Reference Genes Selection and Validation for Cinnamomum burmanni by Real-Time Quantitative Polymerase Chain Reaction.

In recent years, the field of biology has witnessed a surge of interest in genomics research due to the advancements in biotechnology. Gene expression pattern analysis plays a crucial role in this research, as it enables us to understand the regulatory mechanism of gene expression and the associated biological processes. Real-time quantitative polymerase chain reaction (q-PCR) is an efficient method to analyze the gene expression patterns, for which accuracy relies on the standardized analysis of reference genes. However, numerous studies have shown that no reference gene is universal in all conditions, so screening a suitable reference gene under certain conditions is of great importance. Cinnamomum burmannii (C. burmannii) is rich in volatile components and has high medicinal and economic value. However, knowledge of the screening of reference genes for the gene expression analysis of C. burmannii is insufficient. Aiming at this problem, we evaluated and screened the reference genes in C. burmannii under different experimental conditions, including different abiotic stresses (Cold-treated, PEG-treated and Nacl-treated), different tissues, leaves at different developmental stages and different chemical types. In this study, different algorithms (∆Ct, geNorm, NormFinder and BestKeeper) were used to evaluate the stability of the candidate reference genes, and RefFinder further merged the output data to screen out the optimum reference gene under various experimental conditions in C. burmannii. The results showed that the optimal reference gene number for gene standardization was 2 under different experimental conditions. RPL27|RPS15 was the most suitable combination under the Nacl-treated and PEG-treated samples. RPL27|APT was the optimum combination under the Cold-treated samples. The optimal combinations of other samples were EF1α|ACT7 for different tissues, eIF-5A|Gllα for different borneol clones in C. burmannii, RPS15|ACT7 for leaves at different developmental stages and RPS15|TATA for all samples. Additionally, two terpenoid synthesis-related genes (CbWRKY4 and CbDXS2) were standardized to verify the feasibility of the selected reference genes under different experimental conditions. This study will be helpful for the subsequent molecular genetic mechanism study of C. burmannii.

Oxidative Stress-Mediated Repression of Virulence Gene Transcription and Biofilm Formation as Antibacterial Action of Cinnamomum burmannii Essential Oil on Staphylococcus aureus.

This work aimed to identify the chemical compounds of Cinnamomum burmannii leaf essential oil (CBLEO) and to unravel the antibacterial mechanism of CBLEO at the molecular level for developing antimicrobials. CBLEO had 37 volatile compounds with abundant borneol (28.40%) and showed good potential to control foodborne pathogens, of which Staphylococcus aureus had the greatest inhibition zone diameter (28.72 mm) with the lowest values of minimum inhibitory concentration (1.0 µg/mL) and bactericidal concentration (2.0 µg/mL). To unravel the antibacterial action of CBLEO on S. aureus, a dynamic exploration of antibacterial growth, material leakage, ROS formation, protein oxidation, cell morphology, and interaction with genome DNA was conducted on S. aureus exposed to CBLEO at different doses (1/2-2×MIC) and times (0-24 h), indicating that CBLEO acts as an inducer for ROS production and the oxidative stress of S. aureus. To highlight the antibacterial action of CBLEO on S. aureus at the molecular level, we performed a comparative association of ROS accumulation with some key virulence-related gene (sigB/agrA/sarA/icaA/cidA/rsbU) transcription, protease production, and biofilm formation in S. aureus subjected to CBLEO at different levels and times, revealing that CBLEO-induced oxidative stress caused transcript suppression of virulence regulators (RsbU and SigB) and its targeted genes, causing a protease level increase destined for the biofilm formation and growth inhibition of S. aureus, which may be a key bactericidal action. Our findings provide valuable information for studying the antibacterial mechanism of essential oil against pathogens.

Application of Cinnamomum burmannii Essential Oil in Promoting Wound Healing.

