Supplementation with the Traditional Thai Polyherbal Medicine NawaTab Ameliorates Lipid Profiles in High-Fat Diet-Induced Hyperlipidemic Rats

A tablet formulation developed from Nawametho decoction (NawaTab), a traditional Thai herbal mixture described in the Worayokasan scripture, has been used clinically for several years in the management of borderline hyperlipidemic individuals. Nevertheless, scientific evidence supporting its claims has never been identified. This study aimed to describe the antihyperlipidemic properties of NawaTab in a well-described model of high-fat diet (HFD)-induced hyperlipidemic rats. Decoction of Nawametho containing equal quantities of Aegle marmelos (L.), Carthamus tinctorius L., Hibiscus sabdariffa Linn., Phyllanthus emblica L., Piper longum L., Piper nigrum L., Terminalia bellirica (Gaertn.) Roxb., Terminalia chebula Retz., and Zingiber officinale Roscoe were prepared. The HFD-fed rats were administered NawaTab for 4 consecutive weeks starting from the ninth week of HFD treatment at a dose of 125 mg/kg (p.o.). Anthropometric parameters, serum lipid profiles, lipase activity, and liver and renal functional markers were measured. Histopathological examination of the liver and any steatosis was carried out at the end of this study. Consecutive treatment with NawaTab (125 mg/kg/day) in HFD-induced hyperlipidemic rats caused a significant reduction in serum total cholesterol, triglyceride, low-density lipoprotein cholesterol, and very low-density lipoprotein cholesterol levels. However, at the tested dose, NawaTab failed to prevent the onset of hepatic steatosis and adipose tissue accumulation. No adverse events due to the consumption of NawaTab on liver and kidney function markers were noted. These findings are the first suggestive evidence for the lipid-lowering capability of NawaTab. However, to promote the use of this formulation in the management of borderline hyperlipidemic patients, elucidation of the underlying mechanisms of action, quantification of biological markers, and clinical trials of NawaTab are urgently needed.


Introduction
Cardiovascular diseases (CVDs) are the leading cause of mortality and contribute substantially to decreased quality of life worldwide, particularly in low-and middle-income countries [1][2][3]. According to a recent systematic review, CVD caused approximately 17.8 million deaths worldwide in 2017, an increase of 21% since 2007 [1]. Moreover, ischemic heart disease and stroke were ranked as the frst and second causes of disability-adjusted life-years in both the 50-74 years and ≥75 years age groups in 2019 [2]. As a major preventable risk factor for CVD, the constantly increasing incidence of hyperlipidemia in several countries has become the main threat to public health [3][4][5].
Statins successfully reduce total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels in patients with elevated LDL-C, thereby reducing the risk of cardiovascular development and mortality. Te administration of statins was found to be associated with elevated liver enzyme transaminases, increased blood glucose and glycated hemoglobin levels, and the occurrence of musclerelated symptoms [6]. Nonadherence to this medication was also noted as a limitation of statins and has been associated with an increased risk of CVD and mortality. Studies have revealed that only half of the patients continue statin therapy through the frst year, and discontinuation rates were approximately 75% in the second year [6]. Terefore, efective treatment of hyperlipidemia with fewer side efects is urgently required to control raised LDL-C levels and thereby limit the morbidity and mortality of CVD.
For many decades, medicinal plants and polyherbal formulations have been used throughout the world for their hypolipidemic activity [7,8]. Tese antihyperlipidemic efects, particularly those of individual medicinal plants, have been tested using both animal and clinical studies. In contrast, only limited information has been established regarding polyherbal formulations/mixtures, which are regularly utilized in several traditional medical systems. Some traditional Chinese medicinal formulas with hypolipidemic efects have been reported, such as Danggui Buxue decoction supplementation of diabetic atherosclerosis rats and hyperlipidemic model rats [9] and Shengmai-san [10], which resulted in a remarkable decrease in TC and LDL-C levels. Furthermore, treating postmenopausal women with hypercholesterolemia with the Danshen-Gegen formula resulted in a signifcant improvement in intima-media thickness and a considerable reduction in TC and LDL-C levels [11]. Based on these earlier fndings [9][10][11], it is interesting to note that traditional polyherbal medicines can be valuable and promising sources for antihyperlipidemic drug discovery in addition to the utilization of single herbs or plant-derived compounds.

