MATERIALS AND METHODS

Plants: Flowers, aerial parts, fruits, roots and seeds of different plants were collected from various provinces of Iran or purchased from the medicinal herbal markets in Kerman city. Scientific names of the plants were authenticated by Dr. Mirtajaldini, Department of Botany, Bahonar University and Kerman, Iran (Table 1). A voucher specimen from each plant was deposited at the herbarium of the Herbal Medicines Research Center, Faculty of Pharmacy and Kerman University of Medical Sciences, Iran. Each plant material was air dried and grounded into fine powder. The powdered material (20 g) was extracted with 200 mL of absolute methanol for 24 h. The suspensions were filtered and air-dried; these air-dried samples were stored at -20°C until use (Sharma et al., 2005). Solution of 5 mg mL^-1 of each extracts in 0.05 M phosphate buffer was prepared just before enzyme assay.

Enzyme assay: Pancreatic lipase activity was measured by turbidimetric method, used by Vogel and Zieve (Shihabi and Bishop, 1971; Burtis et al., 2006). The assay was based on the reduction in turbidity of a triolein emulsion by porcine pancreatic lipase (5 unit, Sigma, USA) at 340 nm, pH 8.9 and 37°C (Carrere et al., 1987; Han et al., 2001; Yamada and Fujita, 2007). Ten microliter of each preparations containing 50 μg crude extract was added to the reaction mixture including; Triolein (0.3 mmol L^-1), sodium deoxycholate (16.7 mmol L^-1), colipase (4 mg L^-1), calcium chloride (0.04 mmol L^-1) and 0.05 M tris buffer (final volume of 1 mL). The mixture was incubated at 37°C for 15 min and absorbance at 340 nm was determined spectrophotometrically (Han et al., 2001; Yamada and Fujita, 2007). Triolein was solubilized in 0.2% Triton X100 (Rocha et al., 1999; Tashiro et al., 1992). The active extracts re-examined by commercial lipase assay kit (Randox, Laboratories. LTD. UK).

Pancreatic lipase inhibitory activity was calculated according to the following formula (Huerta et al., 2007):

Table 1:	Anti porcine pancreatic lipase activity of plants extracts
The enzyme activity was measured by turbidimetric assay. Reaction mixture was contained triolein as the substrate, sodium deoxycholate, colipase, calcium chloride and tris buffer. Reduction in turbidity was assayed in 340 nm

Inhibition (%) = [A_Control-A_Extract/A_Control]x100

Kinetic study: In order to measure the inhibition mode by mathanolic extract of Quercus infectoria, Eucalyptus galbie, Rosa damascena and Levisticum officinale, pancreatic lipase activity was assayed with increasing concentrations of triolein as substrate (0.15, 0.3, 0.625 and 1.25 mM) in the absence and presence of two different concentrations of the extracts (0.05 and 0.15 mg mL^-1). Inhibition mode was determined by double-reciprocal Lineweaver-Burk plot analysis of the data resulted from enzyme assays containing various concentrations of triolein and extracts according to the Michaelis-Menten kinetics (Zhao and Kim, 2004; Won et al., 2007).

Fig. 1:	The Lineweaver-Burk plot of kinetic analysis for porcine pancreatic lipase at two different concentrations of Quercus infectoria (0.05 and 0.15 mg mL-1) in the presence of four different triolein concentrations

RESULTS

Plants with Pancreatic lipase inhibitory effect: Among one hundred extracts; Quercus infectoria, Eucalyptus galbie, Rosa damascena and Levisticum officinale showed 85, 64, 57 and 55% inhibitory effect on pancreatic lipase, respectively.

Extracts prepared from Pistacia vera, Myrtus communis, Otostegia persica, Urtica urens, Cinnamomum zeylanicum, Rheum ribes, Pimpinella anisum, Alhagi camelorum, Nigella sativa, Carthamus oxyacantha, Arctium lappa, Trigonella foenum graecum, Ficus carica and Buninm persicum showed an inhibitory effect between 25-50% on pancreatic lipase. The rest of plant extracts showed less than 25% or no inhibition on the activity of the enzyme in this study (Table 1).