Skin wounds, leading to infections and death, have a huge negative impact on healthcare systems around the world. Antibacterial therapy and the suppression of excessive inflammation help wounds heal. To date, the application of wound dressings, biologics and biomaterials (hydrogels, epidermal growth factor, stem cells, etc.) is limited due to their difficult and expensive preparation process. Cinnamomum burmannii (Nees & T. Nees) Blume is an herb in traditional medicine, and its essential oil is rich in D-borneol, with antibacterial and anti-inflammatory effects. However, it is not clear whether Cinnamomum burmannii essential oil has the function of promoting wound healing. This study analyzed 32 main components and their relative contents of essential oil using GC-MS. Then, network pharmacology was used to predict the possible targets of this essential oil in wound healing. We first proved this essential oil's effects in vitro and in vivo. Cinnamomum burmannii essential oil could not only promote the proliferation and migration of skin stromal cells, but also promote M2-type polarization of macrophages while inhibiting the expression of pro-inflammatory cytokines. This study explored the possible mechanism by which Cinnamomum burmannii essential oil promotes wound healing, providing a cheap and effective strategy for promoting wound healing.

Full-Length Transcriptome Sequencing Combined with RNA-Seq to Analyze Genes Related to Terpenoid Biosynthesis in Cinnamomum burmannii.

Cinnamomum burmannii is a cinnamomum plant rich in natural D-borneol. Natural D-borneol is a bicycle monoterpenoid compound widely used in the food, pharmaceutical, and cosmetic industries. Therefore, analyzing the biosynthesis mechanism of natural D-borneol in C. burmannii at the molecular level is helpful for directional breeding in the future and further development and utilization of C. burmannii and its related gene resources. In our study, 76 genes related to terpene metabolism were analyzed through third-generation sequencing and second-generation sequencing. Of these genes, 57 were associated with the synthesis of the terpenoid skeleton, and 19 belonged to terpenoid synthase, including four monoterpenoid synthases, seven sesquiterpenoid synthases, and eight diterpenoid synthases. Two genes in diterpenoid synthase were differentially expressed in high D-borneol and low D-borneol plants. It was speculated that these two genes might be related to D-borneol synthesis. How these two genes participate in the synthesis of D-borneol needs further study.

Diversity of Fungal Communities on Diseased and Healthy Cinnamomum burmannii Fruits and Antibacterial Activity of Secondary Metabolites.

The composition and structure of fungal communities on healthy and diseased fruits of Cinnamomum burmannii (Nees and Nees) Blume were characterized, with evaluation of the antibacterial activity of secondary metabolites from culturable fungi following the first identification of secondary metabolites in the fungus Medicopsis romeroi (Esf-14; GenBank accession number OK242756). These results are significant for understanding the functional variation in bioactivity in fungal communities and developing a broader range of bioactive resources. High-throughput sequencing results indicated that the fungal community in diseased fruit differed from that in healthy fruit at the phylum, class, order, or genus level, with significant differences in the species and relative abundance of the dominant flora. A total of 49 (healthy fruit) and 122 (diseased fruit) artificially cultivable endophytic fungi were isolated, and 41 different strains (11 from healthy fruit and 30 from diseased fruit) were successfully identified by morphological and molecular biological analyses, which were classified into 8 groups and 23 genera by phylogenetic tree analysis, with Pleosporales, Glomerellales, and Hypocreales being the dominant groups at the order level and Colletotrichum being the dominant group at the genus level. The results of the antibacterial assay demonstrated that the secondary metabolites of all strains had different degrees of antibacterial activity, while the secondary metabolites of endophytic fungi from diseased fruit were generally stronger than those of fungi from healthy fruit, with the active secondary metabolites dominated by small and moderately polar compounds. Combined analysis of fungal communities, phylogenetic tree analysis, and bioactivity analysis of culturable strains revealed strong antibacterial activity of both upregulated and downregulated flora in diseased fruit. Five compounds, including two new (5,6-dimethoxy-[1',1:4,1″-terphenyl]-2-ol [compound 1] and 5-(methoxycarbonyl)-2-methylbenzo[d][1,3]dioxole-2-carboxylic acid [compound 2]) and three known compounds (3,7-dihydroxy-1,9-dimethyldibenzofuran [compound 3], methyl 3-hydroxybenzoate [compound 4], and uracil [compound 5]), were isolated and identified for the first time from the endophytic fungus Medicopsis romeroi. In general, the diversity of fungal communities on diseased fruit was lower than that on healthy fruits, while the antibacterial activity of artificially cultured endophytic fungi on diseased fruits was generally stronger than that on healthy fruits, suggesting excellent promise for the development of secondary metabolites from active strains on diseased fruit as antibacterial agents. IMPORTANCE Powdery fruit disease is a notorious disease of Cinnamomum burmannii that causes severe loss in fruit production. Studies on the function of endophytic fungal communities in healthy plant tissues are not new, while little is known about the functional changes of fungal communities in disease-causing plant tissues. Our results demonstrate that fungal communities in diseased fruits differ from those in healthy fruits at the level of phylum, class, order, or genus, with significant differences in the species and relative abundance of dominant groups. Endophytic fungi in diseased fruits appeared to produce secondary metabolites with stronger antibacterial properties, although the community diversity was not as varied as that in healthy fruits. In addition, secondary metabolites of the Medicopsis romeroi strain from diseased fruits were identified for the first time. These results have important implications for understanding the functional variation of bioactivity in fungal communities and for developing a broader resource of bioactivity.

