1. Introduction
Obesity is defined in terms of body mass index (BMI), which is calculated as body weight divided by square of height, and it is mainly a result of an imbalance between energy intake and expenditure [
1]. The World Health Organization (WHO) has reported that worldwide obesity has more than doubled since 1980 [
2]. In 2008, more than 1.4 billion adults aged 20 or older were overweight, with women outnumbering men by 3:2. More than 40 million children under the age of five were overweight in 2010 and in 2012 overweight and obesity represented the fifth leading risk for global deaths, with at least 2.8 million adult deaths. Many diseases such as hypertension, non-insulin dependent hyperlipidemia, and diabetes mellitus and coronary heart diseases are attributable to overweight and obesity [
2]. In Malaysia, some of the diseases related to obesity are increasing at a rate to such an extent that cardiovascular disease is now the fundamental cause of death [
3].
Overweight and obesity were once considered a high-income country problem, but now these conditions are on the rise in low- and middle-income countries, particularly in urban settings [
4,
5]. It has been reported that among Malaysians, one in four is overweight or obese and the percentage of obese Malaysians is double what it was a decade ago [
5]. A survey of more than 50,000 school children in Malaysia confirmed that a higher percentage of children in urban areas are obese compared to those in rural areas [
6]. Although there appear to be a decline in the rate of increase or even a leveling off in prevalence of obesity, 35% of men and women in the USA were still considered as obese in 2009–2010 with no significant difference in prevalence between men and women at any age [
7].
Lipases (triacylglycerol hydrolase E.C. 3.1.1.3) are enzymes that catalyze the hydrolysis of ester bonds of triacylglycerols (fats and oils) to produce free fatty acids, diacylglycerols, monoglycerols and glycerol. In the small intestine of mammals, the digestion of dietary triacylglycerols (TAG) is essentially due to the action of pancreatic lipase. The end products after they have been absorbed by the body are responsible for the development of obesity. Therefore, if the hydrolysis of TAG, and thus, its movement from the intestinal lumen into the body is stopped or minimized, the prevalence of obesity can be reduced [
8,
9]. For this reason, an inhibitor of digestive lipases could become a useful anti-obesity agent.
One of the approaches to reduce obesity is treatment with synthetic drugs such as sibutramine, rimonabant, phentermine, diethylpropion, zonisamide, topiramate and orlistat [
10]. Orlistat (N-formyl-L-leucine (1
S)-1-[(2
S,3
S)-3-hexyl-4-oxetanyl]methyldodecyl ester, known also as tetrahydrolipstatin) is a unique non-centrally acting anti-obesity compound that acts within the gastrointestinal tract by impeding pancreatic and gastric lipases that play an essential role in the digestion of long chain TAG. At the recommended therapeutic dose of 120 mg three times a day, it inhibits dietary fat absorption by about 30% [
11,
12]. It is also the only anti-obesity drug that is approved for long term weight management [
10]. Sibutramine and rimonabant were recently withdrawn from the market due to adverse side effects. Sibutramine could lead to an increased risk of heart attack and stroke in high-risk cardiac patients, whereas the latter could lead to potentially serious psychiatric disorders. Hence, there is an urgent need for safer and more efficient anti-obesity agents from natural sources, even though they are also toxic, but are of less damaging as compared with the pure synthetic ones [
13].
There have been several reports on the search for anti-lipase inhibitors from natural sources [
14,
15,
16,
17,
18,
19,
20]. Although to date there are no reports on plants with pro-lipase activity, it is possible that some plants may also possess metabolites that will stimulate the activity of pancreatic lipases. Thus, the objective of this study was to examine the crude methanol extracts of ninety eight (98) different parts (seeds, fruits, leaves, stems, flowers, roots) of some medicinal, herbal and aquatic plants largely found in Malaysia for their anti-lipase, pro-lipase activity as well as to identify an anti-lipase compound from one of the plant sources.
3. Discussion
As can be observed, plant parts that possessed inhibitory activity against PPL far outnumbered those that stimulated its activity. The ripe fruits of A. carambola and the leaves of A. jiringa (Jack) I.C Nielsen L., C. cauliflora, and A. moluccana (L.) Willd were found to completely inhibit PPL activity, expressed as orlistat equivalents. These plants have a lipase inhibitory value equivalent to 12.4 µg orlistat/mL. Another 15 extracts reduced the activity of the enzyme by more than 80% and these may also be regarded as potential and useful sources of anti-obesity agents. Only 2% did not possess any inhibitory activity. Interestingly, almost similar degrees of inhibition were observed when comparing extracts from different parts of the same plant. For example, there is no difference in the degree of inhibition between the leaves and seeds of A. bicolor Moon. Similar levels of inhibition of PPL were observed between the extracts of the ripe fruits and leaves of A. carambola L., the ripe fruits and leaves of C. cauliflora, the roots and leaves of C. longa L., the stems and leaves of E. michelii L., the fruits and leaves of M. citrifolia L., as well as the leaves, seeds and stems of P. cordiflora. However, there are also some variations in the inhibitory activity of plant from the same family. For example, plants that belong to the family Acanthaceae like A. bicolor Moon, A. gangetica L. T. Anderson and S. crispus showed low inhibition while those from the family Falaceae were observed to show a wider range of anti-lipase activity. For example, A. jiringa (Jack) I.C Nielsen and C. cauliflora have a significant inhibitory effect on lipase while Leucaena leucocephala (Lam) Dewit, O. bancana (Miq.) Merr., and P. speciosa Hassk showed relatively low inhibitions.
