Antihyperglycemic and cholesterol-lowering potential of dietary fibre from lemongrass ( Cymbopogon citratus Stapf.)

Abstract

BACKGROUND:

The major component of lemongrass by weight is dietary fibre (DF), but no literature has been reported on its DF components and fermentation products when ingested.

OBJECTIVE:

This study analysed DF components of lemongrass, investigated the potential of the major product from total DF (TDF) fermentation to inhibit α-amylase and HMG-CoA reductase, key enzymes of diabetes mellitus and hypercholesterolemia, respectively, and determined the serum glucose- and cholesterol-lowering potential of TDF in an animal model.

METHODS:

Lemongrass DF components were analysed, TDF was fermented in vitro; the major fermentation product was isolated for enzyme inhibitory assays; and postprandial blood glucose- and cholesterol-lowering potential of TDF was determined in Sprague-Dawley rats.

RESULTS:

TDF in lemongrass was 65.7 g/100g and soluble DF, 2.8 g/100 g. Significant amount of propionate (10.9 mM/g TDF) was produced after TDF fermentation; propionate inhibited 20.4% α-amylase activity, and 13.1 % HMG-CoA reductase activity in vitro. TDF further exhibited antihyperglycemic and cholesterol-lowering potential in an animal model.

CONCLUSIONS:

DF from lemongrass was shown to reduce hyperglycemia and hypercholesterolemia in an animal model, through mild inhibition of α-amylase and HMG-CoA reductase. Thus, lemongrass DF may have a significant role in mitigating the risk of type 2 diabetes mellitus and hypercholesterolemia.

Keywords

Lemongrass dietary fibre propionate antihyperglycemic cholesterol-lowering Sprague-Dawley rats

1 Introduction

Diabetes mellitus (DM) has affected about 463 million people aged 20 –79 worldwide in 2019, and an increase to 578 million is projected in 2030. DM is a chronic disease characterized by elevated serum glucose levels due to defective insulin production (Type 1), or ineffective use of insulin (Type 2), or both. Type 2 DM is the prevalent type, with 90% of the worldwide cases classified as Type 2 [1]. Cardiovascular disease (CVD) is the major cause of death worldwide, with an estimated 17.9 million deaths in 2016 [2]. DM is a risk factor for CVD, and around 52% of type 2 diabetics die due to complications related to heart and vascular problems [3]. Insulin resistance in type 2 DM is associated with the release of free fatty acids from fat cells into the blood stream, which leads to secretion of very low-density lipoprotein (VLDL) and LDL cholesterol. Elevated blood sugar levels also cause glycosylation of VLDL and LDL, resulting to formation of plaques in the blood vessels. Patients with DM are prone to CVD as they experience elevated circulating levels of fatty acids, triglycerides, VLDL and LDL cholesterol, but decreased high density lipoprotein (HDL) cholesterol levels [4].

Lemongrass has long been used as a traditional herb in many countries, particularly in Asia and the Philippines. In vitro studies [5] on phytochemicals from lemon grass had been conducted to support traditional claims. Anti-diabetic [6], anti-inflammatory [7], anti-hypertensive [8], and antitumor [9] effects have also been demonstrated in animal models. It has been demonstrated that the extracts contain bioactive compounds, including the essential oil component citral, and the water-soluble components flavonoids, phenolic compounds, and polysaccharides, which were responsible for the antioxidant [10, 11], anti-inflammatory [12, 13], anti-diabetic [14], and anti-cancer activities [15 –17] of the extracts.

In the Philippines, lemongrass is an underutilized plant for all its health benefits. It is mostly used as stuffing and flavor enhancer in native delicacies e.g. roasted chicken or chicken stew. There are few herbal tea products and ready-to-drink juices available, which are expensive. However, more can be produced out of this plant. Fibre is reported to be high [18, 19], but there are no studies yet on the health effects of the dietary fibre of lemongrass or its fermentation products in the colon after ingestion.

Dietary fibre from the family of carbohydrates is non-digestible in the human gut and may be fermented in the colon by beneficial microorganisms. These include cellulose, pectin, β-glucans, fructans, galactooligoasaccharides, and resistant starch [20]. Studies [21] showed that DF is associated with health benefits. Intake of dietary fibre is strongly correlated with weight loss and had been shown to reduce the risk of DM, CVD, and colon cancer. The ability of DF to protect against obesity, diabetes and coronary heart disease (CHD) is attributed to its potential to modulate the expression of key enzymes and hormones related to carbohydrate and lipid metabolism. Sensitivity to insulin may also be improved with DF. Furthermore, DF can be fermented in the colon, resulting in the production of short chain fatty acids (SCFA), and enhanced growth of the beneficial microorganisms in the colon [21].

