anti-mycobacterium and anti-cancer activities of combretin ... · abstract: combretin is the...

Journal of Pharmacy and Pharmacology 4 (2016) 88-98 doi: 10.17265/2328-2150/2016.02.005

Anti-mycobacterium and Anti-cancer Activities of Combretin, an Isolated Steroidal Alkaloid from the Seeds

of Combretum quadrangulare Kurz.

Khesorn Nantachit and Somjing Roongjang Department of Pharmaceutical Science, Faculty of Pharmacy, Chiang Mai University 50200, Thailand

Abstract: Combretin is the steroidal alkaloid isolated from the seeds of Combretum quadrangulare Kurz. by macerated powder of the seeds with 95% ethanol. Purified further by avicel column chromatography and preparative thin layer chromatography. Combretin was investigated for anti-mycobacterium and anti-cancer activities. Combretin was inactive against Mycobacterium tuberculosis but showed anti-cancer activities against human hepatocarcinoma (Hep G2) ATCC HB-8065 and human caucasian colon adenocarcinoma (Caco2) ATCC HTB-39 at concentration greater than 300 mcg/ml in DMSO (Dimethyl sulfoxide). Key words: Combretum quadrangulare, antimycobacterium, anticancer activities.

1. Introduction

Combretum quadrangulare Kurz is found throughout Thailand especially in open wet places. Therapeutic uses of this plant in the country were anthelmintics (the parts used were seeds, roots and leaves) and curing veneral disease (the part used were roots and wood) [1].

Aaemon S et al. 1980 [2] studied the chemical constituents of this plant. They found that alcoholic and other extracts from the roots and seeds could kill earthworms. They also found that crude extracts from the seeds showed antibacterial activity. The flavonoid compound found in this plant is Combretol. They further found new compounds from roots and seeds which were 3 compounds of pentacyclic triterpene carboxylic acid, viz 3β, 6β, 18β, trihydroxy-urs-12-en- 30-ic acid, 3,6-diketo-olean-12-en-28-oic acid and olean-12-en28-oic acid. They also found β-sitosterol and β-sitosteryl, 2 compounds of long-chain alcohol and amino compound.

Ian RC et al. 1985 [3] found three flavonoids from

Correspondence author: Khesorn Nantachit, M.S. in Pharm, Faculty of Pharmacy, Chiang Mai University, Thailand, Natural Product Chemistry.

the flowers of this plant which were 1)5-hydroxy-3,7-dimethoxy-2(3’,4’,5’-trimethoxyphenyl)-4H-1benzopyran-4-one (Combretol) 2)5-hydroxy-2-(3’-hydroxy-4’-methoxyphenyl)-3,7-dimethoxy-4H-1-benzopyran-4-one (Ayanin) and 3) Polymorhic form of 5-hydroxy-2-(4’-hydroxy -3’,5’-dimethoxyphenyl)-3-7-dimethoxy-4H-1-benzopyran-4-one Perapol Y et al 1988 [4] Studied the anthelmintic

activity of seeds of C. quadrangulare Kurz. for roundworms in young buffalo. They found that after young buffalo ate the seeds once, the number of eggs of Neoascaris vitulorum in feces was reduced and completely disappeared within 1-3 weeks. They also studied the toxicity of the seeds of this plant and found that seed extracts were not toxic to albino rat and mice. The dose that they studied did not kill the rat and no side effect was found within 48 hours after giving the extract.

Banskota et al. 1998 [5] found seven novel cycloartane-type triterpenes which were tested for their cytotoxicity against murine colon 26-L5 carcinoma cells. Methyl quadrangulare B and methyl quadrangularate D exhibited potent cytotoxicity

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Anti-mycobacterium and Anti-cancer Activities of Combretin, an Isolated Steroidal Alkaloid from the Seeds of Combretum quadrangulare Kurz

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having ED 50 value 9.54 and 5.42 microM, respectively.

Banskota et al. 2000 [6] found methyl quadran-gularates A-D, two known cyclo-artane-type triterpenes ; methyl 23-deoxy-jessate and 4β-14α-dimethyl-5α-ergosta-9β, 19-cyclo-24(31)-en- β-hydroxy -4α-carboxylic acid and two known flavonoids,5,7,4’-trihydroxy-3,3’-dime-thoxyflavone and 5,4’-dihydroxy-3,7,3’-trimethoxyflavone. All the isolate compounds were tested for their cytotoxicity towards highly liver metastatic murine colon 26-L5 carcinoma cells and the cycloartane-type triterpenes showed various degrees of cytotoxicity, whereas all the flavonoids possessed strong cytotoxicity with ED 50 values equal to a less than 6 microM.

