1 RESEARCH AND DEVELOPMENT OF WEIGHT LOSS PRODUCT WITH LIPASE INHIBITORY ACTIVITY SIRINAN TUUBTHIMTHED Department of Pharmaceutical and Natural Products Thailand Institute of Scientific and Technological Research (TISTR)

2 OUTLINE ● INTRODUCTION ● CHEMICAL STUDY Extraction Chemical analysis ● PHARMACOLOGICAL STUDY lipase inhibitory activity Effect of plant extracts on serum TG levels ● PRODUCT DEVELOPMENT ● SAFETY EVALUATION Acute oral toxicity Subchronic toxicity

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5 Anti-obesity mechanisms for herbal plants included ● reduction in lipid absorption, ● reduced energy intake, ● increased energy expenditure, ● decreased lipogenesis and increased lipolysis ● Decreased energy intake from the gastrointestinal tract is caused by herbal product acting on pancreatic lipase.

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Plant Extraction - Screening of 16 Thai medicinal plants Each plants were - washed, dried, ground into powder - exracted with 95% ethanol by using percolator at room temperature. - concentrated the extract on rotary evaporator under reduced pressure.

PHARMACOLOGICAL STUDY 9 Lipase inhibitory activity (BALB-DTNB method ) Effects of plant extract on serum Triglycerides levels

10 Preparation of pancreatic lipase enzyme The 0.2 g. of rat pancreas was mixed with 10 ml of 0.9% normal saline, spun at 7,000 rpm/min for 10 min. and then the supernatant was kept under -80 ◦C until next experiment. Lipase inhibitory activity (BALB-DTNB method )

Assay for lipase inhibitory activity - The 10 µl amount of each ethanolic extract solution was added to 1 ml of pancreatic lipase enzyme -incubated the reaction at room temperature for 15 min. -The positive control Orlistat 120 mg - a commercial lipase assay kit (BioAssay Systems. USA) was used to determined the activity. The inhibitory activity (I) was calculated according to the following formula. I % = 1 - B-b x 100 A-a Where A is the activity of the enzyme without inhibitor, a is the negative control without inhibitor, B is the activity of the enzyme with inhibitor b is the negative control with inhibitor 11 Clin.Chem.39(5):

12 No. Plants name Plant Part Family %Extract% Inhibition 1 Acmella oleracea (L.) R.K.Jansen APCompositae Camellia Sinensis var.assamica(Mast.) LTheaceae Cinnamomum verum J.Presl. BLauraceae Cissus quadrangularis L. APVitaceae Citrus hystrix DC. LRutaceae Ipomoea aguatica Forsk APConvolvulaceae Nelumbo nucifera Gaerntn LNelumbonaceae Ocimum americanum L. APLabiatae Ocimum basillicum L. APLabiatae Ocimum gratissimum L. APLabiatae Ocimum sanctum L. APLabiatae Piper nigrum Linn. FPiperraceae Solanum torvum Swartz FSolanaceae Solanum stramonifolium Jacq. FSolanaceae Zingiber officinale Roscoe. RZingiberaceae Quercus infectoria G. Olivier FFagaceae Oristat ( 5 µg/ml) Plant Parts: AP: Arial Part, B: Bark, F: Fruit, L: Leaf, R: Root.

13 Groups triglyceride level control172.71±26.89 Orlistat 30 มก./กก.79.57±12.86* Basil ext. 200 มก./กก ±22.31 Basil ext. 800 มก./กก ±35.37 C. Hystrix ext. 200 มก./กก ±22.75 C. Hystrix ext. 800 มก./กก ±26.87 * P<0.05, versus the control group. Effects of plant extracts on serum TG concentrations in normal mice after an oral administration of lipid emulsion International Journal of Obesity (2007) 31, 1023–1029;

Effects of Nutgall extract on serum TG concentrations in normal mice after an oral administration of lipid emulsion Groups Triglyceride level control247.66±46.7 Orlistat 30 mg./kg ±9.1* Nutgall ext. 200 mg./kg ±13.7* Nutgall ext. 800 mg./kg ±16.7* Cinnamon ext. 200 mg./kg.254.5±47.9 Cinnamon ext. 800 mg./kg ±17.8* * P<0.05, versus the control group. Oral administration of nutgall extract at a dose of 200 and 800 mg/kg decreased the serum TG level.

Nut-galls, Quercus infectoria G. Olivier 15 Common name: nut-galls, gall oak, Aleppo Oak, Asian Holly-Oak, Thai name : Ben Ka Nee; เบญกานี

16 Determination of total phenolic and tannin contents Total phenolic content = mg GAE /g dry extract Tannin content = mg TAE /g dry extract) (Folin-Ciocalteu method)

Acute oral toxicity showed that - LD 50 of the product was 9,700 mg/kg body weight. - treated rats did not exhibit abnormal signs of toxicity or deaths Safety evaluation of the product OECD Test Guideline No. 420 : Acute Oral Toxicity – Fixed Dose Method (Limit test) (OECD-420,2001).

Subchronic toxicity - showed no sign of any toxicity. - These results confirmed that the product is safe to be an other food supplement for weight control OECD Test Guideline No. 408 : Repeated Dose 90-day Oral Toxicity Study in Rodents. (OECD-420,1998). Safety evaluation of the product

19 Lipase inhibitory activity (BALB-DTNB method ) Effects of plant extracts on serum TG levels Safety evaluation Development of WEIGHT LOSS PRODUCT

20 The result indicated that Nutgall extract have potential source of lipase inhibitors for the treatment of obesity and the safety evaluation confirmed that this product is safe to be an other food supplement for weight control. CONCLUSION

Tanwarat Kajsongkram Tuanta Sematong Sareeya Reungpatthanaphong Sarunya Laovitthayanggoon Chuleratana Banchonglikitkul Sinn Tangsti Sawai Nakakaew Wichein Khoeynok

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