3. Discussion
Thailand has a tropical climate, and many indigenous plants have potential health benefits, as suggested by a long history of traditional usage. However, the lack of scientific evidence concerning plant health properties has resulted in limited therapeutic applications and improper agricultural management. Some plants are at risk of extinction, and their potential health applications have not been recognized. To resolve this crisis, the Plant Genetic Conservation Project was established under the royal initiative of Her Royal Highness Princess Maha Chakri Sirindhorn (RSPG), with the ultimate goal of sustainable conservation and allocation of plant resources for optimized beneficial utilization. Ten indigenous plants, namely, Alb. lebbeck, Alp. malaccensis, C. arborea, D. esculentum, K. roscoeana, Mi. brandisiana, Mo. charantia, P. emblica, Z. cassumunar, and Z. citriodorum, were collected from the plant conservation area under the RSPG project to investigate their potential health benefits, which can lead to future food applications through proper plant handling. Commonly consumed parts of these plants, including shoots and young leaves (Alb. Lebbeck, C. arborea, D. esculentum, K. roscoeana and Mi. brandisiana), fruits (Mo. Charantia and P. emblica), and rhizomes (Alp. malaccensis, Z. cassumunar and Z. citriodorum), were prepared by freeze- and oven-drying and extracted as tea infusions before investigating antioxidant activities, which were determined by DPPH radical-scavenging, FRAP, and ORAC assays. Inhibitory activities against the key enzymes relevant to obesity (lipase), diabetes (α-glucosidase and α-amylase), and Alzheimer’s disease (AChE, BChE, and BACE-1) and the nonenzymatic glycation reaction were also assessed. These plant medicinal properties might result from the biological functions of their phenolics determined by LC–ESI-MS/MS. Findings suggested that the bioactivities of freeze- and oven-dried samples were similar, while slightly favoring the freeze-drying process. Among the 10 plant extracts, (i) P. emblica exhibited the highest antioxidant activities, TPCs, and anti-glycation activities, (ii) D. esculentum and Mi. brandisiana exhibited the highest inhibitory activities against carbohydrate-degrading enzymes, α-amylase and α-glucosidase, (iii) D. esculentum exhibited the strongest inhibitory activity against the key enzyme interfering with β-amyloid formation, BACE-1, and (iv) C. arborea exhibited the highest inhibitory activity against the fat-degrading enzyme, lipase, and the key enzymes controlling cholinergic neurotransmitters, AChE and BChE.
Interestingly, our results suggested that the drying processes (oven-drying at 60 °C for 5 h and freeze-drying at -50 °C and 0.086 mbar for 72 h) had minimal effects on plant biological properties. These results were valid only for our studied plant samples and their plant parts. Previous studies on thermal treatment of guava suggested that TPCs, total flavonoid contents (TFCs), and antioxidant activities determined by FRAP and ORAC assays of samples that underwent oven-drying (55 °C for 22 h) and freeze-drying (-50 °C and 0.025 mbar for 48 h) were insignificantly different [
4]. An experiment using a tray dryer with suction flow on broccoli suggested that maximal antioxidant activities (through DPPH radical-scavenging assay) were achieved by high-temperature and short-time processes [
5]. This conclusion was supported by a literature review on the effect of drying on antioxidant potential of fruits and vegetables [
6]. However, this review also suggested that each fruit and vegetable under a particular thermal treatment possessed different results. For example, pear underwent thermal treatment of 54 °C with cold-air drying pretreatment, exhibiting reduced TPCs with increased antioxidant activities, while jujube underwent -50 °C for 48 h, exhibiting increased TPCs and reduced antioxidant activities [
6]. Results from our experiments indicated little effect of drying methods on the biological activities of indigenous plants, supporting oven-drying processes with cheaper operational costs.
