NEUROPROTECTIVE EFFECTS OF TRIANTHEMA DECANDRA IN DIABETES-ASSOCIATED COGNITIVE IMPAIRMENT

Abstract

ABSTRACT NEUROPROTECTIVE EFFECTS OF TRIANTHEMA DECANDRA IN DIABETES-ASSOCIATED COGNITIVE IMPAIRMENT The present invention aims to evaluate the neuroprotective potential of Trianthema decandra in diabetes-induced cognitive impairment, which may find clinical application in treating neuronal deficit in the diabetic patients. The protective actions of T. decandra on diabetic dysfunction may be attributed to its multiple pleiotropic effects like antihyperglycemic and anticholinesterase activity. The extract of T. decandra was standardized by TLC and HPTLC methods. To verify the identity and purity of isolated compounds, they were segregated and characterized using various techniques, including UV-visible spectrophotometry, FT-IR, H-NMR, and Mass spectroscopy. α-amylase and α-glucosidase inhibition property of the extracts were assessed in-vitro. The screening of the neuroprotective effects of METD in hyperglycemic rats was done utilizing Morri’s water (MWM) and elevated plus maze (EPM) model, as well as acetylcholinesterase (AChE) activity. (FIG. 6 will be the reference figure)

Specification

Description:NEUROPROTECTIVE EFFECTS OF TRIANTHEMA DECANDRA IN DIABETES-ASSOCIATED COGNITIVE IMPAIRMENT
FIELD OF THE INVENTION
The present invention relates to the evaluation of neuroprotective ability of methanolic extract of Trianthema decandra (METD) against hyperglycemia-related cognitive impairment in rats.
BACKGROUND
Alzheimer-related Diabetes Mellitus, also called as Type 3 Diabetes Mellitus (T3DM) is a type of hyperglycemia (diabetes) along with Alzheimer's disease (AD). The brain is an important part in AD context, i.e., remarkably affected by humulin production, leading to energy deficiency and neuronal damage. Cognitive dysfunction can occur in patients with impairment of glucose metabolism. The studies showed normal glucose metabolism is necessary for working of cognitive function. Embodiments of the present invention address the foregoing and other needs. In the present invention, the effects of hyperglycemia on neurological decline and neuro-protective capability of methanolic extract of Trianthema decandra (METD) against hyperglycaemia-related cognitive impairment in rats are evaluated.
SUMMARY OF THE INVENTION
According to an embodiment of the present invention, quercetin and phytol, two chemical constituents isolated from Trianthema decandra, were standardized. The extracts of Trianthema decandra and its chemical constituents, namely quercetin and phytol demonstrated a significant protective effect on enzymes like α-amylase and α-glucosidase. Methanol and hydroalcoholic extracts have shown the strongest inhibitory activity followed by chloroform extract. Quercetin and phytol were associated with the methanolic and chloroform extracts which were identified using TLC and HPTLC techniques.
According to another embodiment of the present invention, a diabetes mellitus animal model was evolved by giving streptozotocin (STZ) dose which caused the glucose level to increase via destroying β-cells. STZ is a known toxin for β-cells and is utilized for checking medicinal effect of drugs in the experimental model of hyperglycemia to research on antidiabetic drugs. The present invention showed the potency of methanolic extract of Trianthema decandra (METD) for diabetes, it was confirmed by the outcomes of OGTT and BGL. Blood glucose level of diabetes-induced batch when correlated with control batch, it was lowered in METD and standard groups. METD upregulated glucose uptake and downregulated hyperglycemia and reduced BGLs in diabetic rats, same as insulin action.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts, A) Dried crude powder maceration using Methanol B) Filtration of macerates and collection of crude extract C) Partitioning of methanolic extract using pet ether D) Partitioning of methanolic extract using chloroform.
Figure 2 shows all TLC runned in Mobile phase pet ether: ethyl acetate (3:1).
