Plant natural products as an anti-lipid droplets accumulation agent

Recently people often suffer from unhealthy energy metabolism balance as they tend to take more energy than required. Normally, excess energy taken in is converted into triglyceride and stored in adipocyte as lipid droplets. Recent studies have suggested that irregular accumulation of triglyceride in adipocyte might be a cause of many metabolic diseases. Thus, the awareness of the detrimental effects on health of excessive lipid droplets accumulation (LDA) has urged the development or finding of drugs to counter this effect, including those from botanical origins. This review summarized recent progress in this field from the viewpoint of crude drug studies with references to their anti-LDA activity. Possible mechanisms involved in their anti-LDA effect and isolations of the relevant bioactive compounds were also discussed.


Introduction
Excess energy taken into the body is to be stored in an organelle, lipid droplets in the cells called adipocytes.Their major physiological role is to function as reservoir of energy [1]. Lipid droplets in adipocyte are always in a state of flux, when the energy excess is in the surplus, energy is converted into triglyceride, and when the energy is in short supply, the stored triglyceride is re-converted into energy [2]. Biologically, body lipid serves as body energy storage and insulates us from low temperature. But, it can act as a double edged sword [3]. This is because excessive storage of lipid has been recently shown to be a cause of various diseases including type-2 diabetes mellitus, cardiovascular disease, and atherosclerosis [4,5].
The possible detrimental effects on health of excessive lipid droplets accumulation (LDA) have prompted the search for counter measures, including studies of drugs which reduce LDA. A natural product, berberine, has so far been reported to reduce LDA in vivo via down-regulation of peroxisome proliferator-activated receptor protein-c (PPARc) [6,7]. Apart from berberine, other examples of plant-derived compounds with well-known anti-LDA effect includes, genistein, curcumin, and (-)-epigallocatechin-3-gallate (EGCG) [8][9][10]. A long history of the use of plant origin drugs by many nations resulted in the widespread consumers' perception that plant natural origin drugs are safe or had fewer side effects.
Because of the knowledge of the importance of LDA in adipocyte and that a few compounds isolated from plants actually possess such an anti-LDA effect, researchers studying plant natural products with anti-LDA are gaining momentum recently. This review compiles information of botanicals that have been or are being investigated for their anti-LDA activity. Possible mechanisms involved in the anti-LDA process and possible active compounds are also included in this review.