Among all plant extracts, our results indicated that a hot-water extract of
P. emblica fruit exhibited high antioxidant activities, related to its high TPCs, especially gallic acid content. This experiment was aimed at investigating potential antioxidant activities using different mechanisms and the effect of drying processes on antioxidant activity. These antioxidant measurements underwent different mechanisms, in which the DPPH radical-scavenging and FRAP assays followed the single electron transfer (SET) mechanism, while the ORAC assay followed the hydrogen atom transfer (HAT) mechanism. Our results indicated that
P. emblica extract exhibited higher ORAC values than DPPH radical-scavenging and FRAP activities, suggesting that this plant extract might contain antioxidants that follow the HAT rather than SET mechanism. Interestingly, previous studies suggested the effect of extraction solvent on antioxidant activities and TPCs of
P. emblica fruit. The results on
P. emblica fruit from China extracted with methanol, followed by partition with ethyl ether, ethyl acetate, butanol, and water, indicated that the highest TPCs and DPPH radical-scavenging activity were achieved in the ethyl acetate fraction, while those in the aqueous fraction were the lowest [
7]. Extraction using hexane, ethyl acetate, and ethanol suggested that the ethanolic extract of
P. emblica fruit from Indonesia provided the highest TPCs among all extracts [
8]. However, comparing between ethanol and distilled water extraction, the aqueous extract of
P. emblica fruit from India exhibited higher TPCs than its ethanolic extract [
9], while high TPC and DPPH radical-scavenging activities were detected in methanolic extracts of
P. emblica fruit from six regions of China [
10]. A strong correlation between antioxidant activities and TPCs was also observed [
9,
10]. High antioxidant activity in
P. emblica fruit resulted from the biological functions of its phenolics [
11]. Phenolics in
P. emblica fruit from China were geraniin, quercetin 3-β-
d-glucopyranoside, isocorilagin, and kaempferol [
7], while another group reported cinnamic acid, quercetin, 5-hydroxymethylfurfural, gallic acid, β-daucosterol, and ellagic acid [
12]. The former used methanol extraction, followed by partition with ethyl acetate [
7], while the latter employed 90% (
v/
v) ethanol extraction, followed by ethyl acetate [
12]. Gallic acid, corilagin, and ellagic acid were also detected in the aqueous ethanolic extract of
P. emblica fruits collected from 10 different habitats in China [
13]. While our experiments found gallic acid as a predominant phenolic in
P. emblica, it could be concluded that different growing locations yielded different types and quantities of phenolics in the plant samples. Even though, in previous reports, the plant samples were extracted using different solvents, as opposed to the aqueous extraction in our study, these reports found that antioxidative agents contributed to anti-glycation activities, while the ethanolic extracts of
P. emblica fruit collected from Thailand and Saudi Arabia exhibited high antioxidant and anti-glycation activities [
14,
15]. Similar results were observed in the methanolic extract of
P. emblica fruit from Sri Lanka, with the highest anti-glycation activity among nine antidiabetic plant extracts [
16]. The most abundant phenolic detected in our
P. emblica, gallic acid, was also able to inhibit advanced glycation end-products in both cell cultures and animal models [
17,
18,
19].
Advanced glycation end-product formation is highly related to diabetic complications. Unlike
P. emblica with high anti-glycation activities,
D. esculentum and
Mi. brandisiana were found to be the potent plants with high inhibitory activities against the key enzymes relevant to diabetic control (
P. emblica was in third place). Prepared as tea infusions, the results of our in vitro experiments suggest potential antidiabetic activity through key enzyme inhibitions of
D. esculentum and
Mi. brandisiana. These results were supported by a recent review on
D. esculentum [
20], suggesting that the plant extract exhibited both α-amylase- and α-glucosidase-inhibitory activities at more than 50%, even though extraction condition was not reported. Furthermore, its hydroalcoholic extract (500 mg/kg) effectively halved serum glucose in streptozocin (STZ)-induced diabetic rats [
21]. Upon being extracted by hot water (90 °C, 1 h), its α-glucosidase-inhibitory activity was eight times more effective than myricetin, suggesting that this plant extract was a potentially effective α-amylase and α-glucosidase inhibitor [
22]. Several phenolics were isolated from
D. esculentum including ascorbic acid, quercetin, cyanidins,
trans-cinnamic acid, protocatechuic acid, and rutin [
20,
23], while we found quercetin, luteolin, and rosmarinic acid, which might be responsible for α-amylase- and α-glucosidase-inhibitory activities. It was previously suggested that quercetin exhibited the half-maximal inhibitory concentrations (IC
50) of 500 and 7 µM against α-amylase and α-glucosidase, respectively, while those of luteolin were 360 and 21 µM against α-amylase and α-glucosidase, respectively [
24]. Rosmarinic acid, on the other hand, was a less effective inhibitor against both carbohydrate-degrading enzymes [
25]. Unfortunately, no information on the α-amylase- and α-glucosidase-inhibitory activities of
Mi. brandisiana was previously reported, with the focus on bioactive compounds contained in its roots [
26]. Only isoflavones and brandisianins were identified in the leaves of
Mi. brandisiana [
27,
28]. However, we detected trace amounts of luteolin and isorhamnetin; thus, the anti-α-amylase and anti-α-glucosidase agents might be other phenolics.