Figure 3 shows HPTLC Fingerprint profile of Methanol Extract test sample (MT), Hydroalcoholic fraction test sample (HT), Chloroform fraction (CT), Quercetin (Q STD) Phytol (R STD).
Figure 4 shows HPTLC chromatogram of Qucertein.
Figure 5 shows HPTLC chromatogram of Phytol.
Figure 6 shows detection of isolates: (A) Isolation of compd-A using PMA stain (B) Isolation of compd-B using near UV (C) Isolation of compd-B using PMA Stain.
Figure 7 depicts IR Spectrum Analysis of Isolated Compound - Phytol.
Figure 8 shows Mass Spectrum of the isolated compound- Phytol.
Figure 9 shows 1H NMR Spectrum of the isolated compound- Phytol.
Figure 10 shows 13C NMR Analysis of Isolated Phytol Compound.
Figure 11 shows IR Analysis of the isolated compound Quercetin.
Figure 12 shows Mass Spectral Analysis of Isolated Quercetin Compound.
Figure 13 shows 1H NMR Analysis of Isolated Compound B.
Figure 14 shows 13C NMR Spectrum of the isolated Compound-B.
Figure 15 shows effect of METD on OGTT
Figure 16 shows effect of METD on AUC.
Figure 17 shows effect of METD on serum glucose levels.
Figure 18 shows effect of extract on TG levels.
Figure 19 shows effect of METD on TC levels.
Figure 20 shows effect of METD on HDL levels.
Figure 21 shows effect of METD on AST levels.
Figure 22 shows effect of METD on ALT levels.
Figure 23 shows effect of METD on Escape Latency on Morris Water Maze.
Figure 24 shows effect of METD on transfer latency (TL).
Figure 25 shows effect of METD on the brain ACHE levels.
DETAILED DESCRIPTION OF THE INVENTION
Methods
Collection of plant materials: Trianthema decandra leaves were collected at Osmania University in Hyderabad, Telangana, India. Division of Botany, identified and herbarium number was assigned. (Authentication No. 1 for No. 13/Hort/MADP/2011-12. Plant material is then crushed into a fine powder once it has totally dried. Methanolic extract of the shade-dried powder was prepared.
Preparatory qualitative chemical constituents' analysis Chemical constituents were assessed by standard procedures. Alkaloids, saponins carbohydrates, glycosides, flavonoids, phytosterols/terpenes, proteins, and lipids were subjected to analysis. The HPTLC technique was used to standardize the crude methanol extract and its fractions containing quercetin and phytol.
Acute Toxicity Study methanolic extract of Trianthema decandra (METD) was found to be safe in an acute oral toxicity study. Animals were given 2000 mg/kg METD exhibit no lethargy, toxicity, or aberrant way of behaving. We established that the LD50 for METD is more than 2,000 mg/kg (OECD, 2001). Therefore, the ED50 is often said to be 10% of the LD50, the ED50 of METD was explained to be 200 mg/kg and 400 mg/kg thus, these doses were selected for the investigation.
Healthy Wistar rats weighing (200-250 g) were used. Streptozotocin and nicotinamide (STZ-NA) model was used in the study. Diabetes Mellitus (DM) was induced by giving intraperitoneal STZ and NA injections.
Table 1: Experimental design.
Group Name Treatment
I Control group Saline
II Diseased group STZ (50 mg/kg) + NA (110 mg/kg)
III Low dose group Diabetic animal model + extract (low dose)
IV High dose group Diabetic animal model + extract (high dose)
V Standard group Glibenclamide (5 mg/kg b.w.)

Oral glucose tolerance test (OGTT) Oral glucose resistance trial was done for entire night on fasted rats by rupturing the tail. Firstly, fasting blood glucose levels were checked then 2 g/kg body weight glucose was given orally. There after sugar levels were tested at 30, 60 and 120 mins.
The AUC of each group was determined as formula.

A, B, C and D showed plasma glucose level at 0, 30, 60, and 120 min respectively (Kshirsagar et al. 2015).
