Evaluation on Antidiabetic Properties of Medicinal Plants from Myanmar

Objectives To explore the effective and safe medicines for treating diabetes. Methods Hydroalcoholic extracts of 130 medicinal plants belonging to 66 families were evaluated using porcine pancreatic lipase (PPL) inhibition and glucose uptake methods together with a literature review. Results The extracts of 22 species showed the PPL inhibition activity; 18 extracts of 15 species stimulated glucose uptake in 3T3-L1 adipocytes. Among them, Mansonia gagei J.R. Drumm., Mesua ferrea L., and Centella asiatica (L.) Urb. exhibited both activities. The extracts of Caladium lindenii (André) Madison rhizomes and Azadirachta indica A. Juss. leaves presented the utmost lipase inhibitory activity with IC50 of 6.86 ± 0.25 and 11.46 ± 0.06 μg/mL, respectively. The extracts of Coptis teeta Wall. rhizomes and Croton tiglium L. seeds stimulated the maximum glucose uptake. Ten species are reported to have antidiabetic activity for the first time. Flavonoids and triterpenoids are the dominant antidiabetic compounds in selected medicinal plants from Myanmar. Conclusions P. zeylanica, L. cubeba, H. crenulate, M. gagei, C. teeta, and M. ferrea are worthy to advance further study according to their strong antidiabetic activities and limited research on effects in in vivo animal studies, unclear chemical constitutes, and safety.


Introduction
Diabetes mellitus (DM) is a chronic metabolic disease and characterized by hyperglycemia that result from defects in insulin secretion, insulin action, or both [1]. Diabetes includes three main types, type I, type II, and gestational diabetes. Type II diabetes mellitus (T2DM) is the most common and accounts for over 90% of the total cases [2,3]. T2DM has high morbidity and mortality worldwide and brings a large economic burden. Cardiovascular complications are a major cause of morbidity and mortality among people with diabetes [2]. e International Diabetes Federation reported that there were 463 million adults with diabetes in 2019, which have more than tripled over the past 20 years, and diabetes-related healthcare costs (USD 760 billion) are now estimated to account for 10% of the total healthcare budget (https://www.diabetesatlas.org/).
Most patients with T2DM are overweight or obese. Pancreatic lipase is an important enzyme for digestion and absorption of dietary lipids, and lipase inhibition is the most widely studied for the discovery of potential antiobesity agents [4]. Monogenic and common forms of obesity initially cause insulin resistance [5]. Insulin resistance stimulates glucose production in the liver and attenuates glucose uptake in muscle and adipose tissue which is one of the important factors resulting in T2DM [2]. As the behavior of 3T3-L1 adipocytes is similar to primary adipocytes and mostly available muscle cell lines are not insulin sensitive in glucose transport, 3T3-L1 adipocytes have become the most preferred cell line to study insulin-stimulated glucose uptake and an excellent cell model to study insulin action and signaling [6].
Currently, oral antidiabetic medications are mainly classified as biguanides, sulfonylureas, meglitinides, thiazolidinediones, dipeptidyl peptidase 4 (DPP-4) inhibitors, sodium-glucose cotransporter inhibitors, and α-glucosidase inhibitors [1]. ese agents have adverse effects such as leading to hypoglycemia, cardiovascular, and metabolic alterations. People prefer medicinal plants for DM therapy due to low cost, minimal side effects, and inexpensive availability [1,7]. In many parts of the world, traditional knowledge and biodiversity play an import role in health care, and many widely used plant-based medicines are derived from traditional knowledge [8]. However, only parts of medicinal plants have received scientific evaluation for their efficacy [7]. us, it is a challenge and an opportunity to search medicinal plants to treat diabetes and its complications.
Myanmar, a tropical country, possesses a rich resource of medicinal plants, which is widely practiced by the majority of the population [9]. However, there are limited pharmacological studies on traditional medicines. Aumeeruddy et al. reviewed the ethnomedicinal plants for the management of diabetes worldwide but lack of information in Myanmar [10]. To explore the pharmacological application of antidiabetic properties of traditional plants in Myanmar and search for new antidiabetic agents, we investigated three main regions of the medicinal plants source and reported their current research status on antidiabetes. ese species were screened for the antidiabetic activities by porcine pancreatic lipase (PPL) inhibition and glucose uptake in 3T3-L1 adipocyte assays of selected medicinal plants in Myanmar.