Kinetic analysis of porcine pancreatic lipase inhibition: The inhibition mode of the four most active plant extracts was analyzed by double-reciprocal Lineweaver-Burk plot. The enzyme kinetics demonstrated non-competitive inhibition on porcine pancreatic lipase activity by Quercus infectoria (Fig. 1); Eucalyptus galbie (Fig. 2) and Rosa damascene (Fig. 3) and mixed inhibition by Levisticum officinale (Fig. 4). The Km value of triolein for porcine pancreatic lipase was 0.3 mM and V_max value was 0.078 mM min^-1. The V_max values of the enzyme in the presence of Quercus infectoria (Table 2), Eucalyptus galbie (Table 3), Rosa damascene (Table 4) and Levisticum officinale (Table 5) extracts were 0.051, 0.056, 0.049 and 0.068 mM min^-1, respectively. The Ki value of 0.341, 0.383, 0.335 and 0.930 mg mL^-1 was found for Quercus infectoria, Eucalyptus galbie, Rosa damascena and Levisticum officinale, respectively.

Table 2:	Kinetics analysis of porcine pancreatic lipase under different concentrations of triolein and Quercus infectoria extract
The enzyme activity was measured by turbidimetric assay. Reaction mixture contained of triolein, deoxycholate, colipase, calcium chloride and tris buffer. Turbidity determined by measuring the absorbance at 340 nm

Table 3:	Kinetics analysis of porcine pancreatic lipase under different concentrations of triolein and Eucalyptus galbie extract
The enzyme activity was measured by turbidimetric assay. Reaction mixture contained of triolein, deoxycholate, colipase, calcium chloride and tris buffer. Turbidity determined by measuring the absorbance at 340 nm

Table 4:	Kinetics analysis of porcine pancreatic lipase under different concentrations of triolein and Rosa damascena extract
The enzyme activity was measured by lipaturbidimetric assay. Reaction mixture contained of triolein, deoxycholate, colipase, calcium chloride and tris buffer. Turbidity determined by measuring the absorbance at 340 nm

Table 5:	Kinetics analysis of porcine pancreatic lipase under different concentrations of triolein and Levisticum officinale extract
The enzyme activity was measured by turbidimetric assay. Reaction mixture contained of triolein, deoxycholate, colipase, calcium chloride and tris buffer. Turbidity determined by measuring the absorbance at 340 nm

Fig. 2:	The Lineweaver-Burk plot of kinetic analysis for porcine pancreatic lipase at two different concentrations of Eucalyptus galbie (0.05 and 0.15 mg mL-1) in the presence of four different triolein concentrations

Fig. 3:	The Lineweaver-Burk plot of kinetic analysis for porcine pancreatic lipase at two different concentrations of Rosa damascena (0.05 and 0.15 mg mL-1) in the presence of four different triolein concentrations

Fig. 4:	The Lineweaver-Burk plot of kinetic analysis for porcine pancreatic lipase at two different concentrations of Levisticum officinale (0.05 and 0.15 mg mL-1) in the presence of four different triolein concentrations

DISCUSSION

Obesity is becoming one of the biggest complications to global health in this millennium (Birari and Bhutani, 2007). Pancreatic lipase is the key enzyme for lipid absorption that hydrolysis triacylglycerols in the gastrointestinal tract (Jang et al., 2008). Pancreatic lipase inhibitors which help to limit intestinal fat absorbtion at the initial stage, have been proved as useful medications for the treatment of hyperlipidaemia and a great promise as anti-obesity agents (Sharma et al., 2005). Presence of pancreatic lipase inhibitors has been reported in some natural resources (Han et al., 2001; Zhao and Kim, 2004; Sharma et al., 2005; Moreno et al., 2006; Huerta et al., 2007; Won et al., 2007) but due to the problem arisen by these extracts, further investigations for finding new and better pancreatic lipase inhibitors in the nature is a necessity. In this study we demonstrated that galls of Quercus infectoria, leaves of Eucalyptus galbie, flowers of Rosa damascene and roots of Levisticum officinale have strong anti porcine pancreatic lipase activity. We determined their kinetics properties that have not done so far. The most common anti obesity drug available in the market is Orlistate, which was shown to be irreversible inhibitor of pancreatic lipase (Greenway, 1999; Sharma et al., 2005). Platycodin D and tea-saponin have inhibited the pancreatic lipase activity in a competitive manner which is in contrast to our results suggesting a non-competitive or mixed inhibition of pancreatic lipase by our extracts (Han et al., 2001; Zhao and Kim, 2004). However, in accordance with our results, some studies reported non-competitive inhibition of pancreatic lipase (Won et al., 2007; Huerta et al., 2007). A mixed-inhibitor binds at a distinct site from the active site but it can binds to the enzyme or enzyme-substrate complex as well. It will affect Km and Vmax of the reaction. Components of Levisticum officinale, extract which showed mixed type of inhibition (Table 5), can bind to the enzyme or Enzyme-Substrate (ES) complex and block its activity. When inhibitor bind to the enzyme and/or ES complex it defined as non-competitive inhibition which inhibitor affects only on the V_max of the reaction but has no effect on complex formation between the enzyme and the substrates (Nelson and Cox, 2005). Therefore, the three extracts which showed non-competitive inhibition on PL activity, probably have components that bind to E or ES complex (Table 2-4). It had been shown that, Crocin a glycosylated carotenoid, is the major active constituents of Gardenia jasminoids. This compound exhibited potent hypo-triglyceridemic activity. Crocin competitively and reversibly inhibited PL and Crocin's metabolite, crocetin, also potently inhibited PL. Crocin and crocetin also showed potent hypolipidemic activity in Triton WR-133 or corn oil induced hyper-lipidemic mice. Another compound, Dioscin isolated from methanol extract of Dioscorea nipponica was shown to inhibit PL (Birari and Bhutani, 2007). Similar inhibitors might be responsible for anti lipase activities in our study. The active constituents of these plants have not been completely known and further investigation needed to be done.