D. esculentum strongly inhibited BACE-1, the key enzyme in β-amyloid formation, and one hypothesis underlying AD occurrence. Various pharmacological properties of
D. esculentum were previously reported [
29]; however, none concentrated on BACE-1 inhibition. Nevertheless, its aqueous leaf extract significantly elevated locomotor activity and stimulated the central nervous system (CNS) in mice [
30], while a 70% (
v/
v) methanolic extract inhibited AChE with an IC
50 value of 0.27 mg/mL [
31], suggesting its potential as a source of anti-AD agents.
Interestingly, our results indicated that
C. arborea effectively inhibited the fat-degrading enzyme, lipase. Only one previous report on lipase-inhibitory activity of
C. arborea was available, albeit in the Korean language [
32]. High lipase inhibition in
C. arborea might be due to the biological function of kaempferol and quercetin since both were previously reported to be effective lipase inhibitors with IC
50 values of approximately 0.23 mM [
13,
33]. The former acted as a competitive inhibitor, while the latter was a mixed type, close to a noncompetitive inhibitor [
13,
33]. In addition to its potential to reduce fat absorption, our results indicated that
C. arborea is involved in controlling the degradation of cholinergic neurotransmitters through inhibitions of AChE and BChE. However, there are no previous reports on its AChE- and BChE-inhibitory properties. Nevertheless, the methanolic extract of its stem bark caused CNS depressant activity in Swiss albino mice and Wistar albino rats [
34], while its abundant phenolics, kaempferol and quercetin, inhibited AChE with IC
50 values of 3.05 and 3.60 µM, respectively [
35]. The enzyme flavonoid inhibition constants (Ki) of kaempferol and quercetin against BChE were 92.8 and 38.3 µM, respectively [
36], suggesting that kaempferol and quercetin are effective AChE and BChE inhibitors.
To summarize, our results revealed that drying processes, i.e., freeze-drying and oven-drying, of plants in this study played a minor role in the results of this experiment. Therefore, oven-drying would be a more appropriate method for locals due to its lower cost, instrumental availability, and easier and more convenient preparation step. We also found that P. emblica extract was the most potent antioxidant and anti-glycation provider, while D. esculentum extract was the most potent inhibitor against enzymes relevant to AD through β-amyloid formation. Furthermore, D. esculentum and Mi. brandisiana extracts were effective against enzymes relevant to diabetes, while C. arborea extract strongly inhibited enzymes relevant to obesity and AD through cholinergic degradation. Since hot-water extraction as a tea infusion was chosen as a model for easy and convenient preparation in our study, we hope that the knowledge gained from this research will be useful for future food development from these indigenous plants with high phenolics. Nevertheless, these in vitro findings need further testing in vivo and under clinical conditions, as in vitro studies do not cover the aspects of dose and the effect of bioavailability and pharmacokinetics. These plants, however, show potential for future pharmaceutical applications that will greatly benefit their sustainably, maintenance, advantageous utilization, and agricultural management in line with the RSPG objectives.