Possible targets of anti-lipid droplets accumulation agents
Extra energy is stored in specialized cells, adipocytes. Adipocytes are grouped into two types, brown adipocytes and white adipocytes. Brown ones are mitochondria rich and use energy whereas white ones store energy [3,11,12]. The differentiation process of white adipocytes from the precursor cells and its LDA system could be a target in the study to find LDA inhibition (Fig. 1).
Adipocytes originate from the same precursor stem cells as chondrocytes, osteocytes and myocytes, known as mesenchymal stem cells (MSCs) [13]. Differentiation initiates when the pluripotent MSCs receive signals from extracellular stimulating factors such as bone morphogenetic proteins (BMPs), transforming growth factor-b (TGFb), activin, insulin-like growth factor 1 (IGF-1), interleukin-17 (IL-17), and fibroblast growth factors (FGF) 1 and 2 [14][15][16][17][18][19][20][21][22]. It is also been well documented that a mixture of 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), and insulin (MDI inducer) stimulates the mouse embryonic fibroblast cell line, 3T3-L1, to differentiate into adipocytes in vitro [21]. Intensive studies on 3T3-L1 established that the presence of extracellular stimulating factors rapidly caused expression of early adipogenic regulators, and CCAAT/enhancer-binding proteins (C/EBPs) -b and -d. The expressions of C/EBPb and C/EBPd convey the message from these extracellular stimulating factors to activate the master regulators of adipogenesis, PPARc, and C/EBPa. C/EBPb and -d were proved to be essential for the expression of PPARc and C/EBPa, because the cells with C/EBPb and -d knock out have down-regulated in PPARc and C/EBPa expression. PPARc and C/EBPa operates in a self-regulating positive feedback loop system. This feedback loop system increased expression of adipocyte-specific genes, which are important for the proper functioning of adipocytes [22][23][24][25][26][27][28][29][30][31]. Such specific genes includes glucose transporter type 4 (GLUT4), an insulin-regulated glucose transporter protein that enables translocation to plasma membrane to facilitate the uptake of glucose into cells. This protein is expressed primarily in muscles and fat tissues, which makes it an ideal marker for the determination of adipocyte differentiation [23]. In addition to GLUT4, another lipid homeostasis-related gene, lipoprotein lipase (LPL) is also studied. LPL is an enzyme that hydrolyzes triglycerides including very low-density lipoproteins (VLDL) into two free fatty acids and one monoacylglycerol molecule. It is mostly distributed in adipose, heart, and skeletal muscle tissues [24]. The 11b-hydroxysteroid dehydrogenase type 1 (HSD11b1) is another enzyme that is related to the metabolism, which is highly expressed in adipose tissues. This family of enzymes catalyzes the conversion of inactive cortisone to active cortisol, and vice versa [25][26][27]. Fatty acid binding protein 4 (FABP4) also known as adipocyte protein 2 (aP2), is another adipocyte specific protein expressed primarily in adipocyte that functions as a carrier protein [28]. The lengthy process leading to adipogenesis might provide numerous targets to be attacked for the inhibition of LDA.
All mammalian cells are known to contain some lipid droplets [29], but the differentiated cells called white and brown adipocytes possess larger sized lipid droplets in higher numbers [30]. These lipid droplets consist of neutral lipids, triacylglycerols and cholesterol esters encased in a monolayer of phospholipid [1,32]. The esterified fat is on energy demand hydrolyzed to be used as an energy source [3,30]. Of the two types of adipocytes, white ones generally contain larger lipid droplets occupying a major part of the cytosol, whereas the brown ones contain numerous and smaller lipid droplets. The formations of lipids are catalyzed by the enzymes known as acyl-CoA diglycerol acyltranferase (DGAT) and acyl-CoA cholesterol acyltranferase (ACAT) [31][32][33][34]. Although the enzymes responsible for the lipid synthesis are well-elucidated, the formation process of lipid droplets is not clearly established. Two of the most likely models are: (1) formation of tiny lipid droplets by DGAT or AGAT, which are deposited between the membrane leaflets of endoplasmic reticulum (ER), where the lipid droplets gradually increase in size to finally 'bud off' the ER, and (2) lipids accumulate between the luminal and cytoplasmic membranes, which subsequently are encapsulated by the membranes bilayer to formed lipid droplets [31][32][33]. Apart from inhibiting the adipogenesis process, anti-LDA effect may also be achieved by preventing the adipocyte from accumulating lipid droplets. As discussed before, inhibition of enzymes responsible for the lipid formation may provide a tantalizing target for inhibition of LDA. At present, since the exact mechanisms of lipid droplets formation is still under debate, future identification and understanding of the mechanisms involved may provide new anti-LDA targets.
During energy deprivation, lipid droplets stored in adipocyte hydrolyze their content, triacylglycerols to fatty acids and glycerol, under a process named lipolysis [35]. In addition to the inhibition of lipid droplets synthesis, up-regulation of the lipid metabolism or lipolysis process may also be effective in lowering the adipocyte lipid droplets. Lipolysis is known to be catalyzed by many enzymes generally called lipases, of which adipose triglyceride lipase (ATGL or desnutrin) is known to play a major role. The enzyme is responsible for some 75 % adipocyte lipolysis [30, 34-36]. A study indicated that the first step of lipolysis catalyzed by ATGL is the rate-limiting step, a fact which emphasized the importance of ATGL in the lipolysis [37]. Thus, inhibition of lipid droplets synthesis and promotions of lipolysis of adipocyte lipid droplets equally function to reduce LDA in adipocyte.

