.5, Pw-25r Pw-50 and Pw-100 =
Pinus wallichina oil concentration of 12.5,25,50 and 100[micro]g/ml.
[B.sub.1]-50,[UK.sub.13]-50,[I.sub.2]-50,[G.sub.2]-50,[A.sub.2]-50 and
[B.sub.2]-50 = concentration of fractions 50 [micro]g/ml,mitomycin-C
1 x [10.sup.-5] and Pacilitaxel-1 x [10.sup.-5]=Mitomycin-C and
Pacilitaxel concentration of [10.sup.-5] [micro]g/ml. The results were
expressed a Mean [+ or -]S.E.M. Test and standard groups (Paclitaxel and
Mitomycin-C) were compared with control group (DMSO), statistically
analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's
test. * p<0.01.

Table 4

Anti-proliferative accivity (ICso)rfPimts waUichina essential oil and
its various fractions on human cancer cell lines.

Materials             Leukemia (THP-1)  Lung(A-549) (a)  Liver (HEP-2)
                                   (a)                             (a)

P. wallichina oil     5.6 [+ or -] 1.4     6.1 [+ or -]   9.0 [+ or -]
                                                    0.8            1.5

[B.sub.1]-fraction     18 [+ or -] 7.4  37 [+ or -] 3.6             ND

[UK.sub.13]-fraction   I3 [+ or -] 7.1    17.6 [+ or -]             ND
                                                    2.5

[I.sub.2]-fraction     31 [+ or -] 6.6    28.1 [+ or -]             ND
                                                    1.3

[G.sub.2]-fraction                  ND               ND             ND

[A.sub.2]-fraction     17 [+ or -] 1.1    38.2 [+ or -]             ND
                                                    3.8

[B.sub.2]-fraction                  ND    48.3 [+ or -]             ND
                                                    1.8

Materials             Ovary (ICR-OV-l)   Prostate;PC-3)
                                   (a)              (a)

P. wallichina oil     9.9 [+ or -] 1.9     6.9 [+ or -]
                                                    1.2

[B.sub.1]-fraction     49 [+ or -] 0.6  21 [+ or -] 4.4

[UK.sub.13]-fraction                ND  39 [+ or -] 6.1

[I.sub.2]-fraction     25 [+ or -] 2.5  24 [+ or -] 6.6

[G.sub.2]-fraction                  ND               ND

[A.sub.2]-fraction                  ND    48.2 [+ or -]
                                                    2.5

[B.sub.2]-fraction                  ND    36.5 [+ or -]
                                                    1.8

(a.) The [IC..sub.50] value of oil and its fractions against cancer cell
are in [mu]g/ml.


Radical scavenging activity

The radical scavenging potential of essential oil of P. wallichina was determined by two different DPPH assays. The TLC based DPPH assay showed the presence of three yellow spots on spry with DPPH solution. The reduction of DPPH into [DPPH.sub.2] was the indication of oil to scavenge free radicals, independently without any enzymatic contribution. The scavenging effects of essential oil on DPPH were examined at 50 [micro]g/ml. The decrease in absorbance as a result of a color change from purple to yellow indicates the radical is scavenged by oil. The more rapidly the absorbance decreases, the more potent the antioxidant activity of the essential oil in terms of its hydrogen-donating capacity. All the concentrations of oil were able to reduce the stable free radical DPPH to the yellow colored 1,1-diphenyl-2-picrylhydrazyl. P. wallichina oil 50 [micro]/ml showed 61% and standard ascorbic acid at the same concentration showed 82% radical scavenging activity. Essential oil reduced the DPPH radical with efficacy less than ascorbic acid ([IC.sub.50] 11.5 [micro]g/ml), reaching the 50% of reduction with [IC.sub.50] value 28.8[micro]g/ml The results are arranged in form of Fig. 1. [beta]-Pinene was completely inactive at 50[micro]g/m1 against DPPH radical.

Discussion

Currently chemotherapy is regarded as one of the most efficient cancer treatment approach. Although chemotherapy significantly improves symptoms and the quality of life of the patients with cancer, only modest increase in survival rate can be achieved. Faced with palliative care, many cancer patients use alternative medicines, including herbal therapies (Juvekar et al. 2009). Numerous cancer research studies have been conducted using traditional medicinal plants in the form of specific herbal extracts and combinations to treat specific diseases including cancer, in an effort to discover new therapeutic agents that lack the toxic side effects associated with various chemotherapeutic agents (Adebayo et al. 2010).

Worldwide, breast cancer is the second most common type of cancer after lung cancer and the fifth most common cause of cancer death. Leukemia is one of the major disease in children and liver cancer is also growing because of day to day life style (Balijepalli et al. 2010). Hence it was decided to study anti-proliferative effect of P. wallichina oil on these cell lines.