Hepatoprotection of Cinnamomum burmannii ethanolic extract against high-fat and cholesterol diet in Sprague-Dawley rats (Rattus norvegicus).

Background and Aim: The pathogenesis of non-alcoholic steatohepatitis involves non-alcoholic fatty liver, oxidative stress, inflammation, and fibrosis. Although the long-term use of cinnamon bark in larger doses can negatively affect good health, proper use of its extracts effectively and efficiently improves health. Therefore, this study aimed to determine the minimal dose of Cinnamomum Burmannii extract through its activity in inhibiting oxidative stress in rats' livers treated with a high-fat and cholesterol diet (HFCD). Materials and Methods: Forty-two Sprague-Dawley rats (Rattus norvegicus), weighing 200-250 g body weight (BW), were divided into seven treatment groups with six replications: Normal, HFCD, atorvastatin, quercetin, and C. burmannii ethanol extract group, after which they were administered different dosages (i.e., 100, 200, and 300 mg/kg BW). Except for the normal group, rats were concomitantly administered HFCD with each treatment for 21 days. Then, their malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity were assessed using colorimetry. However, their steatosis levels were determined based on histological preparations with hematoxylin-eosin staining. Results: Duncan's multiple range test (DMRT) results indicated that all treatments had a significantly lower MDA than HFCD and normal rats (a=0.01). DMRT results also showed that treating with the C. burmannii ethanol extract at all dosages resulted in a significantly higher SOD activity level in HFCD rats than those treated with quercetin and atorvastatin (a=0.01). Furthermore, results showed that treatment with C. burmannii extracts at a dosage of 300 mg/kg BW incredibly maintained SOD activity as effective as quercetin, atorvastatin, and normal rats. Besides, while steatohepatitis levels of C. burmannii ethanol extract at dosages of 200 and 300 mg/kg BW commensurated with normal rats, steatohepatitis levels were significantly lower than those administered other concentrations or treatments (a=0.05). Conclusion: Ethanolic C. burmannii extracts protected the liver by regulating oxidative stress. Therefore, a 200 mg/kg BW dose is proposed as the minimal hepatoprotection dose to prevent fatty liver formation.

Larvicidal activity and Histopathological changes of Cinnamomum burmannii, Syzygium aromaticum extracts and their combination on Culex pipiens.

In order to develop an eco-friendly botanical larvicide alternative to the synthetic larvicides, extracts were prepared from the Cinnamomum burmannii (C.B.) and Syzygium aromaticum (S.A.) with hexane using a sonicator. The extracts were evaluated for larvicidal activity individually and in combination against the Culex pipiens larvae. The LC50 value of C.B. and the S.A. hexane extracts tested individually were 184.2 and 363.7 µg/mL against Cx. pipiens respectively. All the combinations of the extract of C.B. and S.A. showed synergistic factors higher than one. Among the different ratios of extracts, the SA25%: CB75% extract was found to be more toxic than the other combinations (LC50:125.7 µg/mL). Midgut cells treated with S.A. 25%: C.B. 75% extract showed severe morphological alterations such as degradation of microvilli; degeneration of epithelial cells, and peritrophic membrane; loss of nuclei, irregular and damage of microvilli. The extract has a promising larvicidal potential against Cx. pipiens, However, the extract was toxic against HUVEC cells, as evident from MTT and cell morphology. Further investigation is required to assess the toxicity of the extract on aquatic animals.

Elucidation of the essential oil biosynthetic pathways in Cinnamomum burmannii through identification of six terpene synthases.

