Development of dietary supplement Manose-CT for cancer prevention/treatment from semipurified Job s tear extract (Coix lacryma-jobi)

Transcription

1 Development of dietary supplement Manose-CT for cancer prevention/treatment from semipurified Job s tear extract (Coix lacryma-jobi) Prof.Dr. Jiradej Manosroi Prof.Dr. Aranya Manosroi Manose Health and Beauty Research Center 179 Moo 10 Klong, Cholpratan Road, Suthep, Muang, Chiang Mai, 50200, Thailand 1

2 Introduction Job s tear Job s tear (Coix lachrymajobi), the native plant of Southeast Asia, exists in the wild and cultivated. It is a tall grain-bearing tropical plant of the grass family, Poaceae or Graminaeae. This plant has also been widely used as a diuretic, stomachic, analgesic, arthritis and antispasmodic agent from ancient times. 2

3 Anti-cancer activity of Job s tears Some bioactive compounds in Job s tears, especially coixenolide, inhibited tumors, prevented cancer and protected against viral infection. The methanol extract of Job's tear exerted an anti-proliferative effect on A549 lung cancer cells. The emulsion of Job's tears oil was approved by the Chinese Ministry of Public Health for the anti-cancer activity. This preparation has been used for the treatment of various cancers, including lung, breast and liver cancers. 3

4 Method Collect Job s tears seeds Preparation the crude and semi-purified extracts Bioactivity tests Selected the highest bioactivity semi-purified extracts In vivo anti-cancer activity test in xenograft nude mice Acute toxicity study in animal Development of dietary supplement product Stability study of finished product Calculation of production cost 4

5 Collect Job s tears seeds 5 Types of Job s tear TBP: Thai Black Phayao TBL: Thai Black Loei LBL: Laos Black Loei LB: Laos Black Luang Phra Bang LWL: Laos White Loei 5

6 Preparation the crude extracts Sample of Job s tears parts (Hull,Endosperm and Whole) Processed by Non cooking, Roasting, Boiling and Steaming Solvent I Solvent II Non cooking/roasting = Hot/Cold extraction Boiling/Steaming = Hot/Cold extraction /Water/Hexane Part Non cooking/roasting = Hot/Cold extraction Boiling/Steaming = Hot/Cold extraction /Water Part 180 crude extracts from solvent I 150 crude extracts from solvent II 330 crude extracts 6

7 Preparation the semi-purified extracts Job s tears Solvent II extracts partitioned with various solvents Semi-purified extracts 7

8 Characteristic and %yield Cells Crude extracts Fraction Weight % Yield Appearance and Odor KB M-WTBL-S1 (0.54 g) Fraction A g light yellow oil and light odor HT-29 M-HTBL-N1 (0.2 g) Fraction B g light brawn solid and light odor HeLa M-WLWL-R2 (0.72 g) Fraction C g brawn oil and light odor HepG2 M-HLBL-R1 (0.23 g) Fraction D g black brawn oil and light oder DU-145 H-WLWL-R2 (2.70 g) Fraction E g light yellow oil and light odor 8

9 Bioactivity test Anti-proliferative activity by Sulforhodamine B (SRB) assay Apoptotic activity by Acridine Orange (AO) and Ethidium Bromide (EB) staining Anti-oxidative activities test Free radical scavenging activity by DPPH assay Metal chelating activity Lipid peroxidation inhibition Immunomodulation test by nitroblue tetrazolium (NBT) assay 9

10 Anti-proliferative activity Cells Crude extracts Semi-purified extracts IC 50 (µg/ml) Fold of crude extract Standard drugs Fold of std. KB M-WTBL-S1 (43.61±0.76 µg/ml) Fraction A 5.93± Doxorubicin (0.21±0.73 µg/ml) HeLa M-WLWL-R2 (44.03±0.84 µg/ml) Fraction B 0.97± Doxorubicin (7.59±0.39 µg/ml) 7.82 HT-29 M-HTBL-N1 (53.05±2.61 µg/ml) Fraction C 2.11± fluorouracil (0.02±0.56 µg/ml) HepG2 M-HLBL-R1 (215.54±1.06 µg/ml) Fraction D 3.57± Vincristine (0.22±0.97 µg/ml) DU-145 H-WLWL-R2 (60.15±0.47 µg/ml) Fraction E 47.32± Doxorubicin (1.37±0.58 µg/ml) A549 H-WLWL-R2 (317.42±0.19 µg/ml) Fraction F ± Vincristine (0.36±0.10 µg/ml)