Collection of Plant Materials and Preparation of NawaTab.
Nine well-known medicinal plants, as noted in Table 1, were provided by a licensed local pharmacy (Farshen Orsot Part., Ltd., Phatthalung, Tailand) and then authenticated and verifed by a traditional Tai botanist, Assistant Professor Dr. Katesarin Maneenoon. Botanical reference materials of the medicinal plants were assigned and preserved in Materia Medica at the Faculty of Traditional Tai Medicine, Prince of Songkla University (Songkhla, Tailand). Te cleaned plant parts were oven-dried at 80°C for 48 h, powdered, and passed through 40 meshes. Tese ground plant parts were separately stored in airtight containers, and the same batch was applied for the entire study to prevent batch-to-batch variation.
Nawametho decoction was made according to the traditional procedure used in Tailand as follows: the formulation (100 g), which contained an equal amount of each powdered plant part in a fne muslin container (30 cm 2 ) mixed with 1.5 liters of distilled water, was boiled for 2 h at 98 ± 3°C. Tis decoction was fltered, cooled at room temperature overnight, and subjected to spray drying to obtain a dark brown-colored powder with an extraction yield of 10.33% (w/w; dry weight basis). Liquid chromatographymass spectrometry (LC-MS) analysis was performed to identify the chemical compositions of Nawametho decoction. Te result from the LC-MS analysis is shown in Supplementary information S1. NawaTab, which contains 385 mg of Nawametho decoction per tablet, was prepared as described in petty patent number 1903002586.

Experimental Animal Husbandry and Ethics Committee
Approval. Four-week-old male Wistar rats weighing 100-120 g were ordered from Nomura Siam International Co, Ltd. (Bangkok, Tailand). Te rats were housed in stainless steel wire mesh cages under controlled environmental conditions consisting of a relative humidity of 55 ± 10%, a temperature of 22 ± 2°C, and a 12 h light/dark regime at the Southern Laboratory Animal Facility (Prince of Songkla University). Te animals were provided with free access to an irradiation-sterilized standard diet (No. CP 082, Perfect Companion Group Co., Ltd., Bangkok, Tailand) and fltered water ad libitum. Te animals were acclimatized to the laboratory conditions for one week prior to the experiments. All possible attempts were made to reduce animal cruelty and decrease the number of animals used. Te Animals Ethical Committee of Prince of Songkla University (MOE 0521.11/876) approved the study protocols.
Evidence-Based Complementary and Alternative Medicine protein, and 35.8% from carbohydrate. Te standard diet used in this present work contained 25.5% protein, 5.7% fat, 2.1% fber, 6.33% water, vitamins, and minerals and yielded 4.02 kcal/g and 1.77% total energy from fat. Twenty-four healthy Wistar rats were randomly assigned into four groups after a one-week quarantine and acclimatization period. Te detailed study groups were as follows: Group 1 (the control group) received standard palletized diets and distilled water (vehicle) throughout the experiment. Groups 2-4 were fed a customized HFD for 12 weeks. Rats in Groups 2, 3, and 4 were orally administered vehicle (Group 2), NawaTab (125 mg/kg) (Group 3), or simvastatin (a standard hypolipidemic drug; 10 mg/kg) (Group 4) for 4 consecutive weeks starting from the ninth week of HFD treatment [24,25].

Food Consumption and Anthropometric Assessments.
Food intake was assessed once a day as the diference between the amount of food before reflling and the food remaining on the next day and expressed as g/head/day. Body weight was taken daily, while the waist and chest circumferences were recorded every four weeks for the last 12 weeks. Body weight gain was calculated from the difference between fnal body weight and initial body weight. Te food efciency ratio (FER) was evaluated based on the weight gain of the rat (g/day) divided by its intake of the food (g/day).

Analysis of Fecal Lipids.
Te feces of the rats were collected daily and oven-dried at 45-50°C for 24 h. Te samples were then pulverized and kept at −20°C for analysis of the total lipids. As previously described with slight modifcations [26], 3 g of the feces were extracted with 6 mL of a mixture of chloroform and methanol (1 : 1, v/v) and continuously stirred using an incubator shaker for 2 h. Te resultant extract was collected by centrifugation at 3000 rpm for 30 min. Te supernatant (5 mL) was taken and oven-dried to obtain a constant weight of the amount of lipids.