Evaluation of lipid profile: HDL cholesterol, cholesterol and triglycerides were evaluated utilizing the protocol laid forth for enzymatic kits.
Evaluation of liver parameters: The estimation of AST and ALT followed the protocol profiled for enzymatic kits.
Behavioral estimations were done by using Morris water maze (MWM) and Elevated Plus Maze Test.
Acetylcholinesterase enzyme biochemical estimation: Acetylcholinesterase, a cholinergic marker in the mouse brain as a whole, was determined using the Ellman technique.
Statistical analysis
Data is expressed as Mean ± S.E.M, 6 animals in each group. Two groups were checked using Graph Pad Prism 5's one-way analysis of variance (ANOVA) and Tukey's post hoc test for exponential content. If the value p value was less than 0.05, it is found to be statistically significant.
RESULTS
Extraction and Partitioning
A separation process was employed (Figure 1) to extract the plant material using solvents, one at a time to prepare it for extraction. Methanol was used to extract 80 g of shade-dried powder, which was then soaked for one week with occasionally stirring in 250 mL of solvent. The methanolic extracts produced from triple maceration after filtering the mixture was mixed with Whatman filter paper No. 1 until raw powder have gone.
Using the formula shown below, extractive yield as a percentage was obtained.
Yield% =Weight of the dried extract x 100/ Weight of the dried crude powder
Yield% =24 g x 100/ 80 g = 30%
Using pet ether, water, and chloroform, mother extract (a methanolic extract) was divided into several fractions. Pet ether was used with the methanolic extract to divide the pigments and lipid compounds. The remaining extract was separated using chloroform and water as solvents after the pet ether fraction was removed. The obtained fractions were then dried and labelled as follows: Hydroalcoholic fraction (H1) for the residual water fraction, Methanolic mother extract (M1) for its fractions, Petroleum ether fraction (P1), and Chloroform fraction (C1).
Qualitative analysis
The manufactured extracts and fractions were put through chemical testing to determine the type of phytoconstituents present, as shown in table 2.
Table 2: Qualitative analysis of prepared Trianthema decandra extract/fractions using Chemical tests.
S. No Phyto constituents M1 P1 C1 H1
1 Flavonoids + - + -
2 Alkaloids + - + +
3 Saponins + - - +
4 Phenolic compounds or Tannins + - - +
5 Volatile oils + - + -
6 Steroids and triterpenoids + + + +
7 Glycosides + - + +
8 Anthraquinone + - - +
9 Protein + + - +
10 Carbohydrates + - + +
11 Lipids + + + -
Whereas, M1 was the methanolic extract and P1, C1, H1 were the fractions of whole parts of Trianthema decandra.
"+" indicates presence; "- "indicates absence of phytoconstituents.
TLC studies
The M1 extract and its fragment (C1, P1, and H1) provided the test samples for our chemical constituents screening. As show in Figure 2, (A) TLC was done using M1 and its fractions P1, C1 and H1 below near-ultraviolet light (B) TLC of the C1 fraction was acknowledge using the PMS spraying reagent in correlation to standards of stigmasterol (STG), quercetin (cru) and phytol (phy). C) TLC analysis of the M1 extract and the C1 and H1 fractions under UV light in comparison to quercetin (Qs) and phytol (Rs). (D) TLC of the M1 extract and C1 fraction in contrast to the visualizing agents Phytol (phy) and Quercetin (Qs) in PMA stain.
HPLC studies (Figure 3, Figure 4 and Figure 5)
Table 3: HPTLC Fingerprint of Methanol extracts of Trianthema decandra.
Component UV254nm Rf value Linearity range (ng/spot)
Methanol Extraction(M) 0.175 200-1400
Aqueous methanol fraction(H) - 200-1400
Chloroform fraction (C) 0.18 200-1400
Quercetin (Q) 0.18 200-1400
Phytol (R) 0.16 200-1400

Methanolic extract exhibited many (around 13-14) different bands under Uv light which showed many phytoconstituents. Hydroalcoholic fraction did not reveal any bands under Uv 254nm. The chloroform fraction showed 8-9 bands, amid which the 4th band from the baseline in the chloroform fraction and the standard quercetin showed an Rf value of 0.18 under UV 254nm. The same spot was also seen in the methanolic extract, which had an Rf value of 0.175, very close to the standard quercetin. Therefore, the existence of quercetin was established using the HPTLC method.
