Introduction
Many medicinal plants have been reported to have multiple beneficial biological properties.
Moringa oleifera,[1] Phyllanthus amarus,[2] Momordica charantia,[3] Gymnema sylvestre,[4] and Grewia asiatica,[5] to name a few, are medicinal plants with various pharmacological properties, including
an antidiabetic property, which have been attributed to the presence of different
bioactive compounds in them. Potentilla fulgens L. is one of these medicinal plants known to possess various biological properties.
It belongs to the Rosaceae family, commonly found in the Western Himalayas. Pharmacologically,
the plant is reported to have hypoglycemic,[6] antihyperglycemic,[6] antitumor,[7] antihypolipidemic,[8] and antioxidant properties,[9]
[10] as well as an antidiarrheal.[11] It is a prophylactic agent,[12] with anthelmintic[13] and wound healing properties,[14] and even improves gum health.[15] Qualitative and quantitative analysis of P. fulgens roots by nuclear magnetic resonance spectroscopy, matrix-assisted laser desorption/ionization
with time-of-flight mass spectrometry (MS), electrospray ionization MS/MS and high-performance
liquid chromatography/ultraviolet have also been reported.[16] Phytochemical investigations of the root parts of P. fulgens have shown the presence of a novel bioflavonoid potifulgene along with epicatechin.[17] It has also been reported that the aerial parts contain two new triterpenes, potentene-A
and potentene-B, as well as three known compounds, afzelchin-4 α→8″ catechin, epiafzelechin,
and rutin.[15]
[18]
[19]
Although the effect of the P. fulgens extract on different parameters[6]
[8]
[9]
[19]
[20]
[21]
[22] under diabetic conditions has been reported previously, its inhibitory effect against
amylase, β-glucosidase, and lipase under in vitro conditions has not been reported
yet. Pancreatic α-amylase and glucosidase are known to lower the level of postprandial
hyperglycemia via the control of starch breakdown.[23] The pancreatic lipase plays a key role in the efficient digestion of triglycerides,[24] and is responsible for the hydrolysis of between 50 and 70% of the total dietary
fats.[25] Elevated triglyceride levels are a common dyslipidemic feature accompanying type
2 diabetes.[26] Therefore, the inhibition of these digestive enzymes is a suitable target for the
treatment of diabetes. Drugs that inhibit these enzymes are already available in the
market, and are used for diabetes treatment.[25]
[27] However, many of these synthetic hypoglycemic agents have their limitations, are
non-specific, produce serious side effects, and fail to elevate diabetic complications.[28] This opens an exciting opportunity for the development of new therapeutic drugs,
especially from plant sources, which are considered safe.[29] Therefore, in the present paper, the in vitro inhibitory effect of P. fulgens extract on the aforementioned enzymes was studied. Although α-glucosidase inhibitory
activity of P. fulgens has been reported previously by Kumar et al.[30], we have performed in vitro α-glucosidase inhibitory activity of P. fulgens extract for comparison with its β-glucosidase inhibitory activity. In addition, the
effect of the plant extract on the ultrastructure of the liver, of the kidneys, and
of the eye lens tissues in diabetic mice was also investigated. Thus, the present
study was designed to provide additional insights regarding the mechanism of action
of this antidiabetic plant.
Materials and Methods
Reagents
Butylated hydroxyanisole, bathocuproine, copper nitrate, lecithin, lipase, potassium
sodium tartarate, sodium acetate, sodium carbonate, sodium chloride, and triolein
were purchased from Hi-Media Laboratories Pvt. Ltd. (Mumbai, Maharashtra, India).
Acetic acid, α-amylase, α-glucosidase, β-glucosidase, dimethyl sulfoxide, dinitrosalicylic
acid, dipotassium hydrogen phosphate, disodium hydrogen phosphate, glycine, n-heptane,
methanol, p-nitrophenyl-α-D-glucopyranoside (pNDG), p-nitrophenyl-β-D-glucopyranoside,
potassium dihydrogen phosphate, sodium dihydrogen phosphate, starch, sodium hydroxide,
and triethanolamine were purchased from Sisco Research Laboratories Pvt. Ltd. (Mumbai,
India). Alloxan and taurocholic acids were purchased from Sigma-Aldrich (St. Louis,
Missouri, USA), acarbose (Glucobay 50) was purchased from Bayer Zydus Pharma (Thane,
Maharashtra, India), and orlistat (Reeshape) was purchased from Meyer Organics Pvt.
Ltd. (Thane, Maharashtra, India).