Investigation and Collection of Medicinal Plant Materials.
is study was carried out in the markets and villages of three regions including Mandalay, Sagaing, and Shan State in 2015-2017. ese places possess affluent medicinal plants according to our survey in Myanmar. Mandalay has the biggest market of medicinal plants, which is Zay Cho market. Shan is the biggest state and the most famous for its traditional medicines in Myanmar [11]. e local name and usage of medicinal plants were recorded in the field survey. Meanwhile, the voucher specimens and materials were collected for identification and further research, respectively. Among them, 93 samples of medicinal plants from Mandalay were screened for antibacterial and anti-T3SS activities in our previous research [12]. e plants were identified by Ms. Jun Yang and Mr. Yu Zhang from the Kunming Institute of Botany based on specimens. e plant names and families were confirmed and conducted at the Plant List (http://www. theplantlist.org).

A Review of Medicinal Plants in Antidiabetic Research.
To make clear the status quo on antidiabetes of these medicinal plants, we performed a thorough literature review by using electronic databases (Web of Science, PubMed, Google Scholar, and SciFinder). Specific keywords include "scientific name of species" and "diabetes" until 2021. e used parts of the species were searched about their antidiabetes including traditional use, in vitro assay, in vivo animal studies, clinical trials, and antidiabetic constitutes. Other parts were continued to search if the collected parts did not report any antidiabetic properties, and other species of the same genus were continued to collect information if the species did not report any antidiabetes. We used rigorous inclusion and exclusion criteria to select antidiabetic compounds with IC 50 ≤ 50 µM, or dose ≤50 µM in vitro, or dose ≤50 mg/kg in vivo, excluding compounds with name or structure inconsistent with the source. Here, we refer to the antidiabetic activity of compounds with IC 50 ≤ 10 µM, dose ≤10 mg/kg in vivo, or dose ≤10 µM in vitro as high and from 11 to 50 as moderate.

Preparation of Plant Extracts.
e dried different parts of the plants were powdered in a grinder and then extracted with 70% ethanol at 60°C once for 30 minutes. After filtration, the solvent was removed under reduced pressure at 60°C in a rotary evaporator. e extracts were stored at 4°C before analysis.

Porcine Pancreatic Lipase (PPL) Inhibition Assay.
Lipase inhibition activity was conducted as described in literature with some modifications [13]. Briefly, p-NPB was used as a substrate. e reactions were carried out in a 96-well microtiter plate where 5 µL of lipase solution (40 U/mL) in Tris-HCl buffer (100 mM Tris-HCl, 5 mM CaCl 2 ; pH 7.0) was used as enzyme buffer.
en, 1 µL of each extract dissolved in dimethyl sulfoxide (DMSO) (20 mg/ mL) and 184 µL of the same Tris-HCl buffer were added and mixed with the enzyme buffer. After incubation at 37°C for 15 min, 10 µL of the substrate solution (10 mM p-NPB in DMSO) was added. en, enzymatic reactions were performed for 15 min at 37°C. Lipase activity was determined by measuring the hydrolysis of p-NPB to p-nitrophenol at 400 nm using a microplate reader, 630 nm as a reference. Orlistat was used as a positive control. e inhibitory activity (I) was calculated according to the following formula: A is the optical density (OD) value of the sample. B is the OD value of negative control. e half-maximal inhibitory concentration (IC 50 ) was calculated by Reed and Muench's method.

Glucose Uptake in 3T3-L1 Adipocytes Assay.
e differentiation of 3T3-L1 adipocytes and glucose uptake assay were carried out using a mild modified method described [14]. e differentiated 3T3-L1 adipocytes were seeded in 96-well plates and preincubated with the same way in 2 e Scientific World Journal reference. After that, they were incubated with 40 μg/mL samples in medium. Insulin (0.1 μmol/L) or berberine (10 μmol/L) was used as positive control. DMSO was added as blank control. After 24 h, 10 μL medium was taken to measure the glucose concentration by the glucose oxidase method. e experiments were repeated three times. Meanwhile, 20 μL MTS was added in the remaining medium of cells and incubated 2 h at 37°C. en, the absorbance at 492 nm was measured to determine the cytotoxicity of extracts. e value of glucose uptake of the sample was calculated using these formulas: e glucose concentration (mmol/L) � sample absorbance/positive control absorbance × positive control concentration.
Glucose uptake (% of difference) � (glucose concentration of blank wells − remaining glucose in the cell-plated wells)/glucose concentration of blank wells ×100%.