Inhibitors of adipogenesis or expression of adipogenic factors
Aristolochia manshuriensis Kom-Aristolochiaceae Aristolochia manshuriensis, a Korean traditional medicinal herb is distributed in Japan, China, and Korea. Preliminary studies showed that a stem extract of A. manshuriensis down-regulates the gene expression of C/EBPb, PPARc, and C/EBPa of 3T3-L1 cells. Further studies on the upstream regulators of C/EBPb, PPARc, and C/EBPa led to the conclusion that the extract disrupts the extracellular signal-regulated protein kinase 1/2 (ERK1/2) and Akt pathway leading to the inhibition of C/EBPb, PPARc, and C/EBPa expression, which eventually leads to the inhibition of the adipocyte differentiation. In addition, gene expression of fatty acid synthase (FAS), adiponectin, LPL, and aP2 is also significantly down-regulated. In an in vitro study, aristolochic acid ( Fig. 2) isolated from the plant is shown to be responsible for the inhibition of triglyceride (TG) accumulation. Oral administration of a stem extract of A. manshuriensis leaves at 62.5 mg/kg/day is reported to significantly decrease the fat tissue weight, total cholesterol (TC) level, and low density lipoprotein-cholesterol (LDLc) level of high-fat diet (HFD)-induced obesity mouse [38], though it is yet not verified if such decreases are due to the effect of aristolochic acid.

Brassica rapa (L.)-Brassicaceae
The roots of Brassica Rapa or commonly known as the turnip are reported to contain licochalcone A (Fig. 3), a major phenolic compound from the root of the Glycyrrhiza plant, commonly known as licorice [39]. This compound was found to suppress the differentiation of 3T3-L1 preadipocytes. Further investigation showed that licochalcone A significantly down-regulates the expression of PPARc,

Camelia sinensis (L.) Kuntze-Theaceae
Leaves of Camelia sinensis, or the tea plant are most commonly consumed in eastern Asia and Europe. Many compounds have been isolated from tea leaves including caffeine, (-)-epigallocatechin-3-gallate (EGCG) (Fig. 4), and (-)-epi-catechin-3-gallate [9]. EGCG was demonstrated to prevent adipogenesis and to cause adipocytes apoptosis, though the details of its mechanism of action are not known yet [41,42]. In another study, the possible mechanism of anti-adipogenesis by EGCG was reported, thus, by the realtime PCR (RT-PCR) analysis of 3T3-L1 cells treated with EGCG it was shown to decrease PPARc and C/EBPa mRNA. Significant decrease in the forkhead transcription factor class O1 (FoxO1) mRNA was also reported, which suggested it to be via PI3-  [54]. Subsequent studies on the protein and mRNA expression suggested that the role ceramicine B in the anti-LDA activity was to inhibit adipogenesis via suppression of PPARc expression. Detailed studies on the upstream regulator of PPARc indicated that ceramicine B interrupts phosphorylations of FoxO1 [55]. Phosphorylations of FoxO1 at Thr 24, Ser 256, and Ser 319 are essential for advancement of adipogenesis and unphosphorylated FoxO1 plays a role in the repression of PPARc mRNA transcription [56,57]. Thus ceramicine B may be said to play an essential role in inhibiting LDA by interrupting phosphorylations of FoxO1 which then leads to suppression of PPARc expression.

Lysimachia foenum-graecum Hance.-Primulaceae
Lysimachia foenum-graecum has been used traditionally as an anti-inflammatory agent and also as a remedy for cold, headache, and toothache. From the whole plant extract of L. foenum-graecum, a series of triterpene saponin, foenumoside A-E and lysimachiagenoside A, C-F have been isolated [58][59][60][61]. By a high-through put screening this plant was demonstrated to suppress adipogenesis and this anti-adipogenesis activity was shown to be due to suppression of PPARc, and C/EBPa protein expression in a concentrationdependent manner, with IC 50 at 2.5 lg/ml, which downregulate the downstream targets of PPARc, and C/EBPa, aP2 and adiponectin [58]. Apart from the down-regulation of the adipogenic marker gene, the whole plant extract of this plant was also shown to suppress induction of gene expression of lipogenesis related genes, FAS, SREBP-1c, acetyl-CoA carboxylase (ACC), and SCD1. It is also to be noted that the lipolytic genes were up-regulated, such as acyl-CoA oxidase (ACO) and carnitine palmitoyltransferase 1 (CPT1). An in vivo study showed also that the body weight of C57BL/6 mice fed with HFD decreased when treated with L. foenum-graecum whole plant extract through oral-gavage at 100 mg/kg. Foenumoside B (Fig. 6) was identified to be responsible for the effect in both in vitro and in vivo studies. It inhibited the differentiation of 3T3-L1 preadipocytes in a dose-dependent manner with an IC 50 of 0.2 lg/ml in the nile red staining assay. In an in vivo assay, foenumoside B was shown to suppress lipid accumulation in white adipose tissues and in the liver, and to lower the blood levels of glucose, triglycerides, alanine aminotransferase (ALT), and aspartate aminotransferase (AST), in HFD-induced mice [62]. More detailed studies using the PPARc reporter gene assay indicated that its extract functions as an antagonist to PPARc activity giving the max PPARc inhibition activity of 87 %. Several compounds were identified in the P. balsamifera, such as salicin and salicortin (Fig. 8), both salicortin isomers showing complete inhibition of PPARc activity [64]. Another study showed that both ethanolic, an extract of P. balsamifera (250 or 125 mg/kg), and salicortin (12.5 mg/kg) effectively and equally reduced the accumulations of fat and liver TG in diet-induced obese (DIO) C57BL/6 mice [65]. Other salicortin-derivatives obtained from twigs of another plant of the same family were shown to inhibit adipogenesis via modulation of the C/EBPa and SREBP-1c dependent pathway (Table 1) [66].