Plant Material
The roots of P. fulgens were collected from Bara bazaar, Shillong, Meghalaya, India (Voucher no: 464, North-Eastern
Hill University [NEHU]). The collected plant materials were washed, shredded, dried
and weighed. Afterwards, they were powdered, homogenized and extracted with an aqueous-methanolic
solution (1:4). The mixture was filtered, and the filtrate was evaporated at 40° C
using a rotary evaporator, (RE300, Stuart, Cole-Parmer, Beacon Road, Stone, Staffordshire,
ST15 OSA, UK) and it was further lyophilized until it was totally dry.[6]
[31] The obtained dried mass was used for the investigation.
In vitro Enzyme Inhibitory Studies
The evaluation of α-amylase inhibition was determined by the method of Kim et al.[32] The plant extract and acarbose (positive control) were prepared in a concentration
range between 0.02 and 1.0 mg/mL. A total of 50 µL of plant extract/acarbose, 250
µL of porcine pancreatic amylase (1 mg/100 mL), and 250 µL of sodium phosphate buffer
(0.02 M, pH 6.9) were mixed and incubated at 37° C for 10 minutes, and 450 µL of 0.5%
starch solution was added to the reaction mixture and incubated at 37° C for 20 minutes.
The reaction was concluded by adding 500 µL of 3,5-dinitrosalicylic acid, then heating
at between 70 and 80° C for 15 minutes, and centrifuging at 650 rpm for 1 minute.
The absorbance was measured at λ 540 nm.
The evaluation of α-glucosidase inhibition was performed using pNDG as a substrate.[32] A total of 50 µL of plant extract/acarbose (0.02–1.0 mg/mL) was mixed with 50 µL
of buffer and 50 µL of α-glucosidase solution (1 mg / 100 mL), followed by incubation
at 37° C for 5 minutes. After the incubation period, 100 µL of pNDG was added and
incubated again at 37° C for 15 minutes. The reaction was stopped by adding 750 µL
of sodium carbonate. The absorption of 4-nitrophenol was measured at λ 400 nm. Acarbose
was used as a positive control.
The β-glucosidase assay was performed by adopting the method of Sánchez et al.[33] with slight modification. A total of 100 µL of p-nitrophenyl-β-D-glucopyranoside,
50µL of plant extract/acarbose (0.02–1.0 mg/ml), and 100 µL of acetate buffer were
mixed and incubated at 37° C for 10 minutes, followed by the addition of 50 µL of
β-glucosidase solution and incubation at 37°C for 30 minutes. After the incubation
period, the reaction was stopped by adding 700 µL of glycine-NaOH buffer. The absorbance
was measured at λ 410 nm. Acarbose was used as a positive control.
A method slightly modified from that of Lin et al[34] was utilized to measure the pancreatic lipase inhibitory activity. A total of 50
µL of pancreatic lipase, 100 µL of substrate, and 100 µL of plant extract/orlistat
(0.02–1.0 mg/ml) were mixed and incubated at 37° C for 30 minutes, followed by the
addition of 3 ml of a chloroform, n-heptane and methanol mixture and centrifugation
at 2,000 rpm for 10 minutes. One ml of copper reagent was added to the lower organic
phase, followed by vigorous shaking for 10 minutes and centrifugation at 2,000 rpm
for 10 minutes. One-half ml of supernatant was transferred to 0.5 ml of chloroform
containing 0.1% weight/volume (w/v) of bathocuproine and 0.05% w/v of 3–2-tert-butyl-4
hydroxylanisole. The absorbance was measured at λ 480 nm. Orlistat was used as a positive
control.
The reaction mixture without the plant extract was used as a control, and the reaction
mixture without the plant sample and the enzyme was used as a blank. The experiments
were performed for five sets, and the data were represented as percentage of inhibition
(mean ± standard error of mean [SEM]). The percentage of inhibition of α-amylase,
α-glucosidase, β-glucosidase, and lipase activities was calculated using the following
formula:
In vivo Studies
Animals and Experimental Designs
Healthy, male Swiss albino mice, weighing between 25 and 30 g, were used for the study.
The mice were kept under controlled conditions, with the temperature maintained at
22°C on a 12-hour light/dark cycle in the animal facility room of the NEHU. The mice
were fed with balanced mice food obtained from Pranav Agro Industries Ltd. (New Delhi,
India). The institutional guidelines were followed during the experimentation. The
animal models were divided into three groups. The normal control group (group I) received
only 2% ethanol intraperitoneally (ip); the diabetic control group (group II) received
only 2% ethanol ip; and the P. fulgens-treated diabetic group (group III) received 250 mg/kg body weight (bw) of extract
ip. After a period of 4 weeks, the mice were sacrificed, and the dissected tissues
(liver, kidneys and eye lens) were studied using a JEOL100 CX II transmission electron
microscope (JEM, JEOL Ltd., Tokyo, Japan).