The anaerobic fermentation of DF in the colon produces various SCFAs, with acetate, propionate, and butyrate, being the most abundant. The amount of SCFA produced was shown to be affected by the amount of soluble DF (SDF) available for fermentation. The SCFA produced was directly related to SDF levels [22]. However, insoluble dietary fibre may also be partially fermented in the colon and may also contribute in the total SCFA produced [23]. The SCFA products from DF fermentation are absorbed by the host and provide energy. About 10% of the daily energy requirement is provided by the SCFA in humans. Acetate and propionate are absorbed by the liver and other organs or cells of the body, and enter various metabolic pathways for energy production while butyrate stays in the colon [24]. In addition, these SCFAs also serve as substrates in the synthesis of biomolecules needed by the body. Acetate is used in the synthesis of cholesterol, long-chain fatty acids, and some amino acids [25]. Propionate is used as a substrate for gluconeogenesis in the liver, and inhibit the limiting enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase for cholesterol synthesis [26]. Butyrate produced remains in the colon since they are the preferred energy source of the colonocytes [27]. The SCFA exerts regulatory effects on fatty acid, glucose, and cholesterol metabolism through various mechanisms [24, 28]. The presence of SFCA in the colon improves blood flow, oxygen uptake, and mucus production; enhances cell differentiation in the colon and proliferation of intestinal cells, as well; SCFA also prevents colon cancer and colitis [27, 29].

Bioactive compounds in functional foods prevent hyperglycemia and diabetes presumably by reducing the absorption of dietary glucose in the blood, either through inhibition of enzymes involved in carbohydrate digestion, e.g. α-amylase and α-glucosidase, or lowering of the glycemic index of carbohydrate-rich foods. The reduction of postprandial blood glucose levels through moderate inhibition of pancreatic α-amylase is a promising treatment for type 2 diabetic patients [30]. On the other hand, bioactive compounds prevent CVD through various mechanisms [31], such as reduction of fat and cholesterol build-up in the arterial and blood vessels; prevention of cholesterol absorption and synthesis; regulation of cholesterol receptors; and increased secretion of bile acids. Inhibition of bile acids reabsorption in the liver also inhibits the formation of cholesterol from the enterohepatic circulation of bile acids [31]. Cholesterol synthesis is reduced when the key enzyme controlling the rate-limiting step, HMG-CoA reductase is inhibited. HMG-CoA reductase is also the target of statin drugs used in treating hypercholesterolemia [32].

There are few functional food products in the Philippines from lemongrass. Thus, research is needed on the dietary fibre component of lemongrass, the major dry weight component, as a potential functional food in preventing the risk for type 2 DM and CVD. A better understanding of the mechanisms of dietary fibre in the potential lowering of serum glucose and cholesterol levels will help promote functional food development based on the major component of lemongrass.

This study was conducted to (1) analyse the dietary fibre components of lemongrass; (2) investigate the potential of the major fermentation product of an in vitro colon model to inhibit α-amylase and HMG-CoA reductase as key enzymes regulating glucose and cholesterol levels, respectively; and (3) determine the potential of TDF in lowering serum glucose and cholesterol levels in an animal model.

2 Materials and methods

2.1 Collection and preparation of lemongrass plant materials

Lemongrass plants were collected from Brgy. Baong, Alimodian, Iloilo, Philippines. Plant samples were submitted for authentication, and a voucher specimen was deposited to the University of Santo Tomas (UST), Philippines herbarium (Certificate Acc. No. USTH 014150). The plant materials were freeze-dried and ground to a fine powder.

2.2 Extraction and analysis of total, soluble and insoluble dietary fibre

Dietary fibre was analysed in duplicates using the enzymatic-gravimetric method of the Association of Official Analytical Chemists (AOAC) Official Method 985.29 and 991.43, Official Methods of Analysis of AOAC International [33]. One gram of the sample was added to 40 mL MES-TRIS buffer pH 8.2, then incubated with 50μL heat stable α-amylase for 35 min with continuous agitation at 95–100°C. The reaction mixture was cooled to 60°C, then 100μL protease was added, and the mixture was incubated further with agitation for 30 min. The mixture was then incubated with 100μL amyloglucosidase for an additional 30 min with agitation. Then 225 mL 95% ethanol was added to the enzyme digest at 60°C, and the mixture was allowed to stand for 1 h at room temperature to precipitate the total dietary fibre. The precipitate was filtered through a pre-weighed crucible containing celite, followed by washing twice with 78% ethanol, 95% ethanol, and acetone, and drying in an oven at 105°C. The residue was analysed for protein and ash content.