Cinnamomum burmannii is a traditional plant that has long been used as a spice, food preservative, and food flavoring. Essential oils in C. burmannii, which mainly consist of mono- and sesquiterpenes such borneol, linalool, and caryophyllene, have impressive pharmaceutical properties. Although the transcriptome-based discovery of (+)-bornyl diphosphate synthase (CbTPS1) from C. burmannii was reported in our previous study, the remaining terpene synthases (TPSs) corresponding to various terpene biosynthesis pathways remain unidentified. In this study, we report the results of RNA-sequencing of a borneol type plant and functional characterization of six additional full-length candidate TPS genes (named CbTPS2-7). Phylogenetic analysis revealed that CbTPS2 and CbTPS3 together with the previously identified CbTPS1 protein belong to the TPS-b subfamily, and enzyme assays using geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) as substrates revealed that CbTPS1, CbTPS2 and CbTPS3 catalyze the formation of monoterpenes. CbTPS4, CbTPS5, and CbTPS6, which belong to the TPS-a clade, generated monoterpenes and sesquiterpenes. CbTPS7, which belongs to the TPS-g clade, showed linalool/nerolidol synthase activity. These CbTPSs identified in C. burmannii produced a total of 10 monoterpenes and 14 sesquiterpenes in an in vitro assay. These findings clarify the biosynthesis pathways of 13 monoterpenoids and 12 sesquiterpenoids in the leaf essential oil of C. burmannii and shed light on terpene biosynthesis in Cinnamomum.

Effect of Aqueous Cinnamon Extract on the Postprandial Glycemia Levels in Patients with Type 2 Diabetes Mellitus: A Randomized Controlled Trial.

Cinnamon is a spice used in traditional cuisine that has been investigated due to hypoglycemic properties. The objective of this study was to investigate the effect of aqueous cinnamon extract on postprandial glycemia levels in type 2 diabetes mellitus (DM2) adults. This clinical trial enrolled 36 adults with DM2, randomly allocated in two groups: the control group (n = 18) took only an oral glucose tolerance test (OGTT) and the intervention group (n = 18) took OGTT immediately followed by aqueous cinnamon extract (6 g/100 mL) ingestion. Blood glucose levels were measured on fasting and after 30, 60, 90 and 120 min in both groups. The chemical analysis of the aqueous cinnamon extract included total phenols content determination and antioxidant activity assessment through FRAP and DPPH methods. The data reveal that aqueous cinnamon extract ingestion did not show a significant difference in the incremental area under the curve (p = 0.834), maximum glucose concentration (p = 0.527) and glucose concentration variation (p = 0.873) compared with the control group. Cinnamon extract possess a total phenol content of 1554.9 mg/L gallic acid equivalent and a strong antioxidant capacity, revealed by the DPPH (5125.0 µmol Trolox/L) and FRAP (3658.8 µmol Trolox/L) tests. Aqueous cinnamon extract did not significantly influence postprandial glucose response in diabetic patients during an OGTT.

Antibacterial activity of clove (Syzygium aromaticum) and cinnamon (Cinnamomum burmannii) essential oil against extended-spectrum ß-lactamase-producing bacteria.

BACKGROUND AND AIM: Extended-spectrum ß-lactamase (ESBL) is an enzyme produced by the family of Enterobacteriaceae, especially Escherichia coli and Klebsiella pneumoniae, which can hydrolyzeß-lactam antibiotics, such as penicillins, cephalosporins, cephamycin, and carbapenem. ESBL-producing bacteria are widely distributed from farms to slaughterhouses until food products originating from animals are available in the market, which plays an important role as a pathway for the exposure and transmission of ESBL-producing bacteria from food products of animal origin to humans. This study aimed to determine the antibacterial activity of Syzygium aromaticum (clove) and Cinnamomum verum (cinnamon) essential oils against strains resistant to ESBL-producing E. coli and K. pneumoniae isolates. MATERIALS AND METHODS: The antibacterial activity of clove and cinnamon essential oils was tested against three strains of tested bacteria using the disk diffusion method. The minimum inhibitory concentration (MIC) of clove and cinnamon essential oils was determined using the broth microdilution method. The minimumbactericidal concentration (MBC) was determined using the MIC. Morphological changes on each tested bacteria were observed through scanning electron microscopy (SEM). RESULTS: Both essential oils exhibited inhibitory effects toward all test organisms, indicated by inhibition zones around the disk. The MIC values of clove essential oil were 0.078% (v/v) for all tested bacteria, whereas the MICs of cinnamon essential oil ranged from 0.039% (v/v) to 0.156% (v/v) for all tested bacteria. MBC values of clove and cinnamon essential oils ranged from 0.078% (v/v) to 0.156% (v/v) for all tested bacteria. There were morphological changes in each tested bacterial cell that was observed through SEM. Each tested bacteria treated with clove and cinnamon essential oils showed shrinkage and cells lysis. CONCLUSION: It was concluded that clove and cinnamon essential oils have emerged as effective antibacterial agents by showing high antibacterial activity against ESBL-producing E. coli and K. pneumoniae isolates, as evidenced by the inhibition zone diameter and MIC value.