Preparation of Diabetic Mice
The diabetic mice were administered ip with alloxan monohydrate prepared in acetate
buffer, as described earlier.[7] Prior to the administration, the mice fasted overnight but had ad libitum access
to water. Mice with more than a three- to four-fold increase in their blood sugar
levels were considered diabetic and used for further tests.
Transmission Electron Microscopy Study
The primary fixation of the isolated tissues of the liver, the kidney, and the eye
lens from all the three groups were made in 3% glutaraldehyde prepared in a sodium
phosphate buffer (200 mM; pH 7.4) for 3 hours at 4° C. The materials were washed with
the same buffer and postfixed in 1% osmium tetroxide and in a sodium phosphate buffer
for 1 hour at 4° C. The tissue samples were then washed with the same buffer for 3
hours at 4° C, dehydrated in a graded ethanol series and then embedded in Araldite
CY212 (Agar Scientific, Essex, UK) sections ranging between 60 and 90 nm. The embedded
tissues were cut on a RMC Ultramicrotome (Powertome-PC, RMC Boeckeler, USA) using
a diamond knife (Ultra 45 degree, Diatome, USA) and the sections were mounted on a
copper grid. Afterwards, the sections were stained with uranyl acetate and Reynolds
lead citrate. The grids were examined using a JEOL100 CX II TEM at the Sophisticated
Analytical Instrument Facility (SAIF), NEHU, Shillong, Meghalaya, India.
Results and Discussion
Diabetes, particularly type 2, is a multifunctional disease.[35] Therefore, a treatment with drugs that have multiple targets has great potential
for tackling diabetes.[36] Medicinal plants are known to contain a complex of phytochemicals and bioactivities
that may have multiple benefits by targeting several metabolic pathways and, essentially,
“killing several birds with one stone”.[3] Drugs with the ability to target more metabolic pathways seem to show more encouraging
results than those that target a single pathway.[37] The purpose of the present study was to explore the effect of P. fulgens root extract on multiple targets other than the known activities as an anti-diabetic
plant. Here, the in vitro inhibitory effect of P. fulgens extract on α-amylase, α-glucosidase, β-glucosidase, and lipase were explored. In
addition, its effect on the ultrastucture of the liver, of the kidney and of the eye
lens was studied as no previous reports have been given on this study.
In vitro Enzyme Inhibitory Studies
The in vitro inhibitory activity of the P. fulgens extract against α-amylase, α-glucosidase, β-glucosidase, and lipase is shown in [Table 1]. The maximum percentage of inhibition of P. fulgens against α-amylase was found to be 37.51 ± 0.750 at 1 mg/mL, which was lower than
the inhibition percentage of 44.02 ± 0.79 at 1mg/ml showed by the positive control.
The percentage of inhibition of P. fulgens against α-glucosidase was found to be maximum at 1 mg/mL (94.57 ± 0.16), whereas
that of the positive control was found to be 45.08 ± 1.91 at the same dose.
