Key words
seaweed -
Caulerpa racemosa
- Caulerpaceae - type 2 diabetes mellitus - antioxidant - anti-inflammatory
ALP Alkaline phosphatase
ALT Alanine transaminase
AST Aspartate Aminotransferase
CaR Caulerpa racemosa
CaRE Caulerpa racemosa ethanolic extract
CAT catalase
CMC carboxymethylcellulose
GSH glutathione
HDL high-density lipoproteins
HPTLC high-performance thin-layer chromatography
IL-4 interleukin -4
LDL low-density lipoprotein
MDA malondialdehyde
NEFA non-esterified fatty acids
SGOT serum glutamate oxaloacetate transaminase
SGPT serum glutamate pyruvate transaminase
STZ streptozotocin
SOD superoxide dismutase
TC total cholesterol
TGA triglyceride
VLDL very-low-density lipoprotein
Introduction
Natural compounds derived from plant, microbial, and marine sources have been
extensively used for therapeutic purposes since time immortal. However, their
extensive use and increasing demand have resulted in insufficient plant-derived
materials to satisfy market demands. Further, phytochemical research has focused
more attention on discovering novel beneficial natural products. This situation has
immensely contributed to stimulating the increasing interest in unconventional and
unexplored sources of natural products. In this context, marine algae have attracted
much attention over the past four decades [1].
Algae are omnipresent globally and are an important part of the aquatic ecosystem.
Several seaweed species are used as therapeutics as well as food. The rheological
properties of seaweeds have made them crucial as food additives. Apart from that,
several bioactive compounds from marine algae are also known to exhibit various
biological activities [2]. However, it has
been observed from the literature that seaweeds are not investigated extensively for
their antidiabetic activity.
Despite the presence of several hypoglycemic agents in the market, type 2 diabetes
mellitus remains a major global health problem. Herbal products may act by several
mechanisms to alleviate the diabetic symptoms, deriving multifaceted benefits [3]. Of all the herbal alternatives, the
therapeutic properties of marine algae are the least explored. The hypoglycemic
potential and bioactive compound present in seaweeds is a new unexplored potential
area [4]
[5].
These marine compounds either exhibit direct hypoglycemic activity or ameliorate
complications associated with type 2 diabetes mellitus through other properties,
viz. antioxidant, anti-inflammatory membrane stabilization, etc. Several animal
studies have indicated an association between oxidative inflammation and the
incidence of type 2 diabetes mellitus. Previous studies have shown that
hyperglycemia and insulin resistance in diabetic patients lead to an elevated
generation of reactive oxygen species [6]
[7]. Furthermore, metabolic syndrome and insulin
resistance are also associated with the generation of acute-phase inflammatory
markers, showing the key role played by chronic low-grade inflammation in type 2
diabetes mellitus progression.
Seaweeds have traditionally been considered active agents for the treatment of
inflammation, cancer, type 2 diabetes mellitus, bacterial infections, and obesity.
CaR (sea grapes) is green seaweed that belongs to the Caulerpaceae family. CaR is
extensively spread in tropical areas, particularly in Indo-Pacific Asia, where it
is
eaten either raw as a salad or cooked in vegetable soups. CaR has been considered
a
rich source of various bioactive components. Chemical analysis of CaR conducted
previously has shown that CaR is rich in chemical constituents such as proteins,
lipids, minerals, pigments, and vitamins [8].
Polysaccharides or polysaccharide extracts from CaR exhibit significant anticancer,
anti-inflammatory, antibacterial, antiobesity, and antidiabetic properties [9]. Previous studies have also shown that CaR
exhibits potent antioxidant activity [9].
Various extracts of CaR have also shown potent larvicidal activity [10]. No toxicity was seen in the sulfated
polysaccharides detected in CaR and these reduced nociception and inflammatory
activities are partly attributed to the activation of the hemoxigenase-1 pathway
[11]. These data suggest that CaR not only
exhibits versatile medicinal properties but it also is safe. Moreover, active
components of CaR have the potential to activate various signaling pathways, such
as
immune signaling pathways, which can modulate the immune response for treatment of
various diseases and disorders. With this background information, CaR was selected
for this study. Phytochemical and acute toxicity studies were performed. Since the
extracts were found to be safe, they were studied for hypoglycemic activity.
Subsequently, its antidiabetic activity was evaluated in an STZ-induced diabetic rat
model, along with its impact on proinflammatory markers, antioxidants, lipid, and
serum biomarkers, and histoarchitecture of the pancreas to understand its underlying
mechanisms of hypoglycemic activity [12]
[13].
Results
The fingerprinting pattern of CaRE was studied using HPTLC. Around 15 different peaks
were noted on the HPTLC fingerprint of CaRE ([Figs.
1] and [2]). The plate was
derivatized with Liebermann-Burchard reagent showed a pink colored compound, which
was identified as β-sitosterol, ([Fig. 3]) and others that saponins (like oleanolic acid, ursolic acid, and
lupeol) as active constituents. Other plates sprayed with Dragendorff reagent showed
only one compound, which presumably belonged to the alkaloid group.
Fig. 1 HPTLC fingerprint of CaRE; solvent system petroleum ether:
acetone (8:2 v/v) (Peak 6: Rf 0.38, peak area 5984.2).
Fig. 2 Peak display of standard ß-sitosterol in CaRE.
Fig. 3 HPTLC pattern of CaRE before and after derivatization at
366 nm.