Measurements of Relative Organ Weight, Adipose Tissues, and Hematological and Biochemical Parameters.
After the 12-week treatment, the rats were fasted overnight and fully anesthetized with 100 mg/kg thiopental sodium (Scott-Edil Pharmacia Ltd., Chandigarh, India). Blood samples were taken directly via cardiac puncture and processed for biochemical and hematological assessments in ethylenediaminetetraacetic acid (EDTA)-coated test tubes and nonheparinized tubes, respectively. All biochemical and hematological parameters were analyzed by National Healthcare Systems Co., Ltd. (N Health Hat-Yai, Songkhla, Tailand) according to standard procedures as described below. Te serum was separated by centrifugation at 3000 rpm for 10 min. Assessment of lipase activity and the levels of TC, triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), LDL-C, and very low-density lipoprotein cholesterol (VLDL-C) were determined using a Cobas ® 6000 analyzer (Roche Diagnostics, Penzburg, Germany). Biomarkers of renal and liver function, including levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine, total bilirubin, direct bilirubin, total protein, globulin, and albumin, were also analyzed.
Blood samples with EDTA were used to evaluate hematological parameters, including the white blood cell count (neutrophil, lymphocyte, monocyte, eosinophil, and basophil counts), red blood cell count, hemoglobin, hematocrit, platelet count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and red cell distribution width, using an ADVA 2120i hematology system (Siemens Healthineers AG, Erlangen, Germany).
At the end of the experiment, internal organs (heart, liver, spleen, and kidney) and brown, subcutaneous, and visceral (epididymal, retroperitoneal, and mesenteric) adipose tissues were carefully dissected, and their relative weights were calculated. Te liver was examined for any gross lesions and kept in 10% bufered formaldehyde solution for fxation preparatory to histopathological examination.

Histopathological Evaluation of Hepatic Steatosis.
Liver tissues were fxed in 10% neutral bufered formalin for 24 h, dehydrated with alcohol, and embedded in parafn wax. Serial sections were cut by a rotatory microtome (Leica RM2035, Nussloch, Germany) with 6 μm thickness. After deparafnization, the sections were stained with hematoxylin-eosin (H&E). Microscopic examinations were conducted at magnifcations of 20× using an Olympus Bx53F2 microscope and digital camera attached to CellSens Standard Software (Olympus Optical, Co. Ltd, Tokyo, Japan), which was used to calculate the percentage of hepatocytes containing lipid droplets per total hepatocyte within the hepatic area of 4.5 × 10 12 mm 2 . Hepatocellular steatosis was categorized as follows: grade 0, 1, 2, and 3 if steatosis occupied less than 5%, 5-33%, 34-66%, and more than 67% of the hepatic parenchyma, respectively.

Statistical Analysis.
Te data are presented as the mean ± the standard error of the mean and analyzed by oneway analysis of variance (ANOVA) using SPSS version 17 software (SPSS Inc., Chicago, IL) followed by Tukey's post hoc test. Diferences were considered statistically signifcant at p < 0.05.

Efect of NawaTab on the Food Consumption, Body Weight, and FER of HFD-Induced Hyperlipidemic Rats.
Based on earlier reports, as shown in Table 1, and the traditional utilization of NawaTab as a hypolipidemic agent, the efects of this herbal formula supplementation and HFD consumption on the body weights and fat accumulation of the experimental rats are reported in Figure 1. During the 12-week experiment, it was found that the rats exposed to the HFD did not have a signifcantly diferent food intake than controls, but had a signifcantly lower intake of water. As expected, FERs, body weights, and body weight gain were signifcantly higher in the HFD group than in the control group. Supplementation with either NawaTab or simvastatin caused a slight reduction in the FERs, body weights, and body weight gain of the animals fed a HFD. Furthermore, NawaTab at a dose of 125 mg/kg/day signifcantly decreased the food consumption of these animals. Te rats exposed to either the standard control diet or HFD did not show signifcant changes in their waist-chest ratio.