11 Apoptotic activity Cells Crude extracts Semi-purified extracts % Apoptosis Fold of std. Standard drugs % Apoptosis KB M-WTBL-S1 Fraction A Cisplatin HeLa M-WLWL-R2 Fraction B Doxorubicin HT-29 M-HTBL-N1 Fraction C Doxorubicin HepG2 M-HLBL-R1 Fraction D Cisplatin DU-145 H-WLWL-R2 Fraction E Doxorubicin A549 H-WLWL-R2 Fraction F Vincristine

12 Anti-oxidative activities Cells Extracts Free radical scavenging activity SC50 (mg/ml) Fold of std. Metal chelating activity MC50 (mg/ml) Fold of std. Lipid peroxidation inhibition activity IPC50 (mg/ml) Fold of std. KB M-WTBL-S1: Fraction A 4.88± HeLa M-WLWL-R2: Fraction B 0.88± HT-29 M-HTBL-N1: Fraction C ± HepG2 M-HLBL-R1: Fraction D 56.55± DU-145 H-WLWL-R2: Fraction E > ± A549 H-WLWL-R2 : Fraction F > ± Commercial (Kanglaite ) > ± Ascorbic acid 0.20±0.02 ND 0.02±0.009 EDTA ND 0.51±0.07 ND 12

13 Immunomodulation test Sample Phagocytotic activation (%) Folds of positive control Positive Control (20 µg/ml LPS in serum) 32.0 ± Separation of whole blood after centrifuged with Polymorphprep Negative Control (PBS) 8.9 ± Negative Control (10 % DMSO PBS) 10.3 ± Kanglaite 52.0 ± M-WTBL-S1: Fraction A 13.0 ± M-HLBL-R1: Fraction B 17.3 ± Positive result of immunomodulation from NBT test M-HTBL-N1: Fraction C 26.3 ± M-WLWL-R2: Fraction D 21.7 ± H-WLWL-R2: Fraction E 11.7 ± H-WLWL-R2: Fraction F 16.7 ±

14 Summary of bioactivity tests Cells Code Fraction SRB IC 50 (µg/ml) DPPH SC 50 (mg/ml) Metal chelate MC 50 (mg/ml) % apoptosis % phagocytosis % yield KB M-WTBL-S1 Fraction A 9.72±0.57 (0.02 Folds of doxorubicin) 4.88±1.73 (0.04 Folds of ascorbic acid) (1.05 Folds of cisplatin) 13.0±1.7 (0.41 Folds of LPS) HepG2 M-HLBL-R1 Fraction B 3.57±0.20 (0.06 Folds of vincristine) 56.55±9.77 (0.003 Folds of ascorbic acid) (1.41 Folds of cisplatin) 17.3±2.1 (0.54 Folds of LPS) 12.3 Selected HT-29 M-HTBL-N1 Fraction C HeLa M-WLWL-R2 Fraction D 2.11±0.41 (0.009 Folds of 5-FU) 0.97±0.82 (7.82 Folds of doxorubicin) 3.69±0.60 (0.05 Folds of ascorbic acid) 0.88±0.44 (0.22 Folds of ascorbic acid) (0.42 Folds of doxorubicin) (0.19 Folds of doxorubicin) 26.3±2.5 (0.82 Folds of LPS) 21.7±3.2 (0.68 Folds of LPS) DU-145 H-WLWL-R2 Fraction E 47.32±0.69 (0.02 Folds of doxorubicin) > ±0.38 (1.02 Folds of EDTA) (0.75 Folds of doxorubicin) 11.7±2.5 (0.37 Folds of LPS) 2.49 A549 H-WLWL-R2 Fraction F ±0.29 (0.001 Folds of vincristine) > ±16.18 ( Folds of EDTA) 0.88 (0.01 Folds of vincristine) 16.7±3.5 (0.52 Folds of LPS)

15 In vivo anti-cancer activity test in xenograft nude mice 1 week 5 weeks Low dose Feed Feed&Treat Sacrify&Observe Scheme of cancer prevention test of semi-purified Job s tear extract in xenograft nude mice Medium dose High dose Kanglaite Distilled water No treatment % inhibition of normalized tumor weight from HeLa xenograft nude mice Tumor from HeLa xenograft 26 nude mice 15