In acute toxicity, the administration of CaRE at various doses did not lead to
mortality until concentrations up to 2000 mg/kg body weight. Even at this dose,
there were no gross behavioral changes or any adverse clinical symptoms, except mild
sedation and analgesia. The rats were monitored once during first 30 min
post-dosing and subsequently up to 24 h, followed by daily observation for
14 days.
A single-dose administration of the CaRE (200 mg/kg) on the 4th day after STZ
administration led to a significant reduction in blood glucose levels at 1 and
6 h (p<0.01). However, at 100 m/kg dose, CaRE did not elicit
any significant blood glucose reduction. Glipizide (5 mg/kg) treatment led
to a greater reduction in blood glucose levels at 6 h compared to CaRE
treatment ([Table 1]).
Table 1 Acute and Sub-acute effects of CaRE on STZ-diabetic
rats.
|
Acute effect of CaRE on STZ-diabetic rats
|
|
Group and Treatment (mg/kg, p.o)
|
Blood glucose (mg%) at different time intervals
(Mean±SEM, n=6)
|
|
|
|
|
0 h
|
1 h
|
3 h
|
6 h
|
|
Normal control
|
62.30±6.30
|
62.83±5.90
|
63.02±7.10
|
63.11±6.90
|
|
Diabetic control
|
492.33±52.30
|
499.40±57.60##
|
433.50±29.60##
|
422.60±44.10##
|
|
CaRE (100)
|
445.20±49.30
|
433.33±47.80NS
|
424.30±42.30 NS
|
438.30±54.30 NS
|
|
CaRE (200)
|
467.30±49.60
|
366.16±38.60 **
|
420.30±52.80 *
|
132.20±22.10 **
|
|
Glipizide (5)
|
467.00±46.00
|
272.50±21.30*
|
268.82±28.60 **
|
69.00±8.90 **
|
|
Sub-acute effect of CaRE on STZ-diabetic rats
|
|
|
|
|
|
Group and Treatment (mg/kg, p.o)
|
Blood glucose (mg%) at various time intervals (days)
(Mean±SEM, n=6)
|
|
|
|
|
10
|
20
|
30
|
|
|
Normal control
|
63.66±2.90
|
62.16±2.40
|
63.33±2.40
|
|
|
Diabetic control
|
389.66±3.80##
|
531.83±3.30##
|
449.16±4.50##
|
|
|
CaRE (100)
|
355.50±4.90 **
|
261.40±3.50 **
|
108.66±4.60 **
|
|
|
CaRE (200)
|
115.16±5.05 **
|
90.83±3.50 **
|
94.33±1.70 **
|
|
|
Glipizide ( 5)
|
80.33±3.50 **
|
125.63±6.00 **
|
91.16±3.60 **
|
|
## p<0.01, when compared to normal control rats;
*p<0.05, when compared to diabetic control rats;
** p<0.01, when compared to diabetic control
rats.
STZ-induced diabetic rats showed a significant elevation in the levels of blood
glucose. Oral administration of CaRE (100 and 200 mg/kg/day)
and glipizide (5 mg/kg/day) to STZ-treated diabetic rats
significantly reduced the levels of blood glucose. In groups treated with
200 mg/kg CaRE and 5 mg/kg glipizide, glucose levels
reduced to almost normal levels. Treatment with both doses of CaRE led to a
significant reduction in levels of blood glucose and its hypoglycemic effect was
comparable to that of 5 mg/kg glipizide on the 30th day ([Table 1]).
In STZ diabetic rats, glucose uptake by the hemidiaphragm was markedly reduced.
Administration of CaRE (100 and 200 mg/kg/day) significantly
improved the glucose uptake via the hemidiaphragm (p<0.01) ([Table 2]). Glipizide-treated rats
(5 mg/kg) did not exhibit increased glucose uptake via the
hemidiaphragm. STZ treatment led to a significant reduction in hepatic levels of
glycogen ([Table 2]). Pretreatment with CaRE
(200 mg/kg/day) significantly prevented depletion of hepatic
glycogen in STZ diabetic rats. The administration of glipizide
(5 mg/kg/day) also significantly alleviated the liver
glycogen reduction (p<0.01) in STZ diabetic rats.
Table 2 Effect of CaRE on various carbohydrate metabolism and
lipid profile parameters in STZ-diabetic rats.
|
Effect of CaRE on various carbohydrate metabolism parameters in
STZ-diabetic rats
|
|
Group and Treatment (mg/kg, p.o)
|
Glycosylated haemoglobin (Hb1Ac%)
|
Glucose uptake by diaphragm (mg/100 mg)
|
Liver glycogen (mg/gm)
|
Glucose transfer by liver (mg/gm)
|
|
|
(Mean±SEM, n=6)
|
|
|
|
|
|
Normal control
|
5.00±0.50
|
18.00±2.10
|
41.00±5.00
|
19.66±1.10
|
|
|
Diabetic control
|
12.00±1.30##
|
4.00±0.60##
|
13.00±1.80##
|
35.66±3.80##
|
|
|
CaRE (100)
|
7.00±0.70 **
|
9.20±1.10*
|
18.00±2.30
|
12.35±1.90 **
|
|
|
CaRE (200)
|
6.00±0.80 **
|
12.60±1.80 **
|
28.00±3.40 **
|
13.16±1.05 **
|
|
|
Glipizide (5)
|
8.00±0.90*
|
6.00±0.50
|
34.00±4.60 **
|
17.33±1.50 **
|
|
|
Effect of CaRE on lipid profile in STZ-diabetic rats
|
|
Group and Treatment (mg/kg, p.o.)