NawaTab Supplementation Improves the Lipid Profle of HFD-Induced Hyperlipidemic Rats.
Except for fecal lipid contents, the activities of lipase and the levels of TC, TG, HDL-C, LDL-C, and VLDL-C of the HFD-fed group were higher than those of the rats fed the standard diet, indicating that the animal hyperlipidemia model that was developed was accurate ( Table 2). Supplementation of the hyperlipidemic rats with this herbal formula at a dose of 125 mg/ kg/day for 4 weeks signifcantly reduced serum TC, TG, LDL-C, and VLDL-C levels. It should be further noted that serum TG and VLDL-C levels of rats in the HFD group receiving NawaTab were similar to those of the negative control group. However, treating the hyperlipidemic animals with this polyherbal decoction did not cause serum HDL-C changes. Although reductions in serum TC, TG, HDL-C, LDL-C, and VLDL-C levels in HFD-fed animals were observed after treatment with simvastatin at a dose of 10 mg/kg/day for 4 weeks, only the levels of TC, HDL-C, and LDL-C were found to be signifcant. In the present study, there were no signifcant changes in the fecal lipid content or the lipase activity of the hyperlipidemic rats supplemented with either NawaTab or simvastatin.
Te results illustrated in Table 2 show that feeding animals a HFD caused a signifcant elevation in the accumulation of visceral and subcutaneous white adipose tissues compared to the negative control group, which received the standard diet. Oral administration of either NawaTab or simvastatin to rats fed a HFD did not signifcantly reduce adipose tissue accumulation. A slight reduction in the amount of retroperitoneal, mesenteric, and subcutaneous adipose tissues was found in the HFD-fed group treated with this herbal decoction. In the hyperlipidemic rats administered simvastatin, a small decrease in the amount of mesenteric and subcutaneous adipose tissues was observed. It has been well-documented that brown adipose tissue plays an important role in thermoregulation, body weight regulation, glucose, and lipid homeostasis [27]. However, treatment with either NawaTab or simvastatin in rats fed a HFD did not afect the level of brown adipose tissue accumulation.

Te Administration of NawaTab Did Not Protect against HFD-Induced Liver Steatosis in Rats.
Feeding a HFD for 12 weeks signifcantly induced liver steatosis in rats, by 33fold compared to the negative control group, as presented in Table 2. Compared to the vehicle-treated group, NawaTabtreated HFD-induced liver steatosis rats did not experience a signifcant change in the percentage of hepatocytes containing lipid droplets or the grading of hepatic steatosis. Conversely, simvastatin administration appeared to significantly reduce the severity of HFD-induced liver steatosis, from grade 3 (92.4 ± 3.9%) to grade 1 (13.0 ± 2.8%).
Pathological images of liver tissue stained with H&E revealed abundant cytosolic lipid droplets in the hepatocytes and both macro-and microvesicular steatosis in the HFD group ( Figure 2). As expected, treatment with simvastatin exhibited signifcant efcacy in defense against these pathological features. However, the consumption of NawaTab at a dose of 125 mg/kg/day for 4 weeks appeared to fail to protect against these changes (Figure 2).

Efect of NawaTab on the Relative Weight of Organ and Biochemical and Hematological Parameters of HFD-Induced
Hyperlipidemic Rats. Any undesirable efects of the plant extract on rats' major organs would likely cause alterations in the organ/body ratio, hematological parameters, and biochemical parameters, as illustrated in Table 3. No such changes were noted in this study, which confrms the nontoxicity of NawaTab. No major changes were observed, except in the kidneys and in the weight of the liver, spleen, and heart of HFD-induced rats. Te results showed that feeding a HFD signifcantly reduced the weight of the kidney, whereas treatment with NawaTab and simvastatin did not cause a notable improvement in this organ weight. Furthermore, there were no alterations in any tested biochemical parameters of vehicle-treated, simvastatintreated, or NawaTab-treated hyperlipidemic rats. In contrast, the percentages of lymphocytes, monocytes, and eosinophils were signifcantly changed in the vehicletreated HFD-fed rats relative to the rats in the normal control group. Supplementation with either simvastatin or NawaTab failed to cause an improvement in these hematological parameters.

Discussion
CVD is one of the most important causes of death in several countries, and the rising number of people with hyperlipidemia as a major risk factor for this disease has become a global concern. In Tailand, six in ten Tai people live with at least one form of dyslipidemia [28]. Analysis has shown that approximately 30%, 47%, and 39% of Tai individuals have high LDL-C, low HDL-C, and high TGs, respectively. Poor public awareness of dyslipidemia should be considered a factor, but a correlation between CVD markers, carotid intima-media thickness, and hyperlipidemia among the Tai population has been demonstrated [29]. Furthermore, previous fndings indicated that 27.1% of the Tai worker population and 38.7% of chronic disease patients were selfprescribed herbal medicine for dyslipidemia [30,31]. However, there is limited scientifc information to show the efciency and safety of traditionally used medicines. Table 2: Efect of NawaTab on serum lipid profles, the relative weight of adipose tissue, hepatic steatosis, fecal lipid excretion, and lipase activity of high-fat diet (HFD)-induced hyperlipidemic rats. Te results are given as means ± SEM (n � 6). Values in the same row sharing a common superscript are not signifcantly diferent (p < 0.05). TC, total cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; VLDL-C, very low-density lipoprotein cholesterol. Terefore, in the present work, we demonstrated for the frst time that oral administration of NawaTab (125 mg/kg/ day) for only 4 weeks efectively reduced plasma TC, TG, LDL-C, and VLDL-C levels in HFD-induced hyperlipidemic rats. Medicinal plants have been highlighted as alternative sources of antihyperlipidemic agents. According to an intensive review by Bahmani and his colleagues [7], some wellknown medicinal plants, such as Cynara cardunculus (artichoke), Trigonella foenum graecum L (fenugreek), Allium sativum L (garlic), Glycine max (soybean), and Commiphora mukul (guggul, gugulipid) were able to alleviate TC, TG, and LDL-C levels in clinical studies. However, it should be noted that their treatment periods were between 6 and 12 weeks.