As shown in Figure 6, compound A and compound B were detected and isolated on TLC plates.
Compound A (Figure 6 to 10)
Yield: 85 mg (1.7%w/w), acquired as a colourless liquid at a B.P. of 204°C. With the Libermann butcher test, it gave a red colour; with the Salkowski test, no green colour; and with the ferric chloride test, a bluish green tint. Terpenoidal alcohol has been recognized as compound A. The TLC examination gave a blue spot after being sprayed with PMA reagent when the petroleum ether: ethyl acetate (7:3) solvent combination was used. It had an Rf value of 0.54. Compound A's UV spectra in methanol showed a peak in absorption at 258 nm. Compound A's infrared (IR) spectrum exhibited a number of different peaks. A peak at 2900 cm-1 indicated Alkyl C-H stretching, a broad range of 3250 cm-1 to 3500 cm-1 corresponding to OH- Stretching, a peak at 1460 cm-1 manifested the existence of a C=C bond (arising from -OH), and a peak at 1004 cm-1 confirmed C-O stretched all desirable similarity to the reference compound Phytol. Compound A's ESI-MS analysis (positive ion mode) of m/z 299 revealed a molecular ion [M+2] + peak. Compound A's NMR spectra exhibited the alcohol CH2 protons at 4.18 ppm and double bond hydrogen at 5.45 ppm. Compound A was confined from the chloroform fraction of Trianthema decandra. The persistent methyl protons were at 0.87 ppm and allylic methyl protons were at 1.69 ppm. At 1.3-1.07 ppm, aliphatic methylene CH2 protons were discovered. All of these bonds are suitable with common phytol, a hydrocarbon with the chemical formula C20H40O. Thus, Phytol was recognized as isolated unknown molecule A.
The chemical shifts of numerous hydrogen and carbon atoms found in compound A and phytol are shown in table 4.
Table 4: 1H and 13C NMR Resonances of Phytol (32) and Isolate Compound A.
Carbon No. Chromophore 1H NMR Chem shifts a (ppm)
Phytol Compound A 13C NMR Chem Shifts b (ppm)
Phytol Compound A
1 -C-OH 4.14 4.18 59.39 59.3
2 =CH 5.39 5.43 123.09 123.18
3 =C< - - 140.23 140.04
4 >CH2 1.97 1.90 39.85 39.88
5 >CH2 1.40/1.36 1.40/1.31 25.12 25.14
6 >CH2 1.24/1.05 1.24/1.09 36.65 36.68
7 >CH 1.35 1.31 32.67 32.74
8 >CH2 1.23/1.03 1.24 37.35 37.53
9 >CH2 1.29/1.15 1.25/1.07 24.45 24.47
10 >CH2 1.23/1.03 1.25/1.08 37.41 37.53
11 >CH 1.35 1.31 32.77 32.74
12 >CH2 1.23/1.03 1.25/1.07 37.28 37.19
13 >CH2 1.25 1.25 24.79 24.79
14 >CH2 1.11/1.03 1.10/1.07 39.35 39.37
15 >CH 1.50 1.52 27.95 27.97
16 -CH3 0.84 0.85 22.60 22.66
17 -CH3 0.84 0.86 22.69 22.71
18 -CH3 0.83 0.85 19.69 19.70
19 -CH3 0.82 0.85 19.72 19.74
20 -CH3 1.65 1.69 16.14 16.14
a 500MHz, CDCl3, b 125 MHz, CDCl3
Compound B (Figure 6 and Figure 11 to 14)