|
Total cholesterol (mg/dL)
|
Triglycerides (mg/dL)
|
VLDL (mg/dL)
|
LDL (mg/dL)
|
HDL cholesterol (mg/dL)
|
|
Normal control
|
71.20±5.29
|
98.60±10.03
|
12.20±2.03
|
37.32±3.13
|
58.26±61.91
|
|
Diabetic control
|
146.40±10.10##
|
212.60±23.51##
|
44.70±6.88##
|
177.70±22.51##
|
21.30±1.17##
|
|
CaRE (100)
|
92.00±9.45 **
|
178.70±23.63 **
|
28.52±3.83 **
|
97.99±10.13 **
|
39.80±6.70 **
|
|
CaRE (200)
|
88.40±9.58 **
|
146.60±22.67 **
|
19.96±2.89 **
|
82.24±8.57 **
|
45.90±4.17 **
|
|
Glipizide (5)
|
101.80±12.50*
|
189.80±14.05*
|
31.06±4.85*
|
130.36±14.16*
|
32.60±5.68*
|
## p<0.01, when compared to normal control rats;
*p<0.05, when compared to diabetic control rats;
** p<0.01, when compared to diabetic control
rats.
STZ-induced diabetic rats also exhibited a marked increase in the levels of HbA1c.
Treatment with CaRE significantly reduced, dose-dependently, the elevated HbA1c
levels ([Table 2]). A similar reduction in
the blood glucose level was also observed after glipizide (5 mg/kg)
treatment. Treatment with both CaRE and glipizide produced significantly
(p<0.01) inhibited glucose transport in liver slices of STZ diabetic rats
([Table 2]). STZ diabetic rats exhibited
significantly elevated levels of TGA, TC, VLDL, and LDL, and a decrease in the level
of HDL in the serum (p<0.01). Treatment with CaRE (100 and
200 mg/kg/day) significantly ameliorated the effects of STZ
([Table 2]). Administration of glipizide
also produced a similar effect on serum lipid levels.
The degree of lipid peroxidation was evaluated in terms of MDA formation. Increased
MDA levels were observed in the liver andpancreas of STZ diabetic rats ([Table 3]). In addition, a concomitant
depletion of endogenous antioxidants enzymes, viz. CAT, GSH, and SOD, was observed
in the liver and pancreas of STZ diabetic rats. Pretreatment with CaRE (100 and
200 mg/kg/day) significantly prevented MDA formation and
restored the depleted levels of GSH, CAT, and SOD in the liver and pancreas of STZ
diabetic rats ([Table 3]). Treatment with
200 mg/kg CaRE led to a greater restoration of depleted antioxidants
compared to treatment with glipizide in STZ diabetic rats.
Table 3 Effects of CaRE on liver MDA and pancreatic MDA
antioxidant enzymes in STZ-diabetic rats.
|
Effects of CaRE on liver MDA and antioxidant enzymes in
STZ-diabetic rats
|
|
Group and Treatment (mg/kg, p.o.)
|
Antioxidants enzymes (Mean±SEM, n=6)
|
|
|
|
|
MDA (nM of MDA/gm)
|
GSH (mg GSH/gm)
|
CAT (µM of H2O2/gm)
|
SOD (U/mg)
|
|
Normal control
|
7.20±0.80
|
1.60±0.30
|
36.32±3.10
|
66.10±8.20
|
|
Diabetic control
|
14.60±2.40##
|
1.10±0.20#
|
17.30±1.30#
|
35.00±2.30#
|
|
CaRE (100)
|
10.10±1.20*
|
1.40±0.20*
|
26.90±1.30*
|
46.80±2.30*
|
|
CaRE (200)
|
8.83±1.00 **
|
1.50±0.30 **
|
32.87±4.70 **
|
56.50±6.30 **
|
|
Glipizide (5)
|
11.20±1.40
|
1.20±0.20
|
24.30±3.50
|
44.90±6.80*
|
|
Effects of CaRE on pancreatic MDA and antioxidant enzymes in
STZ-diabetic rats
|
|
Group and Treatment (mg/kg, p.o.)
|
Antioxidants enzymes (Mean±SEM,
n=6)
|
|
|
|
|
MDA formation (nM of MDA/gm)
|
GSH (mg GSH/gm wet tissue)
|
CAT (µM of
H
2
O
2
/gm)
|
SOD (U/mg of tissue)
|
|
Normal control
|
3.2±0.4
|
1.6±0.4
|
13.2±2.2
|
20.2±3.2
|
|
Diabetic control
|
8.6±0.8#
|
0.5±0.2#
|
6.5±0.7#
|
9.1±1.3#
|
|
CaRE (100)
|
6.3±0.2
|
1.2±0.2*
|
9.5±1.1*
|
18.6±1.9*
|
|
CaRE (200)
|
4.3±1.0*
|
1.4±0.3 **
|
11.4±2.2 **
|
19.4±5.3 **
|
|
Glipizide (5)
|
7.2±0.6
|
0.6±0.2
|
7.8±2.6
|
13.5±2.4
|
## p<0.01, when compared to normal control rats;
*p<0.05, when compared to diabetic control rats;
** p<0.01, when compared to diabetic control
rats.