In many cases, the pharmacological activities of these plants are attributable to the presence of secondary metabolites such as polyphenols, saponins, tannins, terpenes, flavonoids and alkaloids which are active inhibitors of pancreatic lipase [
22,
23]. For example, saponins, tannins, alkaloids and flavonoids have been reported in
A. carambola L. fruits [
15], while
A. moluccana (L.) Willd has been shown to contain bioactive diterpenoids, moluccanic acids, 3,4-secopodocarpane trinorditerpenoids, moluccanic acids, 6,7-dehydromoluccanic acids, and moluccanic acid methyl ester [
22]. Therefore, one reason why some of the plant extracts used in the study demonstrated high anti-lipase activity could be due to higher contents of bioactive compounds in their tissues. To date, there have been no reports on the phytochemicals of
A. jiringa and
C. cauliflora although there are some reports on their biological activities [
24,
25].
Apparently plants are not only sources of anti-lipase compounds as 12.2% of the plant extracts screened were found to promote the activity of the enzyme instead (
Table 2). Of these, the seeds of one plant,
P. anisum L., caused 186.5% activation of PPL activity compared to the control. Seven (7) other extracts showed less than 20% activation of lipase activity, and the rest exhibited between 20% to 40% activation. It should be noted that while the leaves of
M. hastata (L.) Solms inhibited PPL activity, the root enhanced the activity slightly. The therapeutic effects of
P. anisum have been reported such as being gynaecologic, neurologic and hypothermic, having the ability to delay the onset of picrotoxin-induced seizures in mice, and being a muscle relaxant and anticonvulsant [
26,
27]. The ethanolic extract of this plant was also reported to contain methylchavicol, eugenol, psedoisoeugenol, anisaidehyde, caffeic acid derivatives, flavonoids, polyacetylene and polyenes as the major constituent compounds.
In this study, an in vitro analysis of anti-lipase activity of C. cauliflora leaves was conducted in order to confirm its traditional use as an anti-obesity plant. Results obtained show that the EtOAc fraction of the crude methanol extract of the plant was found to be the most active followed by the n-BuOH fraction. Bioassay activity-guided fractionation of this fraction by the use of successive column chromatographic methods and hydrophobic Sephadex chromatography have led to the isolation and identification of a flavonoid type compound that is responsible for the inhibition of pancreatic lipase activity. The structure of the compound was elucidated to be kaempferol-3-O-rhamnoside by a comparison of one and two-dimensional NMR data which entirely match the previously reported values.
In the search for effective anti-obesity compounds from natural sources, several extracts from plants, and bacterial, fungal, and marine species have been screened in order to find new compounds with pancreatic lipase inhibitory activity. Most of the common compounds that are found in several plants species with anti-lipase activity are polyphenols, saponins and terpenes [
28]. For example, licochalcone A, isolated from the ethyl acetate/
n-hexane fraction of ethyl acetate extract of the roots of
Glycyrrhiza uralensis, was shown to significantly inhibit the activity of pancreatic lipase at an IC
50 value of 35 µg/mL [
29]. Phenylboronic acid was the potent inhibitor of lipase isolated from
Oryza sativa [
30] while carnosic acid, a diterpine, was the one isolated from the methanolic extract of the leaves of sage,
Salvia officinalis [
31].
Flavonoids such as kaempferol
-3-O-rhamnoside, which are usually found in the plant kingdom, are widely dispersed and there have been numerous reports on their antioxidant, anti-inflammatory and anti-carcinogenic activities [
32]. Flavonoids have been used for the treatment of patients with intolerance to radiation therapy, vascular diseases, liver diseases, and dementia [
32]. There exist many pharmacological activities for kaempferol
-3-O-rhamnoside which are of vital important to health. For example, Nazemiyeh
et al. [
33] reported that this compound has a free radical scavenging ability in animals. This was basically attributed to the existence of a phenolic moiety in the structure. The compound, among other flavonoids, was also found to be active against antibiotic-resistant bacteria [
34].