Table 1
Percentage (%) of inhibition of Potentilla fulgens extract/positive control against α-amylase, α-glucosidase, β-glucosidase, and lipase
|
Concentration
(mg/mL)
|
α-amylase
|
α-glucosidase
|
β-glucosidase
|
Lipase
|
|
Positive control (%)
|
Plant extract (%)
|
Positive
control (%)
|
Plant extract (%)
|
Positive
control (%)
|
Plant extract (%)
|
Positive
control (%)
|
Plant extract (%)
|
|
0.02
|
15.58 ± 1.81
|
7.75 ± 2.01
|
12.11 ± 1.03
|
24.62 ± 1.85
|
16.55 ± 1.38
|
6.34 ± 1.82
|
10.68 ± 1.30
|
5.52 ± 0.30
|
|
0.04
|
16.11 ± 1.59
|
14.95 ± 2.86
|
14.86 ± 0.99
|
27.56 ± 1.87
|
18.19 ± 1.74
|
8.73 ± 1.22
|
15.22 ± 1.61
|
8.27 ± 0.33
|
|
0.06
|
16.98 ± 1.12
|
17.22 ± 2.33
|
16.21 ± 1.51
|
33.22 ± 1.20
|
20.09 ± 1.42
|
10.92 ± 0.78
|
16.31 ± 1.43
|
10.81 ± 0.33
|
|
0.08
|
17.01 ± 0.94
|
19.77 ± 2.41
|
17.01 ± 1.98
|
38.68 ± 1.49
|
21.98 ± 1.43
|
13.22 ± 1.07
|
18.97 ± 1.08
|
13.60 ± 0.32
|
|
0.1
|
17.55 ± 1.08
|
23.46 ± 2.08
|
18.85 ± 2.01
|
42.78 ± 0.37
|
22.12 ± 1.74
|
14.82 ± 1.10
|
20.70 ± 1.03
|
17.94 ± 0.39
|
|
0.2
|
18.16 ± 0.58
|
26.70 ± 0.89
|
19.97 ± 3.06
|
65.11 ± 0.55
|
24.37 ± 1.70
|
16.44 ± 0.58
|
22.51 ± 0.81
|
21.24 ± 0.52
|
|
0.4
|
24.56 ± 0.80
|
29.36 ± 1.89
|
26.54 ± 2.88
|
90.80 ± 0.93
|
30.14 ± 1.52
|
18.58 ± 0.80
|
24.31 ± 0.75
|
24.87 ± 0.43
|
|
0.6
|
32.18 ± 0.48
|
33.59 ± 0.92
|
34.06 ± 2.62
|
93.38 ± 0.88
|
34.35 ± 1.08
|
25.56 ± 1.63
|
33.96 ± 2.09
|
14.30 ± 0.71
|
|
0.8
|
39.44 ± 0.48
|
35.21 ± 0.85
|
39.16 ± 2.20
|
94.40 ± 0.28
|
40.20 ± 1.30
|
24.40 ± 1.12
|
41.78 ± 1.58
|
11.02 ± 0.86
|
|
1.0
|
44.02 ± 0.79
|
37.51 ± 0.75
|
45.08 ± 1.91
|
94.57 ± 0.16
|
45.73 ± 1.32
|
23.06 ± 1.72
|
52.58 ± 1.20
|
7.37 ± 0.99
|
Note: Values are represented as mean ± standard error of mean where n = 5.
Thus, the P. fulgens extract showed an even higher inhibitory activity than the positive control at the
same concentration. The highest percentage of inhibition of β-glucosidase by the P. fulgens extract was found to be 25.56 ± 1.63 at 0.6 mg/mL, which was lower than that of the
positive control. The maximum percentage of inhibition against lipase of P. fulgens and positive control was found to be 24.87 ± 0.43 and 24.31 ± 0.75 respectively,
at the corresponding concentration of 1 mg/mL. In the present study, the plant extract
showed an inhibitory effect on all of the studied enzymes. However, the highest percentage
of inhibition was shown against α-glucosidase. This suggests that the P. fulgens extract may contain several inhibitory substances, thereby resulting in the inhibitions
of these enzymes to different extents under the assay conditions used.
In vivo Studies
Marked differences were observed in the ultrastructural features of the nucleus and
of the mitochondria in the liver and in the kidneys of diabetic mice compared with
the normoglycemic mice, as shown in [Figs. 1]–[2]. The nucleus is one of the most prominent cellular organelles, and its shape and
size play an important role in cellular function.[38] The normal liver and kidney cells revealed normal features of euchromatic nucleus
(that is, with normal chromatin distribution), with fewer nuclear heterochromatic
contents. The contours of the nuclei were round, with the nuclear membrane showing
normal structures, such as regular outline and the absence of any prominent membrane
protrusion or invagination ([Figs. 1A] & [2A]). The mitochondria were well-maintained in the form of typical oval and elliptic
shapes, with smooth surfaces and clear outlines. The outer and inner mitochondrial
membranes were intact, without breakage. The cristae were arranged in the form of
a concentric ring or a vertical line, congested and clear. No abnormalities, such
as membrane distortion or vacuolization, were observed in the mitochondrial matrix
([Figs. 1D] & [2D]). The electron microscopic observations of the diabetic liver and kidney sections
showed evagination and invagination of the nuclear envelope, as well as apoptotic
cell nuclei with peripheral heterochromatin condensation and margination ([Figs. 1B] & [2B]). This provides enough evidence that alloxan-induced diabetes is likely to cause
apoptosis in liver and kidney cells, as previously reported.[39]
[40] Hyperglycemia-mediated apoptosis has also been well-documented in several previous
studies.[41]
[42]
[43] The mitochondria are considered to be pivotal organelles in determining cell destiny,
and act as an ‘on–off’ switch, modulating autophagy and apoptosis in the process of
cell death.[44]
[45]
[46] In the present study, some mitochondria exhibited breakage of the outer membrane
at places, while some showed distortion of the inner cristae in the liver and in the