STZ diabetic mice exhibited significantly elevated TNF-α and IL-4
levels in serum. Treatment with CaRE prevented the elevation of TNF-α
and IL-4 levels in serum. The inhibitory effect of CaRE
(200 mg/kg/day) on serum TNF-α and IL-4
levels was greater than that observed after glipizide
(5 mg/kg/day) treatment ([Table 4]).
Table 4 Effects of CaRE on serum nitric oxide (NO), cytokines
and serum biomarkers in STZ-diabetic rats.
|
Effects of CaRE on serum nitric oxide (NO) and cytokines in
STZ-diabetic rats
|
|
Group and Treatment (mg/kg, p.o.)
|
NO (mcg/mg protein) (Mean±SEM, n=6)
|
Serum Cytokines (pg/mL) (Mean±SEM,
n=6)
|
|
|
|
|
|
TNF-α
|
IL-4
|
|
|
|
Normal control
|
39.00±4.00
|
154.90±65.41
|
182.00±30.79
|
|
|
|
Diabetic control
|
89.00±9.00##
|
387.60±50.24##
|
472.60±60.16##
|
|
|
|
CaRE (100)
|
70.00±8.00
|
291.30±31.97
|
389.40±44.74
|
|
|
|
CaRE (200)
|
56.0 0±8.00*
|
182.00±30.39 **
|
302.00±31.16*
|
|
|
|
Glipizide (5)
|
49.00±6.00 **
|
193.80±32.03 **
|
231.30±20.27 **
|
|
|
|
Effect of CaRE on serum biomarkers in STZ-diabetic
rats
|
|
Serum Biomarkers
|
Group and Treatment (mg/kg, p.o)
(Mean±SEM, n=6)
|
|
|
|
|
|
Normal
|
Diabetic control
|
CaRE (100)
|
CaRE (200)
|
Glipizide (5)
|
|
AST (U/L)
|
22.2±3.5
|
154.3±16.5##
|
82.6±6.8 **
|
45.6±4.5 **
|
111.3±12.3*
|
|
ALT (U/L)
|
12.3±2.1
|
80.4±12.3##
|
37.2±1.5 **
|
22.6±5.2 **
|
52.3±6.5
|
|
ALP (U/L)
|
10.3±1.6
|
57.6±9.3##
|
26.3±6.5 **
|
21.1±2.3 **
|
33.1±4.7*
|
|
Serum creatinine (mg/dL)
|
0.8123±0.0100
|
1.5570±0.0600##
|
0.8205±0.0100*
|
0.9027±0.0200*
|
0.9862±0.0100*
|
|
Blood urea nitrogen (mg/dL)
|
20.60±1.07
|
41.60±1.72##
|
22.20±1.24 **
|
23.80±1.06 **
|
30.20±1.41*
|
|
Uric Acid (mg/dL)
|
1.97±0.05
|
2.94±0.08##
|
2.11±0.02*
|
2.12±0.04*
|
2.43±0.07*
|
|
Total protein (g/dL)
|
7.58±0.23
|
3.97±0.17##
|
7.01±0.33 **
|
7.26±0.16 **
|
4.95±0.08*
|
|
Albumin (g/dL)
|
5.44±0.50
|
1.93±0.10##
|
4.15±0.36 **
|
4.47±0.26 **
|
2.44±0.06
|
## p<0.01, when compared to normal control rats;
*p<0.05, when compared to diabetic control rats;
** p<0.01, when compared to diabetic control
rats.
STZ diabetic mice exhibited significantly elevated levels of serum liver biomarkers
(AST, ALT, and ALP). Treatment with CaRE (100 and
200 mg/kg/day) for 30 days significantly prevented the
elevation of these biomarkers. Glipizide (5 mg/kg/day)
treatment also reduced the levels of serum biomarkers significantly; however, the
degree of reduction was less compared to that obtained after CaRE treatment ([Table 4]). The depleted serum levels of
albumin and total protein were also restored significantly after treatment with CaRE
and glipizide ([Table 4]).
Control groups exhibited no abnormalities in the histoarchitecture of the pancreas
([Fig. 4a]). However, STZ diabetic rats
exhibited moderate to severe necrotic alterations in β-islets of
Langerhans ([Fig. 4b]). In a few mice, only a
sporadic presence could be observed. CaRE-treated
(200 mg/kg/day) mice exhibited mild to moderate necrosis in
β-islets ([Fig. 4d]),
which was comparable to that observed in glipizide-treated
(5 mg/kg/day) groups ([Fig.
4e]). Furthermore, CaRE (100 mg/kg/day) treatment
led to mild improvement in the necrosis of β-islets ([Fig. 4c]); however, the reduction in the
number and size of β-islets was not significantly improved.
Fig. 4 : 4a – Effect of vehicle on histopathological changes
in pancreas of normal rats; 4b – Effect of vehicle on
histopathological changes in pancreas of diabetic rats; 4c – Effect
of CaRE (100 mg/kg) on histopathological changes in pancreas of
diabetic rats; 4d – Effect of CaRE (200 mg/kg) on
histopathological changes in pancreas of diabetic rats; 4e – Effect
of Glipizide (5 mg/kg) on histopathological changes in pancreas of
diabetic rats.