PPL Inhibition Activity of Plant Extracts.
Twenty-two plant extracts (100 μg/mL) showed the PPL inhibition activity (Table 1 and Table S1). Among them, eight species were tested, with the IC 50 values according to PPL inhibition rate higher than 50% except for Cuscuta sp. in Table 1. Cuscuta sp. showed the PPL inhibition activity with an IC 50 value of 93.52 ± 1.38 μg/mL. Cuscuta sp. was not properly identified because of morphological characteristics without the flower or fruits. It was found in the same spot where C. reflexa was discovered. e ethanolic extracts of C. lindenii rhizomes and A. indica leaves possessed the highest lipase inhibitory activity with IC 50 values of 6.86 ± 0.25 and 11.46 ± 0.06 μg/mL, respectively. e ethanolic extracts of P. zeylanica stems, G. glabra roots, C. pareira aerial parts, M. gagei woods, and C. reflexa whole plants showed moderate lipase inhibitory activity with IC 50 values of 37−95 μg/mL. Fourteen species showed mild lipase inhibitory activity with 30-47% inhibition (Supplementary Materials, Table S1).

Glucose Uptake in 3T3-L1 Adipocytes of Plant Extracts.
Seventeen extracts (40 μg/mL) and one plant extract reduced from 40 μg/mL to 20 μg/mL as cytotoxicity to cells of 15 species stimulated glucose uptake in 3T3-L1 adipocytes ( Figure 1). e ethnobotanical information and pharmacological properties can be found in Table 2. C. teeta rhizomes and C. tiglium seeds (oily and solid phase) stimulated the highest increase in glucose uptake. At concentration 40 μg/mL, their glucose uptakes were higher than positive controls which was insulin (0.1 μM) and berberine (10 μM). Mahonia sp. and L. cubeba fruits stimulated glucose uptake equivalent to berberine (10 μM); M. ferrea leaves were close to insulin (0.1 μM).
e seed extracts of C. tiglium and N. sativa have oily and solid phase; the two phases were separated to screen the antidiabetic activities.
eir oily phase of the extract showed a higher increase in glucose uptake. Mahonia sp. leaves significantly stimulated glucose uptake compared to the control group ( Figure 1). e  Oil can be used for stomach disorders, hypertension, fever, inflammation, infections, and diseases of the throat and ear [8] Antioxidant, antitumor, anti-HIV, anticonvulsant, gastrointestinal, antiinflammatory, and α-amylase inhibitory activities [15,16] Croton tiglium L.
Barks showed antioxidant, antiinflammatory, and tyrosinase inhibitory activities [31]; leaves showed tyrosinase inhibitory activity [32] Hesperethusa crenulata (Roxb.  Wall. were the only reported traditional use for treating diabetes without any report on antidiabetic activity that accounted for 10% of research status (Figure 2(b) , Tables S2 and S3). e Fabaceae family had the largest number of species showed antidiabetic activity (Figure 3). Among them, 7.3% species were already studied in type 2 diabetic patients, 46.9% species were researched antidiabetic activities in vivo, and 11.5% species were only recorded traditional use without any reported pharmacological activities on antidiabetes (Figure 2(c)).

A Literature Review of Antidiabetic Compounds from Selected Medicinal Plants in Myanmar.
Forty of the 82 species were reported antidiabetic components with various types including terpenes, alkaloids, flavonoids, lignans, and other types ( Figure S1). Among them, flavonoids (28 compounds) and triterpenoids (26 compounds) are the dominant compounds accounting for 61.4%. Flavonoids were mainly reported α-glucosidase inhibition and PPARc-ligand-binding activities.

Discussion
e potential species were discussed from pharmacological activities and safety extracts were with IC 50 < 50 μg/mL in PPL inhibition, higher or closely positive in glucose uptake, or showed these two activities.