The insoluble dietary fibre was determined by filtering the enzyme digest followed by washing twice with 78% ethanol, 95% ethanol, and acetone, and drying in an oven at 105°C. Soluble dietary fibre was computed by the difference between the total and insoluble dietary fibre.

2.3 Fermentation of TDF and analysis of SCFA products

In vitro fermentation of TDF was carried out according to McBurney and Thompson [34] and Trinidad et al. [35]. Total dietary fibre (0.5 g) was mixed with 40 mL fermentation media and 2 mL reducing solution in a serum bottle, and flushed with CO₂ gas until colorless. The bottles were sealed and stored overnight at 4°C. The following day, the bottles were placed in a water bath for 1-2 h at 37°C, then 10 mL fresh human faecal inoculum was added. The faecal inoculum was from a healthy male donor who had not taken antibiotics for a year or more. The bottles were stoppered, sealed with an aluminum seal, and incubated at 37°C for 24 h. The fermented digest, or fermentate, was filtered through a 0.2μm nylon membrane and analysed for the SCFA products acetate, propionate, and butyrate, against a volatile acid standard mix (Supelco) using a Shimadzu HPLC 10A System equipped with a UV-Vis detector. Seventy microliters of the fermentate was injected onto a Bio-Rad HPX-87H 7.8×300 mm column and eluted with 0.005 N H₂SO₄ at a flowrate of 0.7 mL per minute.

2.4 Isolation of the major SCFA product from the fermentate mixture

A portion of the fermentate was filtered through 0.2μm nylon membrane and stored at –80°C prior to separation of SCFA products. The pH of the fermentate was adjusted to approximately 2 by addition of 0.1 M H₂SO₄, filtered through a 0.45μm nylon membrane, and loaded on a Supelclean RP LC-18 SPE column (5g/20 mL) previously conditioned with acetonitrile and equilibrated with water containing 0.1% TFA. The column was then eluted with 5% acetonitrile containing 0.1% TFA, and 3 mL fractions were collected. The elution profile was obtained by reading the absorbance of the fractions at 220 nm using a microplate reader (BMG LabTech FLUOstar Omega). The purity and amount of the SCFA were estimated using Shimadzu Nexera X2 HPLC equipped with diode array detector and autosampler against a mix of acetic, propionic and butyric acids prepared from the corresponding pure reagents (Sigma). Aliquots of the fractions were filtered by hand compressor through 0.2μm nylon membrane and 20μL was injected onto a RP Inert Sustain C18 4.6×100mm 5μm analytical column (GL Sciences Inc., Japan). Elution was carried out for 10 minutes at a flowrate of 1 mL per minute using 5% acetonitrile containing 0.1% TFA, and the eluent was monitored at 214 nm.

The fractions from the SPE column containing the SCFA were pooled together and subjected to preparative HPLC (Waters, Ireland) equipped with photodiode array detector (Waters 2998), quaternary gradient module pump (Waters 2545), autosampler (Waters 2707) and fraction collector with parameters scaled up from analytical HPLC analysis. Batches of 5 mL fractions were loaded onto a RP Spherisorb ODS 20×250 mm 5μm Semi-preparative column (Waters, Ireland) and eluted with 5% acetonitrile containing 0.1% TFA at a flowrate of 18 mL per minute for 25 minutes. The eluent was monitored at 214 nm and fractions were collected at a threshold value of 3 mAu. The fractions containing the individual peaks corresponding to the SCFA were pooled together, adjusted to pH 12, and lyophilized. The dried samples were reconstituted in a small amount of ultrapure water (Type I 18.2 MΩcm at 25°C) for enzyme inhibitory assays. An aliquot was analysed for SCFA concentration and purity as described above. A small amount of the dried samples were submitted for Fourier Transform Infrared (FTIR) analysis in comparison with the standards, to provide confirmation of identity.