Yield: 70 mg (1.4%w/w), acquired a yellow amorphous powder, M.P. 316.3°C. Schinoda test showed pink coloring, while ferric chloride test revealed blue green coloration. Flavonoidal alcohol has been recognized as Compound B. The TLC examination showed a blue spot after being sprayed with PMA reagent when the petroleum ether: ethyl acetate (7:3) solvent combination was used. It has an Rf value of 0.54. Compound B's UV spectra in methanol shows maximal absorption at 401 and 261 nanometers. The infrared (IR) spectrum of chemical B revealed multiple distinctive absorption bands. A peak at 3288.04 cm-1 was recognized in infrared spectrum (IR) analysis as stretching vibration of hydroxyl group (-OH) in phenol. The vibration of carbon-carbon (C-C) bond in the aromatic ring was identified as source of a peak at 1613.23 cm1. Additionally, the stretching vibration in carbon-oxygen (C-O) bond in aryl ether functional group was assign to the absorption band at 1245.53 cm-1. The carbon-oxygen-carbon bond in the ketone group's stretching and bending vibrations were both attributed to a peak at 1141.71 cm-1. Finally, two bands at 822.28 cm-1 and 612.01 cm-1 were known and identified as the bending vibrations of the aromatic hydrocarbon's carbon-hydrogen (C-H) bonds. The molecular ion peak [M+1]+ at m/z 303.0514 was found during the +ve ion mode ESI-MS investigation of chemical B. the 1H-NMR spectra showed aromatic hydrogen groups ranging from 6.19 to 7.68 ppm and phenolic-OH groups ranging from 9.32 to 12.50 ppm. All of these bonds were suitable with the common form of quercetin, a hydrocarbon with the molecular formula C15H10O7. Thus, it was determined that isolated unknown component B was quercetin.
Table 5: Chemical Shifts of 1H and 13C NMR Signals in Quercetin and Isolated Compound B.
Carbon No. Chromophore Quercetin 1H NMR Chem Shifts a (ppm) Compound B 1H NMR Chem Shifts a (ppm) Quercetin 13C NMR Chem Shifts b (ppm) Compound B 13C NMR Chem Shifts b (ppm)
1 C-1 12.16 (s) - 177.2 176.30
2 C-2 6.88 (d, J = 8.7 Hz) 7.68 (d, J = 1.8 Hz, 1H) 158.8 156.60
3 C-3 7.50 (d, J = 2.1 Hz), 6.97 (d, J = 8.7 Hz) 6.89 (d, J = 8.5 Hz, 1H) 93.9 93.80
4 C-4 6.55 (d, J = 2.1 Hz) - 160.4 161.19
5 C-5 7.57 (dd, J = 8.7, 2.1 Hz) - 99.3 98.64
6 C-6 6.98 (d, J = 8.7 Hz) - 157.7 156.60
7 C-7 7.86 (d, J = 8.7 Hz) - 121.5 120.43
8 C-8 6.81 (d, J = 8.1 Hz) 6.41 (s, 1H) 145.4 145.52
9 C-9 7.47 (d, J = 2.1 Hz), 6.99 (d, J = 8.7 Hz) 6.19 (s, 1H) 94.1 93.80
10 C-10 6.01 (d, J = 1.8 Hz), 6.18 (d, J = 1.8 Hz) - 105.9 103.47
11 C-1' 6.77 (d, J = 2.1 Hz) - 115.3 115.53
12 C-2' 7.56 (dd, J = 8.4, 2.1 Hz) - 131.9 136.19
13. C-3' 7.47 (d, J = 8.1 Hz) 114.2 115.53
14. C-4' 6.80 (d, J = 8.1 Hz) 152.7 156.60
15. C-5' 6.80 (d, J = 8.1 Hz) 118.8 116.06
16. C-6' 7.56 (dd, J = 8.4, 2.1 Hz) 145.6 145.52
17. C-7' 7.56 (dd, J = 8.4, 2.1 Hz)
a 500MHz, CDCl3, b 125 MHz, CDCl3
For, quercetin and compound B the 1H and 13C NMR chemical shifts are different in the following table. The carbon atom in the molecule is recognized by the number Carbon No. The term "chromophore" mentioned the functional group or component of a molecule that is accountable for light absorption and the creation of colour. For each carbon atom, the chemical shifts (in ppm) of quercetin and compound B are reported. The 13C and 1H NMR chemical shifts are introduced in (a) and (b), respectively.