Banokota AH et al. 2000 [7] found that ‘quadrangularol B, methyl quadran-gularate I, kamatakenin, 5,7,4’-trihydroxy-3,3’-trimetho-xy-flavone and isokaemferide showed strong inhi-bitory effect on TNF-alpha-induced cell death with IC50 values of 34.3, 33.7, 13.3, 22.4, 13.4 and 22.8 microM, respectively. Methyl quadrangularates A and N, norqua-drangularic acid B and vitexin also showed potent inhibition on TNF-alpha-induced cell death with IC50 values of 45.7, 89.3, 67.6, and 40.1 microM, respectively. The flavonoids and some of the cycloartane-type triterpenes appeared to be the hepatoprotective principles of the leaves of Combretum quadrangulare Kurz.

Adnyana IK et al. 2000 [8] isolated five new triterpene glucosides, quadranoside I - V from MeOH extract of the seeds of Combretum quadrangulare. Three new triterpene gluco-sides showed significant hepatoprotective effects against D-Gal N (D-galactosamine)/ TNF-alpha (tumor necrosis factor)-induced cell death in primary cultured mouse heap-tocytes.

Adnyana IK et al. 2000 [9] found that the MeOH extract of Combretum quadrangulare Kurz seeds showed the strongest inhibitory effect on

D-GalN/TNF-alpha-induced cell death (IC50, 56.4 μg/ml). Moreover, the MeOH extract also significantly lowered the sGPT (serum glutamic pyruvic transaminase) level on D-GalN/ LPS (lipopolysaccharide)-induced liver injury in mice. Among the isolated triterpene glucosides, lupine-type (1-3; IC50, 63.1, 59.8 and 76.2 microM, respectively) and ursane-type (IC50; 30.2, and 34.6 microM, respectively) compounds exhibited strong hepato-protectively activity, 1-O-Galloly-6-O-(4-hydroxy-3, 5-dimetho-xy) benzoyl-beta-D-glucose (26; IC50, 7.2 microM), methyl gallate (28; IC50, 19.9 microM), (-) 4 epicatechin (31; IC50, 71.2 microM) also had a potent hepatoprotective effect on D-GalN/TNF-alpha-induced cell death in primary cultured mouse hepatocytes.

Adnyana IK et al, 2001 [10] had test for hepatoprotective activities of three new triter-penes of the lupane type, 2-α, 6β-dihydroxy-betulinic acid and 6β-hydroxyhovenic acid, and on oleanane type, 6β-hydroxyargunic acid isolated from methanolic extract of the seeds of Combretum quadrangulare Kurz. against D-GalN/TNF-alpha-induced cell death in primary cultured mouse hepatocytes.

Adnyana IK et al, 2001 [11] had isolated a new gallic acid derivative, 1-O-gallory-6-O-(4-hydroxy-3, 5-dimethoxy) benzoyl-β-D-glucose from an water-fraction of MeOH extract of Combretum quadrangulare Kurz seeds which exhibited potent hepatoprotective activitiy against D-GalN/TNF-alpha-induced cell death in primary culture mouse hepatocytes with IC50 of 3.3 microM.

Toume K et al. 2001 [12] had isolated nine new cycloartane triterpenes, combretanones A-G, combretic acid A and combretic acid B from MeOH extract of Combretum quadrangulare Kurz leaves. The known oleanane triterpenes and six flavonoids were also isolated. Compounds 7, 9, 12, 16 and 17 enhanced DR5 (death-receptor 5) expression, and 16 showed TRIAL-resistance abrogating activity.


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From our preliminary work Khesorn N. et al (2006) [13] found that crude methanolic extract from the seeds of C. quadrangulare showed MIC against Acenobacter baumannii at 937.50 mcg/ml and showed MIC against Staphyllococus aureus ATCC 25923 at 312 mcg/ml and also showed antibacterial activity against Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Klebcilla pneumonia ESBL non-producing strain. Pure compound, combre-tin (Fig. 3) also showed MIC against E. coli ATCC 25922 at 2625 mcg/ml, MIC against P. aeruginosa ATCC 27853 at 10,500 mcg/ml and showed no effect against S. aureus ATCC 25923. (Khesorn et al, unpublished data)

The objective of this investigation is to isolate pure compound, combretin that might show antibacterial activity against Mycobacterium tuberculosis and Anti-cancer activities.