2.5 Enzyme inhibitory assays

2.5.1 Alpha-amylase

Alpha-amylase inhibition assay was carried out according to Yu et al. [36] and Hu et al. [37], with modifications. Briefly, 100μL of sample or control was incubated with 50μL α-amylase from porcine pancreas (Sigma, 1 U/mL) in a water bath for 15 min at 37°C. The negative control (blank) was buffer while positive control was acarbose (Sigma) as standard treatment drug. At timed intervals, 500μL 1% starch previously equilibrated to 37°C was added, and the mixture was allowed to react for 5 min. The reaction was terminated by the addition of 550μL DNS reagent. The mixture was boiled for 15 min, cooled to room temperature and diluted with 2 mL ultrapure water. Aliquots of 200μL were transferred to a microplate and the absorbance was read at 540 nm. The % inhibition was computed as follows: $% Inhibition = \frac{Ab s_{Blank 540} - Ab s_{sample 540}}{Ab s_{Blank 540}} \times 100$ (1)

2.5.2 HMG-CoA reductase

The assay for the inhibition of HMG-CoA reductase employed the assay kit CS1090 (Sigma-Aldrich, Singapore) and was based on the decrease in absorbance of NAPDH as oxidized by the enzyme. One microliter of sample was added to 181μL of buffer, followed by addition of 12μL HMG-CoA, 2μL human HMG-CoA reductase enzyme, and 4μL NADPH. The mixture was shaken vigorously and the decrease in absorbance was monitored every 10 s for 10 min at 37°C. The inhibitory activity (IC₅₀) was expressed as the amount which inhibits 50% of the enzyme activity. Pravastatin was used as reference standard treatment drug. The % inhibition was computed as follows: $% Inhibition = \frac{Δ Ab s_{Control 540} - Δ Ab s_{sample 540}}{Δ Ab s_{Control 540}} x 100$ (2)

where Control is the assay mixture without inhibitor.

2.6 Animal model

The protocol was evaluated and approved by the UST Institutional Animal Care and Use Committee (IACUC) under Code No. RC2017–950925 at the UST Graduate School Research Center for the Natural Sciences, España Manila on January 22, 2018. Fifteen Sprague-Dawley rats of mixed sexes, 6–8 weeks old weighing 150–180 g were acquired from an accredited breeder, and allowed to acclimatize for one week. They were housed in steel cages lined with wood shavings as beddings, exposed in a well-ventilated room with a daily cycle of 12 hours light and 12 hours dark, room temperature of 22–25°C and humidity of 55–60%. The males were in separate cages from the females. They were fed with standard rat pellets ad libitum, and allowed free access to purified drinking water. Cages were cleaned twice a week by flushing with detergent and water. After acclimatization, the diet was then modified to a high cholesterol, high sugar diet consisting of 60% standard pellets, 15% lard, 10% egg yolk powder, and 15% sucrose and given ad libitum in the next four weeks. After two weeks of feeding with high cholesterol, high sugar diet, the rats were fasted overnight, and blood samples were drawn from the tail vein for analyses of fasting blood sugar (FBS), total cholesterol (TC), high density lipoprotein (HDL) cholesterol, and low density lipoprotein + very low density lipoprotein (LDL+VLDL) cholesterol. The rats were grouped into three consisting of 5 animals each (consisting of a mix of male and female animals), and age-matched per group. One group was given TDF while the positive control group was given both acarbose and pravastatin. The untreated group served as the negative control.

The treatments were administered daily for two weeks, via oral gavage in 0.5% saline vehicle; the rats were maintained on the high cholesterol, high sugar diet throughout the treatments. The body weight (BW) of the animals were measured every 4 days for dosing of the treatments. TDF extracted from lemongrass was dosed at 400 mg/kg BW based on the Food Nutrition Research Institute –Department of Science and Technology (FNRI-DOST) Philippines recommended intake [38]. The treatment for the positive control group, acarbose and pravastatin, were administered separately at the recommended dose of 40 mg/kg BW and 10 mg/kg BW, respectively [39, 40]. The untreated group received saline only. At the end of the treatment, the rats were fasted overnight and anesthesized with 0.1 mg/kg Zoletil. Blood samples were collected for FBS, TC, HDL and LDL+VLDL cholesterol analyses via intracardiac puncture, and the animals were sacrificed by Zoletil overdose. All blood samples were submitted for analysis of FBS, TC, HDL and LDL+VLDL, using the standard hexokinase method for FBS, enzymatic colorimetric CHOD-PAP method for TC, and direct measure-PEG for HDL; while LDL+VLDL was computed as the difference between TC and HDL.