For several carbon atoms, Compound B's 1H NMR chemical shifts differ from quercetins. Compound B, for example, 1H NMR signal at carbon number 2 at 7.68 ppm (d, J = 1.8 Hz, 1H), whereas quercetin showed a signal at 6.88 ppm (d, J = 8.7 Hz). Similar to this, Quercetin exhibited a signal at 6.81 ppm (d, J = 8.1 Hz) at carbon number 8 as Compound B showed a signal at 6.41 ppm (s, 1H).
Quercetin and Compound B showed similar 13C NMR chemical shifts for the majority of carbon atoms, proving that they shared a same carbon skeleton.
Table 6: IC50 Values for In-vitro hindrance of α-Amylase and α-Glucosidase through Fractions of Trianthema decandra.
Sample IC50 αamylase IC50 αglucosidase
Acarbose (standard) 2.54± 0.4 μg/mL 2.81± 0.5 μg/mL
Methanol extract 180.7± 8.7 µg/mL 20.5± 1.7 µg/mL.
Aqueous methanol 96.3± 2.7 μg/mL 102.6± 2.7 μg/mL
Chloroform 56.2± 2.7 μg/mL 47.3± 2.7 μg/mL
Pet ether extract 83.5± 2.7 μg/mL 150± 2.7 μg/mL
Isolate Compound A Phytol 233.6 ± 22.1 μg/mL 198.4 ± 15.1 μg/mL
Isolate compound B Quercetin 11.4 ± 0.3 μg/mL 7.4 ± 0.6 μg/mL
Trianthema decandra IC50 assessment indicated as mean ± SEM, where IC50 shows the inhibitory concentration.
The IC50 values for the in-vitro obstruction of the enzymes by several Trianthema decandra fractions, as well as by two isolated chemical constituents, phytol and quercetin are shown in the following table (Table 6). The IC50 value is the amount of a chemical required to reduce the target enzyme's activity by 50%. The table exhibited that Trianthema decandra's methanol extract and aqueous methanol fractions crucially obstructed the enzyme activity.
Effect of METD on OGTT
The OGTT range in normal control category 260 ± 29 mg-h/dL, that was notably (p < 0.001) increased to 589 ± 5.5 mg-h/dL in hyperglycemic control group. These increased levels were crucially decreased by the therapy with METD and glibenclamide (p < 0.001) (Figures 15 & 16). Sugar levels recorded were given in table 7.
Table 7: Effect of METD on OGTT levels.
Groups 0 min 30 min 60 min 120 min AUC
NC 87 ± 4.6 144 ± 4.4 119 ± 3.2 88 ± 3.5 260 ± 29
DC 266 ± 9.5α 329 ± 3.8α 286 ± 4.1α 287 ± 5.5α 589 ± 3.9α
METD 200 230 ± 11a 272 ± 13a 239 ± 8.3a 222 ± 11a 484 ± 7.0a
METD 400 192 ± 7.5a 235 ± 6.1a 198 ± 6.7a 163 ± 8.5a 396 ± 9.5a
Standard 94 ± 5.6a 203 ± 13a 118 ± 1.8a 102 ± 4.1a 264 ± 11a
αp < 0.001 i.e., related to NC group; ap < 0.001 when matched with DC category.

Effect of METD on serum glucose levels
The DC category exhibited an important rise in glucose levels during the experimental period. The study of serum sugar levels at the end in DC batch were (p < 0.001) rise to 308 ± 9.3 mg/dL as compared to the NC batch. The increased hyperglycemic level was notably lowered by giving therapy with METD and glibenclamide (p < 0.001) (Figure 17 and Table 8).