2. Materials and Methods

This research was conducted as an experimental study to isolate a new compound from the seeds of Combretum quadrangulare for anti-mycobacterium and anti-cancer activities.

2.1 Sample Preparations

Combretum quadrangulare speci-mens were collected from around Chiang Mai Province in 2010. Mature seeds were collected (Nantachit, voucher No.2) and kept in CMU’s Pharmacy Herbarium. The seeds were dried at 40°C and powdered. One kilogram of powder was macerated with 3 litres of 95% ethanol. Each replicate was macerated for 1 day, filtered and repeated 2 times. The filtrate was evaporated in a vacuum. The residue (crude extract) was brownish black. The percent yield of material collected was 14.38.

2.2 Equipments

1. NMR (Nuclear Magnetic Resonance) spectra were recorded on a Bruker FT NMR 500 MHz

spectrometer. 2. Mass spectrum was recorded by a micrOTOF

mass spectrometer.

2.3 Chemical reagents

1. Column chormatography adsorbent —Microcrystalline cellulose powder Merck 70-230

mesh (Avicel) 2. Preparative chromatography adsorbent —Microcrystalline cellulose powder Merck

(Avicel)

2.4 Purification of Crude 95% Ethanolic Extract in 2010

Crude 95% ethanolic extract 1.5 g was purified by column chromatography. Avicel was used as the adsorbent and the column was eluted with 50% methanol in water. A total of 3 fractions 10 ml each, were collected. Each fraction was sound to produce the same spot in thin layer chromatogram. Each fraction was vacuumed by vacuum pump in order to remove methanol in a cool condition because the sample fractions might be heat-labile. The remaining water residue was removed by freeze-drying and the residues combined and further purified with preparative thin layer chromatogram (PTLC) twice. Avicel was used as an adsorbent with 1 mm. thickness and developed with 80% methanol in water. The pure compound obtained from second PTLC.

2.5 Physical and Spectroscopic Properties of Pure Compound

2.5.1 Physical Data The pure compound was pale yellow solid.

Molecular formula of the pure compound is C52H83ON and molecular weight is 737.4804. The pure compound was soluble in water, methanol and ethylacetate. Melting point could not be determined because %yield was very low.

2.5.2 Spetroscopic Data 1H NMR spectrum


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Structure of isolated pure compound based on 1H NMR data was detailed as follwed

First part, 52 protons (Steroidal part), of 33 protons, 15 protons of side chain (C23-C29)’’, δ = 4.9 ppm, J = 1455.38 Hz coupling with 3 protons; 2 protons of methylene groups at C2” positon and 1 proton of methane group at C1” position, δ = 4.5 ppm, J = 1369.73 Hz and coupling with 1 proton of amino group in aziridine ring, δ = 5.3 ppm, J = 1,600 Hz (FigS. 1 and 3)

Second part, 31 protons (Steroidal skeleton), 20 protons of methylene groups, δ = 1.3 ppm, J = 380.92 Hz coupling with 2 protons of methylene groups of C19’ position, δ = 2.0 ppm, J = 612.48 Hz and coupling with 1 proton of olefinic carbon C4’ position, δ = 2.3 ppm, J = 694.82 Hz. And, these 20 protons of methylene groups also coupling with 8 protons of angular methine groups, δ = 0.8 ppm, J = 264.70 Hz and 257.40 Hz (4 protons were coupling each other). (Figure 2 and 3)

Mass Spectrum There were four fragment ions that refered to pure

compound. Fragment ion I, m/z = 457.3318, Fragment ion II, m/z = 301, Fragment ion III, m/z = 369.2695 and Fragment ion IV, m/z = 341.2439. (Fig. 4)

Stereochemistry of ether group was at β-position and aziridine was at α-position (Fig. 5) which was the results of substituted group at 3’, 4’ position of ring A’ (like hormone androgen e.g. 4-Dihydrotes- tosterone usually was double bond and hydroxyl group). Aziridine ring substituted at 3-position was

low stability so the steric hindered at 3-position was easily occurred so fragment ion I was happened. (Fig. 4)

Stereochemistry of first part of the pure compound, A-B, B-C, C-D and D-E ring junction should be all trans-configuration in order to form more stability because the first and second part of the pure compound were large molecules which steric hindrance should be occurred easily (Fig. 3). From the introduction pentacyclic triterpene carboxylic acid, β-sitosterol and β-sitosteryl were isolated. It showed that chemical constituents of C. quadrangulare consisted of terpene and sterol compounds but there was no terpene skeleton at position 3 and 3’. This is the information which confirms that pure compound is a steroroidal compound.