kidneys of diabetic mice ([Figs. 1E] & [2E]), and these features have been reported in the pancreas of alloxan-induced diabetic
mice.[47] Apoptosis is widely assumed to involve the mitochondrial permeability transition
pore complex (MPTPC), which opens a small inner-membrane pore that leads to the influx
of water, ions, and small molecules, causing the mitochondrial matrix to swell and
the outer membrane to rupture.[48]
[49] Vacuolization is also observed in the liver and in the kidneys of diabetic mice,
which indicates an autophagic cell death, morphologically characterized by an accumulation
of vacuoles.[50] Previous reports demonstrated that autophagy normally removes the aggregated or
misfolded proteins induced by diabetes to defend against diabetes-induced mitochondria
damage.[51] The effect of the P. fulgens extract treatment was clearly visible, with an improvement in the ultrastructural
features of the liver and of the kidney sections in diabetic mice, with a pronounced
normalized appearance of the nuclei, as seen in the normal mice ([Figs. 1C ] & [2C]). The ultrastructural abnormalities observed in the mitochondria were improved in
the treatment with the P. fulgens extract ([Figs. 1F] & [2F]). In previous reports, the P. fulgens extract has shown to normalize the lipid profile[8] and the level of enzymes such as hexokinase,[8] aldose reductase,[19]
[20]
[21] sorbitol dehydrogenase,[22] and the antioxidant enzymes[9] in diabetic mice. This suggests the possible reason for its beneficial effect on
the ultrastructural changes of the studied tissues observed in diabetic mice.
Fig. 1 Transmission electron micrograph showing the nuclei (N) in the liver of normal mice
(A), diabetic mice (B), and Potentilla fulgens extract-treated diabetic mice at magnification x 5,000 (C). Transmission electron
micrograph showing the mitochondria (M) in the liver of normal mice (D), diabetic
mice (E), and P. fulgens extract-treated diabetic mice (F) at magnification x 40,000.
Fig. 2 Transmission electron micrograph showing the nuclei (N) in the kidneys of normoglycemic
mice (A), diabetic mice (B), and Potentilla fulgens extract-treated diabetic mice (C) at magnification x 8,000. Transmission electron
micrograph showing the mitochondria (M) in the kidneys of normal mice (D), diabetic
mice (E), and P. fulgens extract-treated diabetic mice (F) at magnification x 40,000.
The lens of the eye is comprised of highly ordered fiber cells that are covered anteriorly
by a monolayer of epithelial cells. Fiber cells are hexagonal in cross-section, and
are arranged in a honeycomb-like pattern, forming an array of regularly aligned, concentric
rings that comprise the bulk of the lens.[52] The fiber lens are developed all around the lens equator, eventually meeting and
forming end-to-end associations with corresponding fibers from other segments of the
lens. Any disruption to this organization impairs light transmission and lens function,
because a loose but intimate and regular bonding of lens fibers is essential for the
normal functioning of the lens ([Fig. 3]).
Fig. 3 Transmission electron micrograph showing the eye lens fibers of normoglycemic mice
(A), diabetic mice (B), and Potentilla fulgens extract-treated diabetic mice (C) at magnification x 10,000.
Mature lens fibers lack cellular organelles and contain a highly concentrated protein
solution to enable light refraction.[53] The transmission electron micrograph of the cross sections of the lens revealed
disorganized fiber patterns in diabetic mice, in which the arrangement of the fibers
was distorted compared with that observed in the normal lens fibers. This is in line
with previous reports that hyperglycemic states adversely affect the lens fiber and
morphology patterns, resulting in structural alterations.[54] In diabetic mice treated with the P. fulgens extract, a regular alignment of the lens fibers was observed. Although the lens segments
were not as compact as observed in normal lenses, the honeycomb-like pattern with
a regularly aligned array was seen to be regular, and the alterations in the fiber
pattern were minimized in P. fulgens extract-treated diabetic mice. Many other plants are known for their ability to restore
the alloxan-induced morphological damage in different organs.[47]
[55]
Conclusion
In conclusion, the experiment performed in the present study revealed that the P. fulgens root extract could inhibit enzymes such as amylase, α-glucosidase, β-glucosidase,
and lipase under in vitro conditions, and exhibit protective effects against the ultrastructural
changes observed in the liver, in the kidney and in the eye lens of diabetic mice.
Studying these effects has added new elements to the understanding of the antidiabetic
property of this plant extract.