Table 8: Effect of METD on serum glucose levels.
Day NC DC METD 200 METD 400 Standard
0 96 ± 7.8 280 ± 9.4 276 ± 9.6 282 ± 7.3 282 ± 11
7 96 ± 7.9 288 ± 9.1α 256 ± 8.7 242 ± 8.1b 222 ± 7.5a
14 98 ± 8.1 295 ± 9.5α 246 ± 8.3b 200 ± 6.6a 137 ± 5.0a
21 100 ± 6.8 301 ± 9.6α 242 ± 9.6b 178 ± 12a 124 ± 8.3a
28 101 ± 6.7 308 ± 9.3α 183 ± 6.9a 159 ± 14a 107 ± 8.1a
αp < 0.001 correlate to the NC group; ap < 0.001, and bp < 0.01, when analyzed to the DC group.
Effect of METD on lipid parameters
Triglyceride levels
The serum triglyceride range of the NC group was 68 ± 3.7 mg/dL, which (p < 0.001) increased to 124 ± 3.3 mg/dL in the DC group (Table 9). The growth serum triglyceride level was decreased by treatment with METD 200, METD 400, and glibenclamide (p < 0.05, p < 0.01, and p < 0.001) (Figure 18 and Table 9).
Cholesterol levels
The serum total cholesterol level of the NC group was 77 ± 3.8 mg/dL, which was importantly (p < 0.001) raised to 157 ± 6.5 mg/dL compared NC group. These increased levels were lowered by therapy with METD 400 and glibenclamide (p < 0.01 and p < 0.001) while METD 200 exhibited no decrease in total cholesterol levels when correlated to the DC group (Figure 19 and Table 9).
HDL-c levels
Diabetes induction produced visible (p < 0.001) decline in serum HDL-c levels by 27 ± 1.5 to 11 ± 1.0 mg/dL when related to the NC group. METD 200, METD 400, and glibenclamide when treated created increase in serum HDL-c range (p < 0.01, p < 0.001, and p < 0.001), when compared to DC group (Figure 20 and Table 9).
Table 9: Effect of METD on serum lipid profile.
Parameters NC DC METD 200 METD 400 Standard
TG (mg/dL) 68 ± 3.7 124 ± 3.3α 104 ± 5.1c 97 ± 4.5b 80 ± 3.7a
TC (mg/dL) 77 ± 3.8 157 ± 6.5α 143 ± 3.6 130 ± 3.7b 86 ± 3.8a
HDL (mg/dL) 27 ± 1.5 11 ± 1.0α 18 ± 0.95b 21 ± 1.1a 26 ± 1.0a
αp < 0.001, it is correlate to the NC category; ap < 0.001 and bp < 0.01, cp < 0.05 when matched to DC batch.
Effect of METD on Liver Parameters
Effect of extract on the aspartate transaminase (AST) activity
Initiation of diabetes made a significant (p < 0.001) increase in liver AST levels from 42 ± 3.7 U/L to 168 ± 7.0 U/L when correlated to the NC group. The AST levels were lowered by treatment with METD 200, METD 400, and glibenclamide compared to DC group (p < 0.05, p < 0.001, and p < 0.001) (Figure 21 and Table 10).
Effect of extract on the alanine aminotransferase (ALT) activity
Induction of diabetes leads to (p < 0.001) increased liver ALT that ranges from 19 ± 2.8 U/L to 167 ± 5.1 U/L relative to NC group (Table 10). ALT levels were dropped by therapy with METD 400 and glibenclamide (p < 0.001), whereas METD 200 didn't showed any decrease in the ALT levels comparatively to the DC category (Figure 22 and Table 10).
Table 10: Effect of METD on liver parameters.
Group AST (U/L) ALT (U/L)
NC 42 ± 3.7 19 ± 2.8
DC 168 ± 7.0α 167 ± 5.1α
METD 200 144 ± 6.5a 153 ± 4.8b
METD 400 113 ± 6.4a 129 ± 5.6a
Standard 56 ± 3.5a 56 ± 4.8a
αp < 0.001, when compared to NC group; ap < 0.001 and bp < 0.01, when correlate to DC category.