2.6 Determining Anti-mycobacterium Tuberculosis Activity of Crude 95% Ethanolic Extract of C. quadrangulare Seeds [14, 15]

Anti-TB (Anti-Mycobacterium tuberculosis) H37Ra strain was determined by GFP-MA (Green fluorescent protein microplate assay), using microplate reader (Synergy MX). Concentration of crude 95% ethanolic extract used was 300 mcg/ml in DMSO (Dimethyl sulfoxide) Table 1.

2.6.1 Preparation of Sample Samples are prepared to routine stock of 10 mg/ml

with 100% DMSO and stored in freezer until used. Before assayed, samples are diluted to 0.5 mg/ml with sterilized water and followed by 2-fold serially dilution with 5% (v/v) DMSO.

Fig. 1 Structure of the first part of pure compound.


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Fig. 2 Structure of the second part of pure compound.

Fig. 3 Molecular ion of pure compound.

Fig. 4 Fragment ions I,II, III and IV.


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Fig. 5 Stereochemistry of ether group and aziridine ring.

Fig. 6 1H NMR spectrum of pure compound.

Fig. 7 Mass spectrum of pure compound.


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Table 1 Anti-Mycobacterium tuberculosis (Anti-TB) H37Ra strain test of crude 95% ethanolic extract.

Item Screening code Sample code

Final concentration (μg/ml)

Fluorescence unit at Day 0

Fluorescence unit at Day 10 % Inhibition Activity MIC

(μg/ml) Average SD Average SD Negative Cell+DMSO 0.5% DMSO 2629 141 5392 660 0.00 - - Positive 1 Rifampicin 0.025

0.0125 0.0063 0.0031 0.0016 0.0008

2866 2798 2830 2864 2875 2809

96 202 158 163 130 127

1935 3537 3972 4321 4428 4632

48 256 401 300 283 612

133.67 73.16 58.67 47.25 43.80 34.04

Active Inactive Inactive Inactive Inactive Inactive

0.0250

Positive 2 Ofloxacin 3.13 1.56 0.781 0.391 0.195 0.098

2884 2921 2980 2707 2786 2882

37 38 104 94 69 42

2090 2064 4661 4756 4829 4892

39 24 558 603 823 621

128.72 131.03 39.15 25.86 26.05 27.27

Active Active Inactive Inactive Inactive Inactive

1.56

Positive 3 Streptomycin 1.25 0.625 0.313 0.156 0.078 0.039

2769 2763 2872 2778 2800 2905

33 70 127 55 91 121

2110 2984 3287 3956 4714 5032

48 232 463 684 730 377

123.86 92.00 84.97 57.37 30.71 23.03


0.625

Positive 4 Isoniazid 0.375 0.188 0.0938 0.0469 0.0234 0.0117

2955 2839 2830 2907 2842 2878

131 181 36 133 133 52

2017 2011 2020 2030 4362 4506

65 51 52 55 210 96

133.92 129.99 129.33 131.73 44.98 41.06

Active Active Active Active Inactive Inactive

0.0469

Positive 5 Ethambutol 3.75 1.88 0.938 0.469 0.234 0.117

2967 2935 2736 2808 2815 2790

107 135 122 111 111 70

2120 3185 4127 4181 4659 4738

47 133 235 126 188 334

130.65 90.96 49.64 50.29 33.26 29.50


1.88

1 V9507* Crude 95% ethanolic extract

300.00 150.00 75.00 37.50 18.75

2181 2508 2594 2768 2884

56 143 102 105 41

4160 5294 4645 4875 4887

185 310 214 97 903

28.37 35.48 25.77 23.75 27.51

Inactive Inactive Inactive Inactive Inactive

-

Notes: Negative control : 0.5% DMSO MIC of positive control: Rifampicin = 0.025 mcg/ml, Ofloxacin = 1.56 mcg/ml, Streptomycin = 0.625 mcg/ml, Isoniazid = 0.0469 mcg/ml, Ethambutol = 1.88 mcg/ml. V9507*, crude 95% ethanolic extract = Inactive at concentration 18.75-300 mcg/ml in DMSO.