2.7 Statistical analysis

Differences between the changes in the TC, HDL and LDL+VLDL of the treatment groups were determined using analysis of variance and Duncan’s multiple range test using SPSS Software (IBM SPSS Statistics version 16).

3 Results

3.1 DF content and fermentation products

The result show that total dietary fibre comprise around 66% of the weight of lemongrass, with the insoluble DF contributing much to this content (Table 1). Soluble DF is low, with ratio SDF: IDF of 1: 22. Fermentation of the TDF in vitro resulted to propionate as the predominant SCFA produced, in comparison with the SCFA standard concentrations. Acetate and butyrate concentrations were below detection limits (Table 2).

Table 1
Dietary fibre content of lemongrass, Mean±SEM. SEM stands for the standard error of mean

g/100g DW

Soluble DF 2.83±0.45

Insoluble DF 63.09±0.06

Total DF 65.68±0.26

	g/100g DW
Soluble DF	2.83±0.45
Insoluble DF	63.09±0.06
Total DF	65.68±0.26

Table 2

SCFA products from in vitro fermentation of TDF fibre isolate, Mean±SEM. The products acetate, propionate and butyrate were analysed using HPLC against known concentrations of SCFA standard mix

	mM/g fiber isolate
Acetate	N.D.
Propionate	10.88±0.42
Butyrate	N.D.

N.D. not detected; below detection limits.

The major product of fermentation, propionate, was further isolated from the fermentate mixture to determine its inhibitory effect on α-amylase and HMG-CoA reductase, key enzymes implicated in diabetes mellitus and hypercholesterolemia, respectively.

3.2 Inhibitory activity of propionate from DF fermentation on α-amylase and HMG-CoA reductase

The FTIR profile of the isolate confirmed the separation of the propionate product from the fermentate mixture. Figure 1b shows the profile of the propionate product from DF fermentation, which is comparable to the propionate standard (Fig. 1a) under the same analytical conditions. The basic fundamental vibrations of the carboxyl O-H, C=O, and C-O stretches are seen in both standard and isolate, and slight differences may be due to the equilibrium between the acid and ionic forms [41]. The major product of DF fermentation, which is the propionate isolate, was used in the succeeding inhibitory assays of α-amylase and HMG-CoA reductase enzymes, alongside the propionate standard.

Fig. 1

FTIR spectrum of (a) propionate standard, and (b) isolate, showing the fundamental vibrations of the carboxyl O-H, C=O, and C-O stretches in both standard and isolate.

Table 3 shows the inhibitory activity of propionate standard and propionate isolate from DF fermentation against the α-amylase, in comparison with treatment drug acarbose. It should be noted that inhibition of α-amylase is one of the strategies in the control of diabetes by decreasing the complete digestion of carbohydrates and therefore its absorption thereby decreasing post-prandial concentrations of glucose.

Table 3

A comparison of the inhibitory activity of the propionate standard and isolate from fermentation, and control drug against α-amylase from porcine pancreas, Mean±SEM

Samples	Final Conc, mM	% Inhibition	IC₅₀^*
Standard propionate	1.58	28.76±0.07	5.14±0.07
Isolate	1.58	20.43±3.75
Acarbose	1.58	>100	2.15×10^- 3±0.1
Soluble DF	12.5–108.3 ppm	Nil

^*The inhibitory activity (IC₅₀) was expressed as the amount which inhibits 50% of the enzyme activity.

The standard propionate and propionate isolate both exhibited mild inhibitory activity of α-amylase, with 28.76% and 20.43% inhibition, respectively, at 1.58 mM concentration. IC₅₀ for the propionate standard against α-amylase was determined to be 5.14 mM. Acarbose inhibited 50% of α-amylase activity at 0.002 mM, which is consistent with published reports, with 0.05 mM acarbose inhibiting 88% of porcine pancreatic amylase activity [42].

Table 4 shows the inhibitory activity of the propionate isolate from DF fermentation against the enzyme marker of CVD, HMG-CoA reductase, in comparison with the propionate standard and treatment drug pravastatin. The propionate isolate inhibited human HMG-CoA reductase enzyme, with 13.11% inhibition at 0.1 mM, while the standard propionate exerted 8% inhibition. The IC₅₀ of propionate standard against HMG-CoA reductase was determined to be 19.24 mM, which is consistent with the results of Lin et al. (1995) where propionate inhibited 50% of cholesterol synthesis in human hepatocytes at 20 mM [26]. Statins or the HMG-CoA reductase inhibitor drugs, which are widely-prescribed for cholesterol-lowering, have their inhibitory values at the nanomolar range [43]. Pravastatin, in this study, inhibited 50% of enzyme activity 0.00057 mM.