Afromomum meleguetta is an African plant which its extract has shown the most potent inhibitor of PL (Ekanem et al., 2007). The methanol extract from the pericarps of Sapindus rarak was found to have pancreatic lipase inhibitory activity (Morikawa et al., 2009). Pribitkin and Boger (2001) asserted that Zingiber officinale inhibits platelets function and Apium graveolens, Foeniculum vulgare, Levisticum officinale and Ficus carica have photosensitizing effect. Here we showed the anti PL activity of these plants (Table 1). Jang et al. (2008) isolated a new pancreatic inhibitor from roots of Actinidia arguta. Slanc et al. (2009) showed that Crocus sativa did not show any inhibition on PL and Menha piperita showed below 40% inhibition on activity of PL. This is in contrast with our results. Also, they asserted that Pimpinella anisum, Arctium lappa, Origanum majorana, Althaea officinalis, Ficus carica, Citrus sinensis and Urtica dioica showed PL inhibitory activity below 40% that are along with our results. Arctium lappa and Linum usitatissimum showed slight PL inhibition in our study while Peter et al. (2009) showed that Arctium lappa and Linum usitatissimum have strong inhibitory effect on PL.

The galls of Quercus infectoria and flowers of Rosa damascenea had shown non-competitive inhibitory effect on Alpha mannosidase activity (Gholamhoseinian et al., 2008a). Quercus infectoria, Rosa damascena and Levisticum officinale showed anti Alpha glucosidase activity, a target enzyme with therapeutic potential in the treatment of diabetes, metastasic cancer and lysosomal storage disease and also showed antiviral effects against human immunodeficiency virus and hepatitis C virus infection (Matsui et al., 2004; Chapel et al., 2006; Gholamhoseinian et al., 2008b). Galls of Quercus infectoria possess many therapeutic activities such as antidiabetic, anti-parkinsonian, anti-tremorine, anti-inflammatory, antiviral, anti- microbial, antifungal and larvicidal activity (Kaur et al., 2004; Basri and Fan, 2005) and showed high potential in skin whitening and antioxidant properties (Pin et al., 2006). We showed that Eucalyptus galbie is one of the porcine pancreatic lipase inhibitors. It had shown that Eucalyptus galbie is an effective agent in reducing the growth of fungus and also has anti diabetic activity (Grover et al., 2002; Joseph et al., 2008). These plants could represent active source of new anti-lipase activity. Further in vivo study on animal model must be done in order to confirm these results. These four plants will be examined in order to isolate, identify and characterize the active ingredients to establish the nature of their lipid lowering activity. This study may serve as a foundation for comprehensive and safe therapeutic strategies to the management of obesity and other related diseases.

ACKNOWLEDGMENT

This study was supported by the research funds provided by the Vice Chancellor for Research and the Kerman Physiology Research Center, Kerman University of Medical Sciences, Kerman, Iran.