Evaluation of EETPAE from Tridax procumbens and Andrographis echioides in The Management of Diabetic Neuropathy

Long standing diabetes mellitus leads to multiple organ damage and it is associated with an increased prevalence of micro vascular disease (nephropathy, neuropathy& retinopathy) and macro vascular diseases (peripheral vascular disease, ischemic heart disease& stroke). Poor glycemic control, a factor that has been observed in the Indian population with diabetes put them at risk of complication including neuropathy-24.6%, cardiovascular disease - 23.6%, kidney problem - 21.1%, retinopathy- 16.6% and foot ulcer- 5.5% 4.  Globally diabetic neuropathy affects approximately 132 million people as of 2010 (1.9% of the population). Diabetes is the leading known cause of neuropathy in developed countries, and neuropathy is the most common complication and greatest source of morbidity and mortality in diabetes.

It is believed that neuropathy affects approximately 25% of individuals diagnosed with diabetes. Diabetic neuropathy is involved in 50–75% of amputations that are not caused by trauma. The primary cause of diabetic neuropathy is high blood sugar. In the DCDT (Diabetes Control and Complications Trial, 1995) study, the yearly occurrence of neuropathy was 2%, but it significantly decreased to 0.56% with intensive treatment of type 1 diabetics. The advancement of neuropathy is influenced by the level of blood sugar control in both type1 and type2 diabetes. The length of time a person has diabetes, their age, smoking habits, blood pressure, height, and high levels of lipids in the blood are all factors that increase the risk of developing diabetic neuropathy. Diabetic neuropathy is a condition that occurs when nerves are harmed as a result of prolonged high blood sugar levels or hyperglycemia, which is a complication of diabetes. Diabetic neuropathy can impact various body parts, such as the legs, feet, bladder, heart, gastrointestinal system, and reproductive system.

Diabetic neuropathy typically progresses gradually over several months, caused by long-term elevated blood sugar levels that harm the nerves in the body. Diabetic neuropathy can manifest as a range of sensations, such as pain, numbness, tingling, or prickling, which typically starts in the feet. In advanced stages of diabetic neuropathy, the hands can also be impacted. In certain instances of diabetic neuropathy, the abnormal sensations can also affect the arms, legs, and torso. Several medications are used to relieve nerve pain, but they don't work for everyone and most have side effects that must be weighed against the benefits they offer.

Fig: 1 Diabetic Neuropathy

2. PLANT PROFILE:

2.1 TRIDAX PROCUMBENS L:

Tridax procumbens, commonly called coat buttons or tridax daisy, maybe a species of flowering plant within the daisy family. It's best called a widespread weed and pest plant. Tridax procumbens is traditionally used ayurvedic medicine for hair growth, liver disorder, diabetic and is sometimes dispensed in situ of Bhringraj. The current study was aimed to assess the efficacy of Tridax procumbens’s use in hair growth-promoting activity.

Fig: 2 Plant Profile of Tridax Procumbens L

2.2 ANDROGRAPHIS ECHIOIDES NEES:

Scientific classification of plant can be defined as “The arrangement of entities of that plant” in a hierarchical series of nested classes, in which similar or related classes at one hierarchical level are combined comprehensively into more inclusive classes at the next higher level. The scientific classification of Andrographis echioides is as follows.

Fig: 3 Plant Profile of Andrographis echioides

3. MATERIALS AND METHODS:

3.1 COLLECTION OF SELECTED HERBS:

Tridax procumbens L, Andrographis echioides L collected from in around Thiruvannamalai district, Tamilnadu. Collected herbs were authenticated by SIDDHA CENTRAL RESEARCH INSTITUTE, Arumbakkam, Chennai.