Table 4

Inhibitory activity against HMG-CoA reductase, Mean±SEM

Samples	% Inhibition^*	IC₅₀
Standard propionate	8.00±.53	19.24±0.8
Pravastatin	>100	5.7×10^- 4±0.5
Isolate	13.11±1.35

^*The final concentration of each of the samples was 0.1 mM.

3.3 Effect of TDF on FBS and cholesterol levels of rats

The effect of TDF was evaluated on Sprague-Dawley rats previously fed with a high sugar, high cholesterol diet for two weeks. The flowchart of the study design and applied protocol in the animal model is shown in Fig. 2. The levels of serum FBS of the rats fed with TDF in comparison to the untreated group and positive control group, Acarbose+Pravastatin, is shown in Fig. 3. All groups experienced increased levels of serum FBS while maintained on the high sugar, high fat diet for another two weeks. However, the group fed with TDF experienced the least increase of serum FBS levels, while the untreated group had the greatest increase in serum FBS, which is significant at p < 0.05. On the other hand, the elevation of FBS levels of the group treated with TDF compared to Acarbose+Pravastatin control group is not statistically different. These result show that dietary fibre fed to the rats could help prevent the rise in FBS levels, even if the rats were on a high sugar, high fat diet.

Fig. 2

Flowchart of study design and applied protocol in animal model.

Fig. 3

Effect of Total Dietary Fibre (TDF) on Serum Fasting Blood Sugar (FBS) of Rats. Connecting bars above each treatment indicate an increase in FBS mean values before and after treatment. ^a–bMean values above connecting bars with unlike letters were significantly different (p < 0.5).

The serum cholesterol levels of the rats are shown in Fig. 4 before and after TDF treatment. Figure 4a shows that all groups experienced a decrease in total cholesterol levels after two weeks of administering the treatments. It is however interesting to note that the animals treated with TDF showed the most lowering of cholesterol level which is higher (p < 0.05) than that of the Acarbose + Pravastatin group. However, this is not significantly different from the untreated group. HDL-cholesterol levels decreased in all groups, but not significantly (Fig. 4b). The LDL+VLDL cholesterol levels increased in all groups (Fig. 4c), with the untreated group having the greatest increase, while the Acarbose+Pravastatin group had the least increase (p < 0.05). The increase in the LDL+VLDL of the TDF group was intermediate between the untreated and the standard drug treated group. Overall, Fig. 4 shows that the TDF of lemongrass has the potential of lowering total cholesterol of Sprague-Dawley rats fed with high sugar, high cholesterol-diet, while minimizing the increase in LDL+VLDL levels.

Fig. 4

Effect of Total Dietary Fiber (TDF) on Serum Cholesterol of Rats. a. Effect of TDF on Serum Total Cholesterol (TC) of Rats. Connecting bars above each treatment indicate a decrease in TC mean values before and after treatment. ^a–bMean values above connecting bars with unlike letters were significantly different (p < 0.5). b. Effect of TDF on Serum High Density Lipoprotein (HDL) Cholesterol of Rats. Connecting bars above each treatment indicate a decrease in HDL mean values before and after treatment; however, the decrease is not significant at p < 0.5. ^cEffect of TDF on Serum Low Density Lipoprotein+Very Low Density Lipoprotein (LDL+VLDL) Cholesterol of Rats. Connecting bars above each treatment indicate an increase in LDL+VLDL mean values before and after treatment. ^a–b Mean values above connecting bars with unlike letters were significantly different (p < 0.5).

4 Discussion

The dietary fibre of lemongrass in this study comprised more than 50% of its weight at 65.68%. Thus, aside from its claimed essential oil and phenolic components, the dietary fibre could contribute to health benefits derived from lemongrass. Intake of DF has been associated with various health benefits, as well as fermentability in the colon, with soluble and insoluble DF being highly and partially fermentable, respectively [20]. Water-soluble polysaccharides have been extracted from lemongrass by boiling in hot water with yield of 10.16% [17] and 1.1% [9], and the presence of significant amounts of mannose in the sugar composition of the polysaccharide has been shown [9, 17]. Mannans were observed to yield high amounts of propionate when fermented in the colon [23]. On the other hand, insoluble fibre from lemongrass consist mainly of cellulose [44], which also yielded good amounts of propionate when fermented in ruminants [45]. Thus in this study, propionate was expected to be the predominant product of lemongrass dietary fibre fermentation in vitro simulating the human colonic conditions; indeed, propionate was observed as the main product of fermentation. It has been demonstrated that inoculum from human feces had potential to degrade fibres of various sugar composition and chemical characteristics. Acetate was produced in higher amounts with polyuronides, e.g. pectin and alginate; propionate levels increased with mannans, fructans, and soy pectin; higher butyrate proportions was obtained with retrograded starch and oat β-glucan [23].