2.6.2 Preparation of TB Culture GFP (green fluorescent protein) expressing M.

tuberculosis H37Ra Strain was established by Changsen, et al (2003). H37Ra gfp is cultivated on 7H10 agar containing 30 μg/ml kana-mycin at 37 °C for 4 weeks or until growth is observed.

For starter cultivation, 2-3 single colonies are inoculated into 10 ml of 7H9 broth supplemented with 0.2% v/v glycerol, 0.1% W/v of casitone, 0.05% v/v Tween 80, 10% v/v Middlebrook OADC enrichment solution and 30 μg/ml of kanamycin. The culture is

then incubated in a rotary shaker 200 rpm at 37 °C for weeks until the optical density at 550 nm (OD550) is between 0.5-1.

For batch cultivation, the starter cultures are transferred at the rate of 1/10 volume to the 7H9 broth and incubated in shaking condition until the OD550 is approximately 0.5 to 1. The cells are pelleted by centrifugation at 8,000 rpm for 10 minutes, washed twice and suspended in PBS buffer before sonicated for 15 seconds each of 8 times to disperse the clumps. TB suspension is then aliquoted and stored at −80°C


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for 2 to 3 months. Each of TB stock is quantified by cfu (colony forming unit) assay. Dilution titer of TB suspension that grows at logarithmic phase on day 10 is selected for an optimal bacterial seeding density in the assay.

2.6.3 Preparation of Assay Plate Test materials are assayed in 384-well plates in

triplicate. Each well contains 5 μl of TB suspension to finally obtain 1x105 cfu/ml. Plate is incubated at 37 °C for 10 days. Fluorescence is measured at 485 nm excitation and 535 nm emission wavelengths by using the bottom-reading mode of fluorometer. The signal is subtracted with that of day 0 before calculation.

2.6.4 Activity Analysis The percentage of Inhibition to the growth of TB is

calculated by the following equation: % Inhibition = [1-(FUT/FUC)]x100 Where FUT and FUC are the mean fluo-rescent unit

of cells treated with test compound and that treated with 0.5% (v/v) DMSO, respectively.

The % Inhibition can be interpreted to the anti-TB activity by this cutoff criteria:

If % inhibition is < 90%, the activity is reported as “Inactive”.

If % inhibition is ≥ 90%, the activity if reported as “Active”.

MIC value can be determined.

2.7 Determing Anti-Cancer Activities of Crude 95% Ethanolic Extract of C. quadrangulare Seeds [16].

Crude 95% ethanolic extracts were determined for Anti-Cancer activities as followed.

Cytoxicity test against human hepato-arcinoma (Hep G2) ATCC HB-8065, Anti-NCI-HB 187-small cell lung cancer and cytotoxicity test against human caucasion colon adenocarcinoma (CaCo2) ATCC HTB-37 were determined by Resazurin. Microplate assay (REMA method). Cytotoxicity test against Vero cells (African green monkey kidney) was determined by GFP (Green Fluorescent Protein)-based assay. Concentration of crude extracts used were 300 mcg/ml in DMSO.

2.7.1 Sample Preparation Samples are solubilized with 100% DMSO to 10

mg/ml and stored in freezer until use. Samples are diluted to 100 μg/ml with culture medium and followed by 2-fold serial dilution with 1% DMSO in medium.

2.7.2 Determining Cytotoxicity against Hep G2 (Human Hepatocyte Carcinoma) Cell Line and Cytotoxicity against Human Colon Adenocar-Cinoma (Caco 2 cell line)

Hep G2 cell line (ATCC HB 8065) is isolated from human epithelial hepatocellular carcinoma cells or

Table 2 Cytotoxicity against human hepatocarcinoma (Hep G2) ATCC HB-8065 test of crude 95% ethanolic extract.