Table 11: Effect of METD on Behavioral Parameters in Morris Water Maze (Figure 23).
Groups Escape Latency (s) TSTQ
Day 1 Day 2 Day 3 Day 4 Day 5
NC 75 ± 0.88 68 ± 1.5 58 ± 0.76 45 ± 1.5 35 ± 0.96 26 ± 1.5
DC 83 ± 1.2α 82 ± 1.2α 78 ± 1.6α 81 ± 1.5α 72 ± 1.6α 5.7 ± 0.88α
METD 200 79 ± 0.71c 80 ± 0.54 77 ± 1.4 67 ± 2.6a 61 ± 0.80a 9 ± 0.58
METD 400 77 ± 0.67b 76 ± 0.76c 70 ± 1.5b 55 ± 2.0a 47 ± 2.0a 16 ± 1.2b
Standard 75 ± 1.3a 68 ± 1.2a 60 ± 1.7a 47 ± 0.39a 35 ± 1.2a 24 ± 2.5a
αp < 0.001 correlate to the NC group; ap < 0.001, bp < 0.01, and cp < 0.05, relative to DC batch.
Effect of METD on EPM (Figure 24) The DC group had a longer transfer delay than control group (p < 0.001). The transfer latency was reduced by METD 400 and Standard group against the DC group (p < 0.01 and p < 0.001 respectively), METD 200 did not show any decrease in transfer latency.
Table 12: Effect of METD on TL on EPM test.
Group TL (sec) on the 0th day TL (sec) on 28th day
NC 23 ± 1.4 18 ± 1.5
DC 22 ± 1.1 28 ± 1.9α
METD 200 22 ± 1.0 24 ± 1.5
METD 400 23 ± 1.2 19 ± 1.2b
Standard 20 ± 0.95 18 ± 1.0a
αp < 0.001, relative to the NC batch; ap < 0.001, bp < 0.01, and cp < 0.05, relative to DC group.

Effect of METD on the brain AChE levels (Figure 25)
AChE levels increased in the DC group after induction of Alzheimer's disease compared to the NC group (p < 0.001). Relative to the NC batch, AChE levels decreased after treatment with METD 400 and Standard (p < 0.05 and p < 0.001), but not after treating with METD 200 compared to the NC group.
, Claims:I/WE CLAIM
1. A method of evaluating the neuro-protective properties of a plant extract comprises the steps of:
a) collecting the leaves of Trianthema decandra;
b) preparing a fine powder once the leaves are totally dried;
c) preparing the extract of shade-dried powder;
d) isolating and identifying the chemical constituents; and
e) characterizing using various techniques, including UV-visible spectrophotometry, FT-IR, H-NMR, and Mass spectroscopy.
2. The method as claimed in claim 1, wherein the solvents used for extraction are methanol, pet ether, water, and chloroform.
3. The method as claimed in claim 1, wherein quercetin and phytol are isolated, segregated and characterized.
4. The method as claimed in claim 1, wherein methanol and hydroalcoholic extracts have shown the strongest α-amylase and α-glucosidase inhibitory activity followed by chloroform extract.
5. The method as claimed in claim 1, wherein the neuro-protective ability of Trianthema decandra is evaluated in hyperglycemia-related cognitive impaired rats.
6. The method as claimed in claim 1, wherein the screening of the neuroprotective effects of the said composition in hyperglycemic rats is done by using Morri's water (MWM) and elevated plus maze (EPM) model.
7. The method as claimed in claim 1, wherein the protective actions of T. decandra on diabetic dysfunction may be attributed to its multiple pleiotropic effects like antihyperglycemic and anticholinesterase activity.
Dated this th November, 2024


Saurabh Kumar Jain
(IN/PA-3637)
Agent for the Applicant

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