The SCFAs were shown to modulate carbohydrate and lipid metabolism through radioactive labelling studies. Anderson & Bridges (1984) demonstrated that propionate lowers the rate of gluconeogenesis in the liver, while increasing the rate of glycolysis; the overall effect leads to lowering of the blood glucose levels [24]. Demigné et al. (1995) observed that propionate effectively inhibited fatty acid synthesis, and to a lower extent, cholesterol synthesis [28]. In this study, the results show that the propionate isolate can modulate both carbohydrate and lipid metabolism through mild inhibition of key enzymes involved, namely, α-amylase and HMG-CoA reductase enzymes, respectively. This suggesting propionate’s contributing factor in the slow release of glucose and cholesterol synthesis. This is desirable since the concerns of hyperglycemia and hypercholesterolemia, resulting from type 2 DM are both addressed. For diabetic individuals, the recommended treatment for hyperglycemia include strict monitoring and control of glycemic levels, while administration of fixed-dosed statins is recommended for treatment of hypercholesterolemia [4]. Propionate’s mild inhibition of α-amylase is one of the strategies in the control of diabetes by decreasing the complete digestion of carbohydrates and therefore its absorption thereby decreasing post-prandial concentrations of glucose. Propionate’s mild inhibition of HMG-CoA reductase also helps in the prevention of high circulating levels of cholesterol in the blood. Thus, propionate derived from the fermentation of lemongrass DF may have a significant role in the prevention for risk of diabetes and hypercholesterolemia. Though these results are consistent with the propionate standard as reported by other authors, the inhibitory activity for both enzymes are low when compared to that of the standard treatment drugs: acarbose having IC₅₀ value for α-amylase in the μM range, and pravastatin having IC₅₀ for HMG-CoA reductase in the nM range. Thus at the nutritional viewpoint, this can be augmented by increasing the amount of fibre intake, which should fall within the recommended daily values, to increase the amount of propionate needed to inhibit 50% of the enzyme activities. The Food Nutrition Research Institute –Department of Science and Technology Philippines recommends a daily intake of 20 –25 g of DF for adult Filipinos [38].

The DF of lemongrass was further tested on Sprague-Dawley rats for its potential to mitigate the increase in postprandial glucose level and cholesterol synthesis an animal model. A high fat and/or cholesterol-enriched, combined with a high sugar diet generally leads to hyperglycemia and hypercholesterolemia in animal models [46], which is the observed trend in this study. After acclimatization of the rats for one week, their diet was modified to a high cholesterol, high sugar diet for two weeks followed by blood collection for FBS, TC, HDL- and LDL+VLDL cholesterol before administration of TDF, and the control treatments. An appreciable variability is observed in the TC levels of the animals after the two week diet. One probable reason for this is that the sudden shift from the standard rat pellets to the high sugar high cholesterol diet may cause variable responses in individual animals. The variability may not be much if a low cholesterol diet was used. It is observed that in rabbits fed with varying cholesterol levels, the group fed with low dietary cholesterol had little variability in plasma TC, e.g. the groups fed with 0.05% and 0.10% cholesterol had plasma TC of 70±7 mg/dL and 109±15 mg/dL, respectively. However, the plasma TC variability response of rabbits increased when dietary cholesterol was increased, e.g. the group fed with 0.20% cholesterol had plasma TC of 1457±347 mg/dL [47]. Also, a high sugar diet further leads to increased levels of serum TC and LDL cholesterol in rats [48], which adds up to the serum TC levels.