P. wallichina oil was first time analyzed and revealed the presence of 17 compounds; some of these were present in major quantity. The oil and its fractions showed anti-proliferative activity against all the cell lines THP-1, A-549, HEP-2, PC-3 and IGR-OV-1 tested. Mitomycin-C and Paclitaxel were used as standards which inhibits the cancer by different mechanisms. Mitomycin-C is a potent DNA cross linker. A single crosslink per genome has shown to be effective in killing bacteria. This is accomplished by reductive activation followed by two N-alkylation's. Both alkylations are sequence specific for a guanine nucleoside in the sequence 5'-CpG-3'. Potential bis-alkylating heterocyclic quinines were synthesized in order to explore their antitumoral activities by bioreductive alkylation. Paclitaxel treated cells have defects in mitotic spindle assembly, chromosome segregation, and cell division. Unlike other tubulin targeted drugs such as colchicine that inhibit microtubule assembly, Paclitaxel stabilizes the microtubule polymer and protects it from disassembly. The inability of the chromosomes to achieve a metaphase spindle configuration leads to a mitotic block in which there is prolonged activation of the mitotic checkpoint with the subsequent triggering of apoptosis or slippage back into the G1 -phase of the cell cycle without cell division. The ability of Paclitaxel to inhibit spindle function is generally attributed to its suppression of microtubule dynamics; but recent studies have demonstrated that suppression of dynamics occurs at concentrations lower than those needed to block mitosis. At the higher antimitotic concentrations, Paclitaxel appears to act by suppressing microtubule detachment from centrosomes, a process normally activated during mitosis. The binding site for Paclitaxel has been shown to reside on the beta-tubulin subunit. P. wallichina essential oil at different concentrations showed more inhibitions as compared to Mitomycin-C and Paclitaxel. However the mechanism of action of P. wallichina essential oil could not follow either of the two Mitomycin-C or Paclitaxel mechanisms, but could be due to synergism of various components present in the oil. The various fractions of oil contained one or more than one component including caryophyllene oxide, [alpha]-cadinol, [alpha]-humulene, [alpha]-pinene, [beta]-pinene, [alpha]-phellandrene and trans-caryophyllene, responsible to exhibit moderate to strong anti-proliferative effects against these cell lines has been previously reported in one or other study to act as good anti-proliferative agents against the same or different tumor cell lines. The fraction G2 contained only caryophyllene oxide 82.7% showed similar results with our previous study against the same cell lines using different method like SRB-assay (Yousuf Dar et al. 2011).

The oil also showed radical scavenging activity with respect to ascorbic acid. The radical scavenging activity in the present study could be also clue to possible role of synergism between the various components of oil, because no phenolic compounds are present in the oil. In addition the major component of the oil [beta]-pinene was also tested and showed no radical scavenging effects. Therefore anti-proliferative activity of oil supports its radical scavenging activity, which are therefore both due to the possible role of synergism.

Conclusion

The results of the current study presented strong antiproliferative and radical scavenging activity against all the five human tumor cell lines tested and against the DPPH radical, respectively. P. wallichina oil could be regarded as promising drugs for cancer therapy, but their toxicity should be addressed. Further investigations are needed to confirm the pharmacological activity of this oil in animal models. This study may lead to utilize the plant in formulation of anti-proliferative agents which could be used to treat lung, liver, ovarian and other cancers.

Acknowledgments

We are grateful to A.H. Malik, Centre for Plant Taxonomy and Biodiversity, University of Kashmir, Hazratbal Srinagar for botanical identification of plant material. The authors also extend their sincere thanks to Dr. A.H. Dar, IIIM Jammu for evaluation of antiproliferative activity of various oil samples and also thank to Dr. Bikram Singh, Head NPP Division IHBT, Himachal Pradesh for analysis and identification of oil and its constituents by GC, GC-MS and [.sup.13]C NMR. In addition there is no conflict to disclose.

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* Corresponding author. Tel.: +91 09697380630.

** Corresponding author. Tel.: +91 09419054484.

E-mail addresses: yousufchemistry@gmail.com. claryousuf_123@yahoo.com (M. Yousuf Dar), doctorwajaht@gmail.com (W.A. Shah).

0944-7113/$--see front matter [C] 2012 Elsevier GmbH. All rights reserved.

http://dx.doi.org/10.1016/j.phymecl.2012.07.015

Mohd Yousuf Dara (a), *, Wajaht A. Shah (a), **, Sofi Mubashird (a), Manzoor A. Rather (b)

(a) Department of Chemistry, University of Kashmir. Srinagar,Jammu and Kashmir 190 006, India

(b) Medicinal Chemistry Division, Indian Institute of Integrative Medicines. Sanatnagar. Srinagar. Jammu and Kashmir, India
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Author:Dar, Mohd Yousuf; Shah, Wajaht A.; Mubashir, Sofi; Rather, Manzoor A.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Geographic Code:9INDI
Date:Oct 15, 2012
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