e Scientific World Journal
H. crenulata is known as anaka, and the stem bark powder is used as a skincare regiment over one thousand years in Myanmar [31]. It is also a common tropical plant species in the Indian subcontinent and Southeast Asia with various medicinal properties, such as purgative, antidote, stomachic, and sudorific [31]. Coumarins and sitosterol were found in its root bark [37]. Extracts from anaka bark showed strong anti-inflammatory, significant antioxidation, and mild tyrosinase inhibition without detectable genotoxicity [31]. We reported the antidiabetic activities of its roots and barks for the first time.
e heartwood of M. gagei showed antibacterial, antifungal, and antioxidant activities [12,17]. e chemical constitutes of M. gagei revealed antiestrogenic, antifungal, and antioxidant, and anticancer activities [17,18]. Coumarins and mansonones are the main constituents and active ingredients in M. gagei [17,18,38,39]. Its antidiabetic activities were reported for the first time which was proved with PPL inhibition and increase the glucose uptake to deserve further study about the activity in vivo and its safety.
e tuber of C. lindenii was used for Stingray wounds in Brazil [40]. e phytochemical investigation of Caladium indicated flavonoids, alkaloids, saponins, cardiac glycosides, carbohydrates, and deoxy sugars [41]. ere is no reported research about the phytochemical and pharmacological research of C. lindenii.
e corm of C. bicolor showed stimulation of glucose uptake in adipocytes and hepatoprotective activity in Hep G2 cells [42]. Methanol leaf extract of C. bicolor showed toxicity to the kidney but no adverse effect on the heart, lungs, spleen, liver, and brain [43].
A. indica, known as neem, is a common traditional medicine for the treatment with diabetes mellitus in Africa and India [44,45]. At 25,48.4,93.5,180.9, and 350 mg/kg body weight, intraperitoneal and oral administration of aqueous leaf extract of A. indica decreased the blood glucose levels in alloxan-induced diabetic mice but not in a dose-related manner [44]. Ten ligands of neem possessed binding properties with T2DM protein enzyme target phosphoenolpyruvate carboxykinase; compound 3-deacetyl-3-cinnamoyl-azadirachtin showed the best binding [46]. Meliacinolin possessed α-glucosidase and α-amylase inhibition activities with IC 50 46.7 and 32.2 µg/mL from chloroform extract of A. indica leaves. And orally administered meliacinolin (20 mg/kg body weight) could be able to revert a set of biochemical parameters of streptozotocin STZ-diabetic mice to respective normal values and its mechanism was deduced via its insulinogenic action [45]. Braga et al. reviewed the safety of A. indica extracts and/or isolated compounds and revealed nontoxicity or less toxicity when orally administered but had acute toxicity by intramuscular injection or via the intraperitoneal route [47]. e roots and leaves of P. zeylanica are traditionally used to treat diabetes in India and China [48,49]. e ethanol extracts of P. zeylanica stem inhibited porcine pancreatic lipase activity with IC 50 39.1 μg/mL (Table 1). Oral administration of ethanolic extract of P. zeylanica roots (100 and 200 mg/kg) decreased blood and urine glucose levels, increased hepatic hexokinase activity, and decreased hepatic glucose-6-phosphatase, serum acid phosphatase, alkaline phosphatase, and lactate dehydrogenase in streptozotocin STZ-diabetic rats [50]. Plumbagin is the major bioactive component for antidiabetic activity (15 and 30 mg/kg body weight) by reducing blood glucose and returning other biochemical parameters to normal and enhancing GLUT4 mRNA, protein expression in diabetic rats from P. zeylanica root. e antidiabetic effect is lower than glibenclamide (4 mg/kg) [49]. Oral administration of aqueous extract was found to be safe up to the dose of 2000 mg/kg during the 14 days of observation by acute toxicity profile experiment [51].
e rhizome of C. teeta along with C. chinensis and C. deltoidea is known as "coptidis rhizome" which has been widely used to treat bacillary dysentery, diabetes, pertussis, sore throat, aphtha, and eczema in China [29]. C. chinensis polysaccharide (200 mg/kg body weight) showed significant inhibition in fasting blood glucose level and triglycerides [52]. Alkaloids are the most abundant components and are considered as the main active ingredients [29]. e methanol extract of C. chinensis rhizome significantly inhibits adipocyte differentiation and lipid contents in 3T3-L1 cells. Five alkaloids, berberine, epiberberine, coptisine, palmatine, and magnoflorine were significantly inhibited lipid accumulation in 3T3-L1 cells without affecting cell viability and reduced the expression levels of several adipocyte marker genes including proliferator-activated receptor-c (PPAR-c) and CCAAT/enhancer-binding protein-α (C/EBP-α) from methanol extract of C. chinensis rhizome [53]. (3β)-Stigmast-5-en-3-ol stimulates glucose uptake by the PI3K-dependent pathway in L6 myotubes and activates GLUT 4 transport [54,55]. Berberine was the main compound from the ethanol extract of C. teeta rhizome [54]. Lan et al. extracted 27 clinical trials (2569 patients) and revealed berberine with comparable therapeutic effects on T2DM, hyperlipidemia, and hypertension with no serious adverse reactions [56].
C. tiglium seeds were traditionally used for stimulating appetite, imbalances in phlegm and gas, jaundice, fainting, and facial paralysis and taken as a laxative to rid the body of impurities in Myanmar. e seeds oil can be used for stomach disorders, hypertension, fever, inflammation, infections, and diseases of the throat and ear [8]. C. cajucara, C. macrostachys, and C. malambo were used to cure diabetes [57].
e extract of C. tiglium inhibited α-amylase with 55.1% at 80 μg/mL [16]. Oral medication can cause severe gastrointestinal syndrome and even mortality because of irritating oils and croton proteins [58]. Tigliane-type diterpenoids are the predominant secondary metabolite constituents in C. tiglium L. [59]. e raw fruits of L. cubeba were traditionally used to prevent and check for hyperglycemia in India. Its methanol extract inhibited α-amylase and α-glucosidase with IC 50 values of 514.9 µg/mL and 1,435.7 µg/mL, respectively. Its ethanol extracts exhibited lower inhibition activity. Phenols and flavonoids were the major phytochemicals in different extracts against diabetes [60]. L. cubeba oil was proved with slightly toxic that the oral LD 50 , dermal LD 50 , and inhalation LC 50 values were approximately 4,000 mg/kg of body weight [61]. e Scientific World Journal e buds of M. ferrea displayed the mild inhibition on α-glucosidase and α-amylase activity with IC 50 values of 128.8 µg/mL and 146.8 µg/mL, respectively [62,63]. Stamens of M. ferrea produced a dose-dependent reduction in blood sugar (hypoglycemia) at a dose of 150 and 300 mg/kg body weight in alloxan-induced diabetic rats [64]. e methanol extract of M. ferrea flower has shown the antidiabetic activity in streptozotocin-induced diabetic rats and deduced the mechanism [65]. e extract of M. ferrea flower was proved no acute toxicity in an animal model but exhibited mild lymphocytic infiltration and hepatocyte degeneration [65,66]. Xanthones and coumarins are predominantly secondary metabolites from Mesua species which display antitumor and antimicrobial, antioxidant, anti-inflammatory, or immunomodulating properties [67]. However, there is not any research reported related with antidiabetic activity of its leaves previously.