Discussion
Alcoholic extracts of marine algae CaRE were studied for fingerprinting patterns
using HPTLC. We used several solvent systems, of which petether:acetone
(8:2 v/v) was found to be most suitable for optimum resolution. Around 15
different peaks were detected and showed Rf at 0.05, 0.07, 0.12, 0.20, 0.38, 0.40,
0.44, 0.53, 0.60, 0.63, 0.78, 0.80, 0.89, and 0.99, which is represented in%
area as 450.3,256.1,956.3,1665.9, 7865.1, 5984.2, 4589.3, 658.1, 125.0, 357.3,
542.0, 170.3, 100.3, 564.2, and 9842.2 respectively ([Figs. 1]
[2]
[3]
). The plate was
derivatized with Liberman-Burchared reagent, revealing a pink colored compound that
comprised sitosterol and saponins as active constituents. When a plate was sprayed
using Dragandorff reagent, only one compound, probably an alkaloid, was
identified.
In this study, we observed that CaRE elicited marked hypoglycemic activity compared
to other extracts and was also found to have a safe toxicity profile. STZ is a
selective pancreatic islets β cell cytotoxic agent and hence widely
used to induce type 2 diabetes mellitus in rodents [14]. Intraperitoneal administration of STZ (55 mg/kg) to
rats simulates conditions similar to type 2 diabetes mellitus, such as the elevation
of blood glucose, glycosylated hemoglobin, and liver glycogen levels, along with a
significant decrease in glucose uptake by the hemidiaphragm, increased glucose
secretion by hepatic cells, and gluconeogenesis (deceased glucose transport for the
formation of amino acids, citrate, etc.) [15].
Treatment of STZ diabetic rats with CaRE (100 and
200 mg/kg/day) and glipizide
(5 mg/kg/day) for 30 days led to a sustained reduction in
blood glucose and maximum reduction was seen on the 30th day, along with increased
formation of and concomitant increase in glucose uptake by skeletal muscles, liver
glycogen (glycogenesis), and gluconeogenesis. The hypoglycemic activity
200 mg/kg CaRE was observed to be comparable to that of
5 mg/kg glipizide.
One of the possible mechanisms linking hyperglycemia and vascular complications is
the increase in non-enzymatic glycosylation. It has been shown in previous studies
that during type 2 diabetes mellitus, excess blood glucose and hemoglobin react in
a
two-step process to form glycosylated hemoglobin (GHbA1c) [16]. This glycosylated HbA1c has been
recommended as a standard of care to monitor type 2 diabetes mellitus. Therefore,
we
assessed the effect of CaRE treatment on the levels of glycosylated HbA1c. Treatment
with CaRE and glipizide reduced the elevation of glycosylated hemoglobin
significantly. The inhibitory effect of CaRE on GHbA1c in STZ diabetic rats was
comparable to that induced by glipizide.
Oxidative stress plays a major role in type 2 diabetes mellitus pathogenesis. It is
often responsible for the tissue damage associated with type 2 diabetes mellitus
[17]. In diabetic patients, non-enzymatic
protein glycation, oxidation of glucose, and increased lipid peroxidation leads to
the generation of free radicals. This leads to the enzymatic degradation of cells
and elevation in insulin resistance. Recent studies have shown that even the
apolipoprotein component of LDL forms insoluble aggregates in the presence of
hydroxyl radicals, leading to oxidative damage [18]. The main source of oxidative stress is the mitochondria. Modulation
of insulin signaling by reactive oxygen or nitrogen species occurs via two ways. In
response to insulin, these reactive oxygen/nitrogen species are produced for
their physiological function and these reactive oxygen/nitrogen species
negatively impact insulin signaling, which leads to insulin resistance [18]. The formation of free radicals may finally
lead to the formation of MDA, a reactive aldehyde responsible for the formation of
covalent protein adducts called advanced epoxidation end products.
Various studies have shown increased levels of MDA in diabetic patients, which has
been associated with a poor prognosis of type 2 diabetes mellitus. A study was
conducted with 120 diabetic patients, and it was found that MDA levels were high in
type 2 diabetes mellitus patients and prediabetic patients compared to control
group, suggesting that MDA levels are augmented during the progression of type 2
diabetes mellitus [19]. The MDA level has also
been associated with insulin resistance. One study aimed to evaluate MDA levels in
96 prediabetic and 101 diabetic patients and establish its correlation to the
Homeostatic Model Assessment of Insulin Resistance for the same subjects. It showed
a statistically significant correlation between the level of MDA and insulin
resistance in obese prediabetic patients [20].
Taking these results into consideration, we assessed if CaRE has potential to reduce
the level of MDA. Superoxide dismutase catalyzes superoxide anions, which induce
oxidative damage to cell membranes. GSH is a non-protein thiol found in all
mammalian tissues. It is an efficient antioxidant that serves as an oxidative stress
biomarker [21]. Depletion of SOD and GSH
levels has been observed in type 2 diabetes mellitus patients [22].
The levels of antioxidant enzymes and lipid peroxidation were assessed in 90 type
2
diabetes mellitus patients and it was found that MDA levels were high in type 2
diabetes mellitus patients and levels of SOD and GSH were reduced [23]. Decreased levels of GSH might be one of
the factors responsible for oxidative DNA damage in type 2 diabetes mellitus. The
depletion of GSH and SOD may be attributed to diabetes-induced oxidative stress and
free radical overproduction. CAT is another antioxidant enzyme found in peroxisomes
and catalyzes H2O2 to H2O and O2. These
antioxidant enzymes work together to protect cells from free radicals induced by
oxidative stress [17]
[24]. In the present study, we assessed if CaRE
treatment has any effect on the levels of MDA, SOD, and GSH. CaRE treatment (30
days) prevented elevated MDA formation in the liver and pancreas of STZ diabetic
rats, and we hypothesized that these effects might have helped restore the impaired
endogenous antioxidant system and reinstate balanced antioxidant homeostasis in
diabetic rats. Such antioxidant effects have likely contributed to its antidiabetic
activity [25].