Citrus unshu Marcovitch-Rutaceae
Fruits of Citrus unshu or Citrus unshiu (Satsuma mandarin orange)are known to contain an active ingredient, psynephrine (Fig. 10) [99]. Juice from unripen fruits of C. unshu was reported to induce lipolysis in fat cells isolated from male Wistar rats in a concentration-dependent manner. To elucidate the mechanism involved in inducing lipolysis by unripen C. unshu fruit juice, an aqueous extract of lyophilized C. unshu fruit juice was added to the isolated fat cells in the presence of b-blocker (inhibitor of lipolysis). Incubation with selective b1-antagonist salicin salicortin (atenolol), b2-antagonist (IC118551), and b3-antagonist (SR59230A) and non-selective b-blocker (propranolol), indicated that an aqueous extract of C. unshu acts as a non-selective b-agonist. Another in vivo study, also showed that its aqueous extract increased lipolysis in rat visceral (epididymal and omental) and subcutaneous (abdominal and femoral) fat cells. Although p-synephrine is reported to be the bio-active ingredient that induces lipolysis, further studies are required to confirm this claim [100]. In another study, C. unshu peel extract was found to reduce as much as 50 % of perilipin, a lipid-associated protein known to be secreted only in adipocytes, and to be responsible for stabilizing lipid droplets. This fact may indicate that C. unshu peel extract is related to the inhibition of the formation of lipid droplets [31,101]. Another in vivo investigation reported that oral administration of a major carotenoid compound from C. unshu peel, b-cryptoxanthin (Fig. 10), effectively reduces visceral adipose tissue, body weight, and abdominal circumference of Tsumura Suzuki Obese Diabetes (TSOD) mice [102]. Capsicum annuum (L.)-Solanaceae Capsicum annuum, more commonly known as paprika or red pepper, contains a well-known compound capsaisin (Fig. 11). Water extract from C. annuum fruits was recently shown to inhibit the action of LPL [103], a major enzyme which reduces lipoprotein into monoacylglycerol and free fatty acid [22]. Capsaisin is known to have an anti-LDA effect via several suggested routes, including promotion of thermogenesis and activation of AMPK [104,105]. Another less well-known related compound of capsaisin, capsiate (Fig. 11), is also reported to increase thermogenesis [106][107][108]. In addition to capsaisin and capsiate, from a methanol extract of the fruit of C. annuum, was isolated 9-oxooctadeca-10,12-dienoic acid (Fig. 11), an inhibitor of ACC, an enzyme that carboxylates acetyl-CoA into malonyl-CoA, an important precursor in the fatty acid biosynthesis [109]. In addition to impairing the biosynthesis of lipid droplets, several studies also indicated that a fruit extract of this plant possesses an anti-adipogenic activity when tested by using 3T3-L1 cells [110,111].