Gastroprotective Effect of DLBS2411 Bioactive Fraction from Cinnamomum burmannii Against Ethanol-Induced Gastric Damage in Rats.

BACKGROUND: The study was carried out to evaluate the anti-ulcerative and gastroprotective effect of DLBS2411, a bioactive fraction from Cinnamomum burmannii (Nees & T. Nees) Blume, in Wistar rats (Rattus norvegicus). METHODS: The rats were divided into five treatment groups, which were the Normal control group, Negative control group (ethanol-induced) and two treatment groups: DLBS2411 at the doses of 25 mg/kg body weight (BW) and 50 mg/kg BW, and the Positive control group treated with sucralfate at the dose of 100 mg/kg BW. Gastroprotective effect was measured by the ulcerative lesion index, ulcer surface area, percentage of lesion area, and cure ratio. Hematological and histopathological analyses were also conducted to gain additional data regarding the gastroprotective effect of DLBS2411 in the rats' stomachs. RESULTS: DLBS2411 was found to contain not less than 15% of total phenolic compounds. Treatment with DLBS2411 at doses of 25 mg/kg BW and 50 mg/kg BW significantly reduced the percentage of ulcer area in rats. The percentage of ulcer area for the Negative control group and both doses in the DLBS2411 treatment group reached 22.64±6.82%, 6.75±4.41%, and 6.18±4.63%, respectively. Ulcer surface area in the treatment groups and Positive control group also decreased. Histopathological data showed that gastric epithelial cells in the Negative control group were more severely ulcerated than in the treatment group of DLBS2411 and the Positive control group. CONCLUSION: This study showed that DLBS2411 at the dose of 50 mg/kg BW was more effective in protecting the stomach lining than DLBS2411 at the dose of 25 mg/kg BW, as measured by percentage of ulceration inhibition and the ulcerative lesion index.

Cinnamomum burmannii (Nees & T. Nees) Blume and Eleutherine palmifolia (L.) Merr. extract combination ameliorate lipid profile and heart oxidative stress in hyperlipidemic mice.

BACKGROUND AND AIM: Hyperlipidemia is an important risk factor for cardiovascular disease. The use of statins has adverse side effects that result in oxidative stress disorders. The objective of this study was to investigate the antihyperlipidemic effect of a combination of Cinnamomum burmannii and Eleutherine palmifolia extract in high-fat diet (HFD)-induced hyperlipidemia mice. MATERIALS AND METHODS: Mice were divided into eight groups (n=4): Control group or healthy mice (normal), HFD-induced hyperlipidemic mice without any treatment (CE0), HFD-induced hyperlipidemic mice treated with 3.6 mg/kg body weight (BW) atorvastatin (atorvastatin), and HFD-induced hyperlipidemic mice treated with a combination of C. burmannii and E. palmifolia in the following ratios: 300:0 (C300), 225:75 (C225), 150:150 (CE150), 75:225 (E225), and 0:300 (E300). Mice were fed a HFD for 4 months to induce hyperlipidemia. Total cholesterol, cholesterol oxidase-peroxidase aminophenazone (CHOD-PAP), triglyceride-glycerine, and fat serum were analyzed with colorimetric method. The measurement of superoxide dismutase was done with the xanthine oxidase method and malondialdehyde measurement was done with the thiobarbituric acid method. RESULTS: Results showed an increase in antihyperlipidemic characteristics as the concentration of E. palmifolia extract (p<0.05) increased. Duncan's multiple range test also showed an increase in anti-stress oxidation as the concentration of C. burmannii extract (p<0.05) increased. CONCLUSION: The E225 group showed the most potential as a safe, antihyperlipidemic agent characterized by improvement in lipid profile and antioxidant balance.