Item Screening code Sample code Final concentration

(μg/ml) Fluorescence unit

% Survival Activity IC50 (μg/ml) Average SD

Negative Cell+DMSO 1% DMSO 23980 1844 100.00 - - Positive Ellipticine 10.00

5.00 2.50 1.25 0.625 0.313

1942 5318 23855 25159 26101 27093

347 660 1404 2775 2426 1966

8.10 22.18 99.48 104.92 108.84 112.98

cytotoxic 3.89

1 V9507* Crude 95% ethanolic extract

300.00 150.00 75.00 37.50 18.75 9.38

11275 15075 17965 20576 22232 24845

469 1343 1004 390 1548 833

47.02 62.86 74.92 85.81 92.71 103.61

cytotoxic 268.24

Notes: Negative control: 1% DMSO positive control Ellipticine, IC50 = 3.89 mcg/ml, cytotoxic V9507*, crude 95% ethanolic extract, IC50 = 268.25 mcg/ml, cytotoxic.


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Table 3 Cytotoxicity against Vero cells (African green monkey kidney) test of crude 95% ethanolic extract.

Item Screening code Sample code

Final concentration (μg/ml)

Fluorescence unit at Day 0

Fluorescence unit at Day 4 % Cell

growth Cytotoxicity IC50 (μg/ml)Average SD Average SD

Negative Cell+DMSO 0.5% DMSO 1448 56 3572 153 100.00 - -

Positive Ellipticine 4.00 2.00 1.00 0.50 0.25 0.13

1447 1467 1462 1454 1479 1435

91 73 54 73 62 54

1499 1724 2213 2896 3346 3468

57 87 144 213 212 259

2.42 12.12 35.35 67.91 87.92 95.70

Cytotoxic Cytotoxic Cytotoxic Non-cytotoxicNon-cytotoxicNon-cytotoxic

1.47

1 V9507 (Blank-well)**

Crude 95% ethanolic extract

300.00 100.00 33.33 11.11 3.70 1.23

1698 1260 1006 914 936 978

82 107 39 73 18 32

2657 1982 1284 1156 1154 1177

69 116 44 17 65 12

- - - - - -

- - - - - -

- - - - - -

1 V9507* (Tested-well)

Crude 95% ethanolic extract

300.00 100.00 33.33 11.11 3.70 1.23

1962 1580 1404 1369 1342 1350

78 35 33 54 55 34

2910 3585 3744 3727 3787 3777

26 143 92 249 138 149

-0.53 60.43 97.05 99.65 104.87 104.90

Cytotoxic Non-cytotoxicNon-cytotoxicNon-cytotoxicNon-cytotoxicNon-cytotoxic

120.035

Notes: Negative control: 0.5% DMSO Positive control Ellipticine, IC50 = 1.47 mcg/ml, cytotoxic **V9507 (Blank-well), crude 95% ethanolic extract, IC50 = Negative. *V9507 (Tested-well), crude 95% ethanolic extract, IC50 = 120.35 mcg/ml, Cytotoxic.

Caco2 cell line (ATCC HTB-37) is isolated from human epithelial colorectal adenocarcinoma cells. Cells are grown and maintained in a complete medium MEM (minimal essential medium) supple-mented with 10% heat-inactived fetal bovine serum, 2 mM L-glutamine, 0.1 mM non-essential amino acid, (1.0 mM sodium pyruvate for Hep G2 or 0.1 IU/ml Insulin-Transferrin-Selenium-X for Caco2) 1.5 g/L sodium bicarbonate, 100 unit/ml penicillin and 100 μg/ml streptomycin) and incubated at 37 °C humidified incubator with 5% CO2. Cells at a logarithmic growth are harvested and diluted to 2x104 cells/ml in complete medium prior to assay.

2.7.3 Assay Description This assay is performed in four replicate wells in

96-well plate. First, plates were seeded with 200 μl of cell suspension or blank medium into well, and incubated at 37 °C humidified incubator with 5% CO2 for 48 hours. Subsequently, culture medium was replaced with 200 μl of fresh medium containing 100 μl/ml of test-compounds or 1% DMSO, and plates were further incubated for 24 hours. After incubation

period, the plates was added with 50 μl of 125 μg/ml resazurin solution and incubated at 37 °C humidified incubator with 5% CO2 for 4 hours. Fluorescence is measured at 530 nm excitation and 590 nm emission wavelengths by using the bottom-reading mode of fluorometer. The signal is subtracted with blank before calculation.

2.7.4 Activity Analysis The percentage of cytotoxicity is calculated by the

following equation: % Cytotoxicity = [1-(FUT/FUC)] x 100 Whereas FUT and FUC are the mean fluo-rescent

unit from cells treated with test compound and that treated with 1%DMSO, respectively.