Coptis chinensis Franch.-Ranunculaceae
Coptis chinensis or Coptis japonica, is commonly known as Huanglian in China, Ouren in Japan or Hwangryunhaedok-tang in Korea. C. chinensis is well-known for containing a yellow isoquinoline alkaloid, berberine (Fig. 12) [112,113]. Berberine itself is isolated from many other plants and was previously suggested to be effective in the treatment of cancer, diabetes, and obesity [114][115][116][117][118][119]. Its use as an agent for treatment of diabetes and obesity might have suggested the possible use of berberine as an anti-LDA agent. Early in vitro and in vivo studies on the effect of berberine indicated that it inhibits ACC, one of the key lipogenesis enzymes which mediate activation of AMPK. AMPK inhibits the ACC activity via phosphorylation of ACC, as demonstrated in myoblasts and adipocyte in vitro and also in db/db mice in vivo [120]. Similar results were also reciprocated by another study, which reported a similar result showing that berberine also causes activation of AMPK in HepG2, the human hepatocellular carcinoma cell line [121]. In addition to the inhibition of lipogenesis, the anti-adipogenesis effect of berberine was also reported. In summary, berberine has been shown to inhibit the expression of PPARc, and C/EBPa while up-regulating GATA binding protein-2 (GATA-2) and -3 that functions as an adipocyte differentiation suppressor [7, 122,123].
Curcuma longa (L.)-Zingiberaceae Curcuma longa, more commonly known as turmeric, is cultivated in many parts of the tropical countries, from the root of which a member of compounds have been isolated, including curcumin, demethoxycurcumin and  (Fig. 13). Studies on the mixtures of the above mentioned compounds have shown that in rats fed with HFD, administration of such mixtures caused reduction in the body weight gain and LDL-c [10]. In another study, an ethyl acetate fraction from a C. longa root methanol extract was shown to cause partial inhibition of lipid synthesis in 3T3-L1 cells, which was suggested to be via the suppression of GLUT4 expression and stimulation of lipolysis via induction of hormone sensitive lipase (HSL) and ATGL, where both lipases played a role as a rate-limiting enzyme in a lipolysis process [124]. Further investigations of those above mentioned, suggested lipolysis activity may be mediated by curcumin. An in vivo study showed that when added as a supplement to HFD at 500 mg/kg, curcumin itself caused the body weight reduction of C57BL/6J mice. Further studies indicated that when curcumin is administrated to C57BL/6J mice fed with HFD at 500 mg/kg, it increased AMPK activity and CPT1 expression while reducing glycerol-3-phosphate acyl transferase-1. Thus, it causes increases in oxidation and decreases in fatty acid esterification. Subsequent confirmation with RT-PCR, showed that curcumin also lowered the expression of PPARc, and C/EBPa [125]. Investigation by another researcher indicated that curcumin also reduced the expression of PPARc, and C/EBPa in 3T3-L1 cells, further supporting the hypothesis of curcumin influence on PPARc, and C/EBPa expression [126]. Participation of the Wnt signaling pathway in the curcumin-induced suppression of adipogenesis in 3T3-L1 cells was reported. Curcumin was shown to dose dependently restored nuclear translocation of b-catenin, a major component of Wnt signaling, though the definite relationship between Wnt signaling pathway and reduction in PPARc, and C/EBPa expression remains unclear (Table 2) [127].

Conclusions
Concerns over various understandable health implications possibly caused by excessive accumulation of lipid droplets have stimulated studies in the search for anti-LDA agents. However the body energy homeostasis system is all too complex to allow easy solution to the problem. This fact, in a way allows researchers to approach this problem from various wider aspects. This paper is a summary of such attempts from the plant chemistry view point. Recent identification of several plants and their contents showing significant potential activity in inhibiting LDA both in vitro and in vivo has been reported. Many studies are still conducted by using crude extracts, but cases supporting their bioactive compounds that are responsible for the expected activity are isolated and, nevertheless, their mechanisms of action involved have also been reported. As to date, the studies on plant-derived anti-LDA agents are considered to be in their early stages and more in-depth studies are necessary. Nonetheless, from the plant chemistry viewpoint, plant derived anti-LDA agents hold great potential to be developed as a remedy for diseases caused by excessive accumulation of lipid droplets in the future.
Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.  Leaf p-coumaric acid Activation of AMPK leading to increase in fatty acid oxidation activity [142][143][144][145] Zingiber mioga Rosc.
Shoot Ethanol extract Reduction TG and G3PDH whose exact mechanism is not yet elucidated [146] 10.