Various studies conducted on human and animal models have supported the fact that
inflammation has a role in the initiation and progression of type 2 diabetes
mellitus [26]. Cumulatively, evidence has
suggested that the activation of inflammatory signaling pathways in the target cells
of insulin action could be a contributing factor towards obesity, resistance to
insulin, and related metabolic disorders, viz. type 2 diabetes mellitus [27].
Various studies have proven that the insulin resistance is associated with abnormal
proinflammatory cytokine secretion and reduced anti-inflammatory cytokine generation
[28]. Type 2 diabetes mellitus is often
characterized by impairment of insulin function, which leads to metabolic syndrome
[29]. TNF-α has been found
to play a crucial role in insulin resistance. It also reduces glucose transporter
type 4 expression [30]. Furthermore,
activation of TNF-α induces the phosphorylation of the insulin
receptor substrate-1, which works as an inhibitor of insulin receptors and
downregulates phosphatidylinositol-3 kinase activation [31]
[32].
TNF-α levels have been observed to be elevated in type 2 diabetes
mellitus patients of all age, disease duration, and ethnicity [33].
IL-4 contributes towards insulin resistance, which leads to increased glycogenolysis
and decreased gluconeogenesis [34]. We
observed a decrease in levels of proinflammatory mediators TNF-α and
IL-4 after CaRE treatment, indicating that CaRE inhibits the secretion of these
cytokines, thereby indicating improvement in insulin sensitivity. Our findings
corroborate those of a previous study wherein improvement in insulin sensitivity was
observed during long-term treatment with anti-TNF-α antibody
infliximab in subjects who were insulin resistant [35].
Lipid abnormalities in diabetic patients, often termed as “diabetic
dyslipidemia”, are characterized by high TC (Total cholesterol), LDL
(low-density lipoprotein), and TG (triglycerides) levels, and low HDL (High-density
lipoprotein) levels [36]. These abnormalities
are common in prediabetic and type 2 diabetes mellitus patients. A recently
published study suggested an association between a risk of type 2 diabetes mellitus
and these lipid parameters [37]. Previously,
hypertriglyceridemia and hypercholesterolemia have been reported in alloxan diabetic
rats [38]. Various factors contribute towards
the alteration in lipid metabolism in patients with type 2 diabetes mellitus, such
as insulin deficiency or resistance, adipocytokines, and hyperglycemia. It has
previously been shown that deficiency or insulin resistance results in the
activation of intracellular hormone-sensitive lipase, leading to the increased
release of NEFAs from the TGAs. This high circulating level of NEFA could increase
the production of TGAs by the liver [36].
Diabetic dyslipidemia is a crucial risk factor of diabetes-associated cardiovascular
disorders. Therefore, the treatment of dyslipidemia is an important strategy for the
management of type 2 diabetes mellitus. We found that CaRE showed hypolipidemic
activity in rats, further suggesting that CaRE could not only lower glucose levels
but could also manage other complications associated with type 2 diabetes mellitus.
The exact underlying mechanisms(s) for CaRE’s hypolipidemic activity
observed in the present experiments is not known. However, it is presumed that CaRE
may directly downregulate NADH and NADPH. The other mechanisms responsible for the
hypolipidemic effect of CaRE include (1) inhibition of lipase or/and an
inhibitory effect on lipolytic hormones on the fat depots and (2) cholesterol
biosynthesis or absorption of lipids from the gastrointestinal tract or uptake
process.
The hypolipidemic activity displayed by CaRE is beneficial and may be useful to
reduce cardiovascular complications in diabetic conditions and may help to
ameliorate the risk of acute myocardial infarction as claimed by the NCEP guidelines
(2002) [39]. Increased hepatic serum markers,
like transaminases, including ALP, have been documented in experimental models of
type 2 diabetes mellitus [40]. The increased
transaminases indicate impaired hepatic function largely due to cellular damage to
hepatocytes. Furthermore, the increased transaminases may serve as a substrate to
amino acids, thereby enhancing gluconeogenesis as well as ketogenesis. Such
observations have also been clinically documented in diabetic patients [41]. In the present study, CaRE (100 and
200 mg/kg) and glipizide treatment in STZ diabetic rats inhibited
the increased serum transaminases, which may be attributed to their hypoglycemic
activity and/or modulation of carbohydrate, lipid, and protein metabolism.
Alternatively, the decreased serum transaminases may be attributed to decreased
oxidative stress and, consequently, reduced cytotoxicity to hepatocytes. Oxidative
stress due to reactive lipid peroxidation recruits inflammatory cells and can be a
potential triggering factor for the increased serum transaminases and enhanced
formation of proinflammatory cytokines (IL-4 and TNF-α) in type 2
diabetes mellitus [42].
STZ treatment produced atrophy of β-islets of Langerhans associated
with severe necrosis represented by pyknotic nuclei and acidophilic cytoplasm in the
necrotic cells along with vascular degenerative alteration, resulting in the
reduction of size and number of β-islets significantly. Only a
sporadic presence was observed in few rats. The treatment of STZ diabetic rats with
CaRE prevented the degree of necrosis dose-dependently. The improvement of necrosis
of β-islets after treatment with 200 mg/kg CaRE was
comparable to that induced by treatment with 5 mg/kg glipizide.