The administration of TDF in rats at a dose equivalent to the recommended daily intake of FNRI-DOST for adult Filipinos for a period of two weeks alongside the high sugar, high cholesterol diet resulted to suppression of increase in serum FBS and LDL+VLDL cholesterol which is comparable to the effect of the Acarbose+Pravastatin control group; and reduction of TC and HDL cholesterol as well. This confirms the in vitro observation of propionate from DF fermentation to exert mild inhibitory action of α-amylase, a key enzyme of DM, resulting to decreased digestion of carbohydrates and lower absorption as well, thus lower post-prandial glucose levels are observed in rats fed with TDF than the untreated group. The mild inhibitory activity of propionate in vitro on HMG-CoA reductase, the key enzyme of cholesterol synthesis, is also confirmed by lower total cholesterol levels in the TDF group, as well as suppressed increase in LDL+VLDL levels.

Cheng & Lai (2000) has demonstrated that the colonic fermentation of DF led to the subsequent increase of propionate levels in serum, further resulting to reduction of serum and liver cholesterol levels in rats [49]. The propionate produced from a high fibre diet has also been shown to reduce incorporation of acetate in serum lipids in humans, which was evident in a longer period of 3-6 months of high fibre intake [50]. Propionate has been associated with cholesterol-lowering effect in rat studies [51] through the inhibition of HMG-CoA reductase. Propionate was also demonstrated to inhibit glucose production and increase glycolysis in the rat hepatocyte [24].

Although the propionate produced from TDF fermentation is one important factor contributing to this effect, it must also be taken into consideration that other mechanisms may enhance this. Dietary fibre is known for its protective effect against diabetes and CVD, though its potential to modulate the expression of key enzymes and hormones related to carbohydrate and lipid metabolism [21]. Dietary fibre is also known to lower the glycemic index of carbohydrate foods and reduce post-prandial glucose absorption, as well as bind cholesterol and bile acids, which may also contribute to the sugar- and cholesterol-lowering effect observed [52].

5 Conclusion

Dietary fibre from lemongrass comprised 65.7% of its weight and produced mainly propionate after simulated colonic fermentation. The propionate isolate from fermentation showed mild in vitro inhibition of α-amylase and HMG-CoA reductase, enzymes that are important in increasing post-prandial glucose and cholesterol synthesis, respectively. Total dietary fibre fed to Sprague-Dawley rats maintained on a high sugar, high cholesterol diet further demonstrated antihyperglycemic and cholesterol-lowering effects; the propionate product of TDF fermentation could be an important contributing factor to this inhibition. Thus, lemongrass dietary fibre and propionate derived from its fermentation may have a significant role in the prevention for risk of diabetes and hypercholesterolemia. This study was the first to provide a better understanding of the potentials of lemongrass as a functional food/ingredient in the prevention for risk of diabetes mellitus and CVD.

Footnotes

Acknowledgments

James David S. Alcantara , for valuable help in DF analyses and TDF extraction.

Niel-Ju Angelle C. Cadiao and Mary Ann Julyn F. Catalan for assistance in the animal study.

Author contribution

MCV, MGN and TPT were involved in conceptualization of the project, write-up, and editing of the manuscript. MCV worked on DF analyses and fermentation, isolation of propionate and enzyme inhibitory assays, conduct of animal study, and data analyses. MGN and TPT supervised the implementation of the project and evaluated data and reports. MCV and TPT acquired the funding of the project.

Funding

Philippine Council for Health, Research and Development – Department of Science and Technology (PCHRD-DOST) for funding of this study.

Conflict of interest

The authors have no conflicts of interest to declare.

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Antihyperglycemic and cholesterol-lowering potential of dietary fibre from lemongrass ( Cymbopogon citratus Stapf.)

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1 Introduction

2 Materials and methods

2.1 Collection and preparation of lemongrass plant materials

2.2 Extraction and analysis of total, soluble and insoluble dietary fibre

2.3 Fermentation of TDF and analysis of SCFA products

2.4 Isolation of the major SCFA product from the fermentate mixture

2.5 Enzyme inhibitory assays

2.5.1 Alpha-amylase

2.7 Statistical analysis

3 Results

3.1 DF content and fermentation products

Table 1 Dietary fibre content of lemongrass, Mean±SEM. SEM stands for the standard error of mean g/100g DW Soluble DF 2.83±0.45 Insoluble DF 63.09±0.06 Total DF 65.68±0.26

5 Conclusion

Footnotes

Acknowledgments

Author contribution

Funding

Conflict of interest

References

Table 1
Dietary fibre content of lemongrass, Mean±SEM. SEM stands for the standard error of mean

g/100g DW

Soluble DF 2.83±0.45

Insoluble DF 63.09±0.06

Total DF 65.68±0.26