Safety assessment of standardised methanol extract of Cinnamomum burmannii.

The present study aims to evaluate the safety of methanol extract of Cinnamomum burmannii (MECB) by acute 14-day (single dose) and sub-chronic 28-day (repeated doses) oral administration to Sprague-Dawley rats. Our results showed that no toxicity was found in either acute or sub-chronic toxicity studies. MECB (containing 0.07% and 0.20% (w/w) of coumarin and trans-cinnamaldehyde, respectively), which was given orally at doses of 500, 1000 and 2000 mg/kg caused neither visible signs of toxicity nor mortality. No significant differences were observed in general condition, growth, organ weight, hematological parameters, biochemical values, or the gross and microscopic appearance of the organs from the treatment groups as compared to the control group. In conclusion, MECB did not cause any mortality nor did it cause any abnormalities in the necropsy and histopathology findings of treated rats. The LD50 for the MECB was found to be more than 2000 mg/kg. No adverse effects were observed in the treated rats at all the doses tested. The no-observed-adverse-effect level (NOAEL) for the 28-day study was determined to be 2000 mg/kg body weight/day.

Inhibitory effects of Cinnamomum burmannii Blume stem bark extract and trans-cinnamaldehyde on nasopharyngeal carcinoma cells; synergism with cisplatin.

Nasopharyngeal carcinoma (NPC) is a malignancy that occurs in the epithelium of the nasopharynx. The standard treatment of NPC patients with locoregionally advanced stages is problematic and is often associated with toxicities. Therefore, it is essential to screen for naturally occurring compounds with strong apoptosis-inducing activity and minimal toxicity. This study investigated the effects of the standardized methanol extract of Cinnamomum burmannii Blume stem bark and its main constituent, trans-cinnamaldehyde (TCA), on human NPC cell lines. The content of TCA in C. burmannii methanol extract was standardized to be 13.61% w/w by means of gas chromatography-mass spectrometry (GC-MS). NPC cell proliferation was clearly inhibited within 24 h of treatment, with TCA exhibiting greater activity than the methanol extract. TCA was more active against NPC cells compared with cisplatin. There was a pronounced downregulation of the proliferation markers, Ki67 and proliferating cell nuclear antigen (PCNA) in the TCA-treated cells; while morphological observation indicated the induction of apoptosis. Caspase activation and prominent DNA damage, which are markers of apoptosis induction were detected. TCA demonstrated the ability to scavenge nitric oxide. The simultaneous combination of TCA and cisplatin produced synergistic anti-proliferative effects. Collectively, these data indicate the potential use of TCA for the treatment of NPC.

Glucose-lowering effect of DLBS3233 is mediated through phosphorylation of tyrosine and upregulation of PPARγ and GLUT4 expression.

BACKGROUND: DLBS3233 is a standardized extract combination containing Lagerstroemia speciosa and Cinnamomum burmannii. The effect of DLBS3233 on glucose uptake, adiponectin secretion, and insulin signaling was examined in this study. METHODS: 3T3 Swiss albino preadipocytes and adipocytes were used to investigate gene expression detected using the reverse transcription polymerase chain reaction method. Immunoblotting assay and in vitro glucose uptake assay were also carried out in the experiment. RESULTS: DLBS3233 was seen to increase phosphorylation at the tyrosine residue of the insulin receptor substrate. DLBS3233 was also found to enhance the expression of genes associated with increased insulin signaling and sensitivity, such as peroxisome proliferator-activated receptor gamma, phosphatidylinositol-3 kinase, Akt, and glucose transporter 4. In addition, glucose transporter 4 protein levels were seen to increase as a result of DLBS3233 administration. The combination of extracts also increased glucose uptake and adiponectin secretion, and decreased resistin secretion significantly relative to control cells. Moreover, DLBS3233 administered to insulin-resistant Wistar rats showed an ability to control blood sugar, insulin levels, and other lipoproteins, including high-density lipoprotein, low-density lipoprotein, triglycerides, and total cholesterol. CONCLUSION: DLBS3233, as a combination of herbal extracts, holds promise in the treatment of type 2 diabetes, and possibly also in prevention of the disease.