Furthermore, the size and number of β-islets also improved after
treatment with 200 mg/kg CaRE and 5 mg/kg glipizide.
The reduction in necrosis and improvement in size and numbers of
β-islet of Langerhans can be attributed to the amelioration of
oxidative stress, which also leads to stabilization of their membranes.
The HPTLC analysis of CaRE revealed the presence of β-sitosterol as an
active constituent. HPTLC analysis also showed the presence of alkaloid and saponin
classes of compounds. It has been reported that β-sitosterol exerts
an inhibitory effect on glycated hemoglobin, thiobarbituric acid-reactive
substances, blood glucose, and nitric oxide, with upregulation in serum insulin
levels and pancreatic antioxidant levels [43].
In agreement with these findings, Bumrela et al. have also confirmed the antioxidant
potential of β-sitosterol both in vivo and in vitro
[44]. HPTLC analysis also demonstrated
the presence of saponins, which could be oleanolic acid, ursolic acid, and lupeol.
Both oleanolic acid and ursolic acid showed significant protection against
chemically induced liver injury in animal models. Oleanolic acid and ursolic acid
have also been long recognized to exert antihyperlipidemic and anti-inflammatory
effects [45]. Various in vitro and
animal studies demonstrated that lupeol possesses a potential cholesterol-lowering
activity [46]. Further, caulerpin, a bisindole
alkaloid, has also been isolated by De Souza et al. Further, caulerpin, a bisindole
alkaloid, has also been isolated by Lucenaet al. from CaRE and found to be a potent
anti-inflammatory agent in various models of inflammation [47].
The antidiabetic and antioxidant activities exhibited by the phytoconstituents
isolated (identified by HPTLC analysis) from the ethanolic extract of CaRE are in
agreement with the various biological activities reported in the literature. The
present experimental findings of CaRE demonstrated significant antidiabetic activity
in STZ diabetic rats. The various biochemical paradigms observed in STZ diabetes
included decreased glucose and lipid metabolism, reduction in levels of
proinflammatory mediators, and oxidative stress alleviation. Histopathological
studies suggested its antidiabetic activity is derived from the antioxidant
activity. Downregulation of proinflammatory mediators improves the histoarchitecture
of β-islets. Such effects may be attributed to the presence of
saponins, β-sitosterol, and other phytochemicals. Further, saponins
and β-sitosterol found in the extract may be responsible for such
beneficial therapeutic effects. Hypolipidemic activity may be beneficial for
patients with type 2 diabetes mellitus. CaRE seems to be safe and doesn’t
affect vital organs adversely. CaRE needs to be further assessed through human
clinical trials to assess its therapeutic efficacy in type 2 diabetes mellitus.
Materials and Methods
Plant material
CaR seaweed was collected from the coastal area of Okha port in the Gujarat state
of India. The collected seaweed sample was authenticated by the Department of
Botany of a seaweed-based organization, Carrag Seaveg Pvt. Ltd., Bhavnagar,
Gujarat, India. A voucher herbarium specimen, number
CSPL/GA/CR07–09, has been preserved at CarragSeaveg.
Drugs and chemicals
Glipizide powder (purity: 99.9%) was obtained from USV Pharma. STZ was
procured from Sigma-Aldrich. Commercial kits used for biochemical analysis were:
glucose reagent kit GOD/POD end point (Biolab), serum glutamate
oxaloacetate transaminase (SGOT/AST) determination kit (Accurex
Biomedical Pvt. Ltd.), serum glutamate pyruvate transaminase (SGPT/ALT)
determination kit (Accurex Biomedical Pvt. Ltd.), alkaline phosphatase
determination kit (Accurex Biomedical Pvt. Ltd.), lipid profile kit (Nirmal
Labs), Keto-Test GK test strips for urine glucose determination (Diascreen),
renal function parameters kit (Nirmal Labs), and reagent strips for urinalysis
(Hypoguard). The control, test item, and reference drug were dissolved in
1% CMC.
Preparation of extract
Samples of dried seaweed were prepared by cutting or crushing the seaweed into
small pieces and then grinding into a coarse powder. Coarse powders
(50 g) were extracted with 500 mL of absolute ethanol
(95%) using a Soxhlet extractor at 500°C. Occasional shaking was
carried out for a period of 72 h. This crude extract was filtered off and
subsequently evaporated to dryness at 45°C. The concentrated dry mass
was used for further investigations. The extract obtained was in ethanol.
High-performance thin-layer chromatography analysis of Caulerpa
racemosa ethanolic extract
CaRE (10 mg) was dissolved in 10 mL of chloroform (final
concentration: 1000 ng/µL) and analyzed using a CAMAG
LINOMATE V automatic sample applicator on a 10× 10 cm HPTLC
plate coated with 250 µm layers of Silica gel G 60 F254 (Merck)
and solvent system. Petether:acetone [(8:2) (v/v)] was used as the
mobile phase. A Scanner-3 (CAMAG) was used to perform densitometer scans at
366 nm. The plates were derivatized in Liebermann-Burchard reagent.
Animals
Adult female mice (Mus musculu Swiss; 18–20 g) and male
mice (Rattus norvegicus Wistar; 200–250 g)
(age:10–12 weeks) were used for the study. They were housed under
standard conditions (room temperature: 24±3°C, relative
humidity: 45–55%, 12:12 dark/light cycle). They were fed
ad libitum and given filtered tap water. Standard pellet diet was provided. The
experimental protocol was reviewed and approved
(CPCSEA/IAEC/SPTM/P-14/2014) by the
Institutional Animal Ethics Committee for Purpose of Control and Supervision of
Experimental Animals.