16 Low dose Medium dose High dose Kanglaite Distilled water (Negative control) No Treatment (Negative control) 16

17 Acute toxicity study in animal After treated with Job s tear extract in 5,000 mg/kg per bodyweight for 24 hr, The result show that no animal die in male and female rat. Therefore, LD50 of semi-purified Job s tear extract was more than 5,000 mg/kg. Control Treated Histology of lung from female rat after treated with semi-purified Job s tear extracts (at 40x magnification) The weight of internal organ from male rat in treated group was not difference from control group. The weight of internal organs from treated female rat such as lung and liver were significant increased (p < 0.05) when compared with control group. Control Treated Histology of liver from female rat after treated with semi-purified Job s tear extracts (at 40x magnification) For the examination of histology in female rat, it found that the tissue of internal organ between treated and control group was not difference. 17

18 Development of dietary supplement product contained with semi-purified Job s tear extract Preparation of dietary supplement product The preparation of capsule product contained with semi-purified Job s tear extract has the component by following: Cap-O-Sil: Oil absorbent from the extracts Corn starch: Diluent Acacia solution: Binder Talcum: Glidant Result: Light brawn granule, Unique odor Disintegration time The disintegration time of capsule product was 3 min 25 second in acetate buffer with similarly gastric condition. The disintegration time of product was pass the USP standard (not more than 30 minutes). Granule of semi-purified Job s tear extract Dietary supplement of semipurified Job s tear extract Analytical balance Disintegration apparatus 18

19 Stability study of finished product T0 4 0 C at week 8 Percentage of linoleic acid remaining in capsule product contained with semi-purified Job s tear extract after kept at 4±2, 25±2, 45±2 and 60±2 o C Temp (0C) 4±2 % Linoleic acid remaining in capsule T0 week 1 week 2 week 4 week 6 week ± ± ± C at week C at week C at week 8 The appearance of granule after kept at 4±2, 25±2, 45±2 and 60±2 o C for 8 weeks - The appearance of granule contained in capsule has no change when compared with the beginning (T0) - The disintegration time of product was pass the USP standard (2-3 minutes in acetate buffer). - After kept at various temperature for 8 weeks, It found that the semipurified Job s tear extract gave high chemical stability because linoleic acid content decrease insignificantly compared with the beginning (T0). Half life (T 50 ) and shelf life (T 90 ) of capsule product contained with semi-purified Job s tear extract Temp ( o C) T 50 (Weeks) T 90 (Weeks) The highest half life (T 50 ) and shelf life (T 90 ) were showed at 4 0 C by the value of weeks (7.13 years) and weeks (1.42 years), respectively 19

20 Price of Job s tear products Products Dose per day Euro per unit Cost per day Folds of cost per day compared with Coix-CT Kanglaite injection Kanglaite soft capsule gel 200 ml / day 0.51 / ml ,249 folds more expensive 24 capsule / day 0.75 / capsule folds more expensive Manose-CT 1 capsule / day 0.08 / capsule Kanglaite injection Kanglaite soft capsule gel Manose-CT 20

21 Manose-CT Kanglaite 2.01 X CA Cervix Dose regimen of Kanglaite Injection: 200 ml/day, by slow IV infusion, once a day. Soft capsule gel: 6 capsule each time, 4 times daily. Indication: treatment for primary non-small-cell lung cancer and primary hepatic cancer. 21 2

22 Competitiveness : 1. Higher Efficacy in cervical cancer model (2X of Kanglaite ) 2. Well known, well advertisement 3. Well support of scientific information 4. Lower cost of long term regimen > Kanglaite injection Manose-CT Kanglaite soft capsule gel 22

23 Conclusion The ethyl acetate solubled fraction from M-WLWL-R2 extract from Job s tear showed potent anti-cancer activity in HeLa cell which was 2.01 and 0.79 fold of commercial product and cisplatin, respectively. The developed dietary supplement containing the ethyl acetate soluble fraction from M-WLWL-R2 extract has high bioactivity and high stability. This work showed the potential of semi-purified Thai Job s tear extract product for cancer prevention and treatment which can be transfered production technology to the entrepreneur. The results of this study increased values of Thai plants, reduced high cost of imported dietary supplements and enhanced the Thai economy. 23

24 Acknowledgements The investigators would like to thank The Thailand Research Fund (TRF), QualiMed Company and Natural Products Research and Development Center (NPRDC), Science and Technology Research Institute (STRI), Chiang Mai University for the financial supports of this work. 24