Conclusion
L. cubeba, H. crenulate, and M. gagei deserve further study because of the potential antidiabetic activity in in vitro assay and unclear active ingredients and mechanism along with research about antidiabetic effects in in vivo animal studies and unclarity in safety except for L. cubeba with slightly toxic activity. Moreover, C. teeta, P. zeylanica, and M. ferrea are also worth giving attention as the low toxicity and antidiabetic activities of these species or its genus level in research status.
e antidiabetic activities and their constitutes of M. ferrea are studied in our research.

Data Availability
e data used to support the findings of this study are included within the Supplementary Materials.

Conflicts of Interest
e authors declare that they have no conflicts of interest regarding the publication of this paper.

Authors' Contributions
Ethnobotanical survey was done by Dongdong Zhang, Yu Zhang, Jun Yang, Pyae Phyo Hein, Aye Mya Mon, and Jianwen Li; specimen identification was performed by Yu Zhang and Jun Yang; Wang Yuehu and Xuefei Yang designed the study; experiment, analysis, and interpretation of the data and drafting of the manuscript were done by Dongdong Zhang; critical revision of the manuscript was performed by Wang Yuehu, Karuppusamy Arunachalam, Dongdong Zhang, Angkhana Inta, and Xuefei Yang. Acknowledgments e porcine pancreatic lipase (PPL) inhibition assay and glucose uptake in 3T3-L1 adipocytes assay were performed by the Service Center for Bioactivity Screening, Kunming Institute of Botany.
is research was supported by the Southeast Asia Biodiversity Research Institute, Chinese Academy of Sciences in China (Grants nos. 2015CASE-ABRIRG001 and Y4ZK111B01) and the National Natural Science Foundation of China (Grant no. 31770368). Table S1. Information with 30-50% PPL inhibition activities of 17 medicinal plants. Table S2. 82 species of 51 families with antidiabetic properties research status. Table S3. Relative plants in antidiabetic reports without any antidiabetes research. Table S4. e information of antidiabetic ingredients. Figure S1. Antidiabetic components from medicinal plants in Myanmar. (Supplementary Materials)