Acute toxicity studies
OECD guideline 423 (OECD 2001) in Swiss albino mice was followed for the acute
toxicity study. The mice were orally administered CaRE
(300–2000 mg/kg) solutions in 1% CMC. The mice
were then kept under observation for 72 h.
Experimental induction of type 2 diabetes mellitus
STZ (55 mg/kg) solution in 0.1 M cold citrate buffer (pH
4.5) was administered intraperitoneally. Blank citrate buffer was used for the
control mice. Initially, Wistar rats were screened for pre-dose glucose levels
and then STZ was administered for inducing type 2 diabetes mellitus. After
72 h, blood was withdrawn from all animals and blood glucose levels were
analyzed again. Mice with fasting blood glucose levels above
300 mg/dL and exhibiting polydipsia and polyphagia were selected
for the 30-day anti-diabetic study.
Antidiabetic activity evaluation in streptozotocin-induced diabetic
rats
Acute dose-response
Overnight fasted STZ diabetic rats were divided into groups of six rats each
as follows: group I comprised normal control rats orally administered with
1% CMC solution (10 mL/kg),
group II comprised diabetic control rats [diabetic rats orally administered
1% CMC solution (10 mL/kg)], group III comprised
diabetic rats orally administered CaRE (100 mg/kg) in
1% CMC, group IV comprised diabetic rats orally administered CaRE
(200 mg/kg) in 1% CMC, and group V comprised
diabetic rats orally administered glipizide (5 mg/kg) in
1% CMC. Fasting blood glucose levels were3 estimated from
retro-orbital sinus prior to and at 1, 3, and 6 h after treatment
[48].
Subacute (30 day) response at double dose
The same animals of the single-dose short-term study were continued at two
different doses of CaRE (100 and 200 mg/kg) or glipizide
(5 mg/kg) orally daily for 30 days. Blood glucose levels
were assayed on the 10th, 20th, and 30th days after drug/CaRE
treatment. Animals were sacrificed by euthanasia on the 31st day and the
liver, pancreas, and diaphragm were extracted, washed with cold saline, and
preserved under -20°C until used for various biochemical
investigations.
Carbohydrate metabolism
Determination of glycosylated hemoglobin (HbA1c)
The glucose level in the blood plasma was evaluated using glucose estimation
kits. Glycosylated hemoglobin (HbA1c) was assessed using a previously
proposed method Parker et al. [49].
Estimation of liver glycogen
The liver glycogen level was estimated using a previously proposed method
[50]. The supernatant was mixed
with 95% ethanol in a 1:5 ratio and incubated overnight for glycogen
precipitation. Then, the glycogen precipitate was dissolved in distilled
water (2 mL). The standard and blank solutions were prepared using
glucose (0.5 mg/mL) and distilled water, respectively. Next,
anthrone reagent (5 mL) was added to all tubes, which were then
incubated in a boiling water bath for 15 min. Then, absorbance was
measured at 620 nm. Results are expressed as mg glycogen/g
tissue [51].
Glucose uptake and glucose transport
The mice were sacrificed under ether anesthesia and their livers and
diaphragm were extracted. The excised tissues were immediately placed in
ice-cold perfusion solution [containing KCl (0.04%), NaCl
(0.687%), MgSO4 (0.014%), NaHPO4
(0.014%), CaCl2 (0.028%), NaHCO3
(0.21%)]. In another batch of perfusate, glucose at a concentration
of 400 mg% was taken. Next, the tissue slices were incubated
at 37°C for 1.5 h with appropriate aeration. Thereafter, the
slices were washed with water and oven-dried at 800°C for
4–5 h. The glucose uptake was measured as mg
glucose/100 mg dry weight of the diaphragm, while glucose
levels in perfusate were measured as mg glucose/g of dry weight of
the liver [52].
Lipid profile
Serum levels of TC, TGA, HDL, LDL, and VLDL were assayed using respective
analytical kits (Nirmal Labs).
Antioxidant, inflammatory, and serum biomarkers
The liver and pancreas were extracted as described previously, washed with
ice-cold saline, and weighed. Tissue homogenates (10% w/v) were
prepared in cold 0.1 M phosphate buffer solution (pH 7.4) and used to
determine MDA formation [53]. Cell debris
was removed by centrifugation (10000× g at 4°C for
20 min) and the supernatant was assayed to determine nitrite levels
[54] and enzyme antioxidants, viz.
superoxide dismutase [55], CAT [56]
, and reduced GSH [57]. The inflammatory biomarkers, including
cytokines, TNF-α and IL-4, serum markers (AST, ALT), and ALP,
were assayed on the 31st day using commercial kits.
Histoarchitecture of the pancreas
Pancreatic tissue was extracted and preserved in 10% formalin. After
fixation, tissues were embedded in paraffin. Then, the
3–4 µm thick sections were cut and stained using eosin
and hematoxylin. The stained specimens were then examined under a light
microscope (40×).
Statistical analysis
The results are expressed as the mean±SEM. Statistical analysis was
performed through ANOVA followed by Dunnett’s test. CaRE- and
glipizide-treated groups were compared with the corresponding normal or diabetic
control groups. Statistical significance was indicated by p<0.05. SPSS
and Graph Pad Prism Version 08 were used for statistical analysis.