Parameters
Several mechanisms involved in the antihyperlipidemic efects of medicinal plants and plant-derived compounds have been established. A pancreatic lipase inhibitor, such as orlistat, leading to a decrease in lipid absorption is an appealing approach for antihyperlipidemic agent discovery. Plant-derived polyphenols have been reported as the most important sources of prospective pancreatic lipase inhibitors to varying degrees [32]. Among these compounds, gallic acid, an active constituent found in P. emblica, T. bellirica, and T. chebula, had remarkable antipancreatic lipase activity [33]. Even though fve out of nine herbal components of NawaTab, including H. sabdarifa [34], P. longum [35], P. nigrum [36], T. bellirica [37], and Z. ofcinale [38], have been shown to produce antipancreatic lipase activity, treatment of HFD-fed rats with NawaTab did not result in signifcant changes in their lipase activity or fecal lipid contents in our study. Te adipogenesis inhibition mechanisms of simvastatin remain unclear, but this HMG-CoA reductase inhibitor is widely used to lower LDL-C in patients with hyperlipidemia and cardiovascular diseases. With the exception of T. bellirica, all herbal components of NawaTab demonstrated an inhibitory efect on adipogenesis in 3T3-L1 preadipocytes [39][40][41][42][43][44][45][46]. Tese medicinal plants were shown to either suppress lipid and TG accumulation, inhibit the proliferation of adipocytes, or initiate fat cell apoptosis. It is speculated that the hypolipidemic efect obtained from NawaTab is at least involved in the inhibitory properties of its herbal component toward adipogenesis and pancreatic lipase activity. However, further examinations are required to elucidate the lipid-lowering mechanisms of NawaTab.
Similar to the hypolipidemic efects of NawaTab, previous in vivo experiments revealed that its individual herbal components also decrease plasma TC, TG, and LDL-C levels. H. sabdarifa [47], P. emblica [48], P. longum [49], P. nigrum [50], and T. chebula [51] were able to improve HDL-C levels and reduce TC and TG levels in diet-induced hyperlipidemia rat models. C. tinctorius [41], H. sabdarifa [43], and T. bellirica [52] also exhibit hypolipidemic efects in other hyperlipidemic animals, including rabbits, hamsters, and mice. Furthermore, oral administration of A. marmelos [53], P. emblica [54], T. bellirica [37], and Z. ofcinale [55] has been found to improve the lipid profles of diabetic animal models. Additionally, consecutive administration of NawaTab for 28 days at a dose of 125 mg/kg/day did not cause undesirable changes in organ or body weight, hematological parameters, or kidney and liver function markers. According to polyherbalism theory, the use of multiple medicinal plant combinations can result in a synergistic efect, thereby ofering extra therapeutic efectiveness and decreasing unpleasant side efects. Te specifc active chemical constituents that might be responsible for the lipid-lowering efects of NawaTab, such as aegeline, halfordinol, ethyl ether aegeline, esculetin, hydroxysafor yellow, delphinidins, digallic acid, gallic acid, piperlonguminine, piperine, pipernonaline, and galanolactone, need to be quantifed.

Conclusion
In conclusion, our fndings demonstrated for the frst time that NawaTab, which is regularly used for the management of borderline hyperlipidemic patients, exhibits antihyperlipidemic efects. Te formulation reduced plasma TG, TC, and LDL-C levels of HFD-induced hyperlipidemic rats at a dose of 125 mg/kg/day for only 4 weeks without adverse events. Although our results provide preliminary support for the traditional utilization of NawaTab, further scientifc investigation is required to describe the mechanisms of action, particularly NawaTab's adipogenesis inhibition efect. Moreover, the active agents need to be determined and further applied as quality markers of NawaTab.