The % cytotoxicity can be used to interpret to the activity level of compounds against to this cell line by this cutoff criteria:

If % cytotoxicity is < 50%, the activity is reported as “Non-cyto-toxic”.

If % cytotoxicity is ≥ 50%, the activity if reported as “Cytotoxic”.

The IC50 value is derived from dose-response-curve that is plotted between % cytotoxicity versus the


97

sample concentrations by using SOFTMax Pro software (Molecular Devices, USA)

2.8 Determing Anti-cancer Activities of Pure Compound, Combretin

Anti-cancer activities of pure compound, combretin were determined against KB-Oral cavity cancer, MCF7-breast cancer, NCI-H187-Small cell lung cancer, human hepatocarcinoma (Hep G2) ATCC HB-8065 and human Caucasian colon adenocarcinoma (Caco2) ATCC HTB-37 by REMA

method. Concentration of combretin used in each test was 100 mcg/ml in DMSO.

3. Results and Discussion

3.1 Anti-Mycobacterium Tuberculosis Activity

Crude 95% ethanolic extract was inactive against Mycobacterium tuberculosis even if crude methanolic extract and combretin showed good antibacterial activities (Khesorn, 2006) because M. tuber-culosis consists of thick and strong cell wall.

Table 4 Cytotoxicity against human hepatocarcinoma (Hep G2) ATCC HB-8065 test of pure compound, Combretin.




Negative Cell+DMSO 1% DMSO 21670 624 100.00 - - Positive Ellipticine 10.00

5.00 2.50 1.25 0.625 0.313

236 400 465 1354 14992 20184

70 137 80 151 1033 1478

1.09 1.85 2.14 6.25 69.18 93.14

cytotoxic 0.733

1 V9460* Combretin 100.00 50.00 25.00 12.50 6.25 3.13

19408 19626 20107 20205 19864 20126

909 842 1134 1368 1176 1214

89.56 90.57 92.79 93.24 91.67 92.88

Non-cytotoxic -

Notes: Negative control: 1% DMSO Positive control Ellipticine, IC50 = 0.733 mcg/ml, cytotoxic V9460*, Combretin, non-cytotoxic, IC50 greater than 100 mcg/ml.

Table 5 Cytotoxicity against human caucasian colon adenocarcinoma (Caco2) ATCC HTB-37 test of pure compound, Combretin.




Negative Cell+DMSO 1% DMSO 4256 472 100.00 - -

Positive Ellipticine 40.00 20.00 10.00 5.00 2.50 1.25

1246 1889 2383 2813 2809 3106

203 140 360 164 214 356

29.27 44.38 56.00 66.09 66.00 72.98

cytotoxic 14.96

1 V9433* Combretin 100.00 50.00 25.00 12.50 6.25 3.13

2971 3750 3554 3708 3915 3762

328 150 518 430 355 411

69.80 88.12 83.51 87.13 92.00 88.39

non-cytotoxic -

Notes: Negative control: 1% DMSO. Positive control. Ellipticine, IC50 = 14.96 mcg/ml, cytotoxicity. V9433*, Combretin, non-cytotoxic, IC50 greater than 100 mcg/ml.


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3.2 Anti-Cancer Activities of Crude 95% Ethanolic Extracts and Pure Compound, Combretin

Crude 95% ethanolic extracts showed cytotoxicity to all cancer cells test at 300 mcg/ml in DMSO. Combretin showed no cytotoxicity against KB-Oral cavity cancer cells, MCF7-breast cancer cells and NCJ-H187-small cell lung cancer cells because crude extract consisted of many constituents that showed cytotoxicity effect. If we raise combretin concentration grater than 100 mcg/ml it may show cytotoxicity against human hepatocarcinoma (Hep G2) ATCC HB-8065 and human caucasian colon adenocarcinoma (Caco 2) ATCC HTB-37 (Tables 4 and 5)

5. Conclusion

Combretin may be used as anti-cancer drug against liver and colon cancer if we modify structure in order to reduce IC50.

Acknowledgement

The author was grateful to Chiang Mai University and Professor Dr.Stang Mongkonsuk who was the first investigator of the work on C. quadrangulare in Thailand.

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anti-mycobacterium and anti-cancer activities of combretin ... · abstract: combretin is the...

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