• AUTHENTICATION CERTIFICATE FOR 1272.29042518-19
• Form No: PCOG002-ACF
• Date: 21-05-2025
• Tridax procumbens L  (Code – T29042518P) (Part – Whole Plant)
• Andrographis echioides (L.) Nees (Code – A29042519E) (Part – Whole Plant)

3.2 EXTRACTION:

The whole plant of Tridax procumbens L, Andrographis echioides L, were dried under shade and then powdered with a mechanical grinder. The powder was passed through sieve No:40 and stored in an airtight container for further use.

Preparation Of Ethanolic Extract: Collect dryed plant of Tridax procumbens L, Andrographis echioides L, were cleaned with water & shade dried until a constant weight was obtained & subsequently powdered & sieved mash no 40. Powdered material 5kg was extracted with of water at 50 degrees in soxhlet apparatus 1L for 72hr. dark brown semi – solid residues. 525g was obtained by evaporating the aqueous extract under reduced pressure.

3.3 PHYTOCHEMICAL TEST:

The aqueous extracts of Tridax procumbens L, & Andrographis echioides L, were subjected to the following preliminary phytochemical analysis

3.4 ACUTE TOXICITY STUDIES:

The procedure was followed by using OECD guidelines (Organization for Economic Co-operation and Development) 423 (acute toxic class method). The acute toxicity study is a Step wise procedure with 3 animals of single sex per step. Depending on the mortality and/or Morbidity status of the animals, on the average 2-4 steps may be necessary to allow judgment on The acute toxicity of the substances. This procedure results in the use of a specified number of Animals while allowing for acceptable data-based scientific conclusion. Albino mice weighed 25-30gms were used for the study.  Body weights of mice before and after administration were observed for morbidity and Mortality. Any changes in skin, fur, eyes, mucous membrane, respiratory, circulatory, autonomic & central nervous system, motor activity and behaviour pattern were observed and also sign of Tremors, convulsion, salivation, diarrhoea, lethargy, sleep and coma were noted.

3.5 EXPERIMENTAL DESIGN:

After six weeks of verifying hyperglycemia in mice, the diabetic-induced mice were assessed for peripheral diabetic neuropathy using von Frey filaments (a tactile allodynia test) and the hot plate test at 55°C (a thermal hyperalgesia test). Once neuropathy development was confirmed, the diabetic mice were distributed into several groups, designated as Group II through, each comprising 5 animals (n=5).

• Group-I (Control group) (n=5) receives normal saline (0.9% Nacl)
• Group-II (Negative Control group) (n=5) receives Streptozotocin (STZ) 45 mg/kg/day for five consecutive days + receives normal saline for 30 days after confirming diabetic neuropathy.
• Group-III (Standard group) (n=5) receives Streptozotocin (STZ) 45 mg/kg/day for five consecutive days + Duloxetine (30 mg/kg/day) for 30 days after confirming diabetic neuropathy.
• Group-IV (Low dose group) (n=5) receives Streptozotocin (STZ) 45 mg/kg/day for five consecutive days + Low dose (250 mg/kg/day) EETPAE (Ethanolic Extract of Tridax procumbens & Andrographis echioides) for 30 days after confirming diabetic neuropathy.
• Group-V (High dose group) (n=5) receives Streptozotocin (STZ) 45 mg/kg/day for five consecutive days + Low dose (500 mg/kg/day) EETPAE (Ethanolic Extract of Tridax procumbens & Andrographis echioides) for 30 days after confirming diabetic neuropathy.

3.6 PHARMACOLOGICAL ACTIVITY:

• Hot Plate Test
• Cold Plate Test
• Von Frey Filament Test
• Formalin Test
• Superoxide Dismutase (SOD) Activity
• Catalase (CAT)
• Glutathione Reductase (GSH)
• Blood Glucose Level
• Free Fatty acid
• Total Cholesterol Level

4. RESULTS AND DISCUSSION:

4.1 EXTRACTION:

Table: 1 Extraction

Fig: 4 Extraction Of EETPAE

4.2 PHYTO-CHEMICAL TEST:

Table: 2 Phyto-Chemical Test

Flavonoids                                 Tannins                                     Phenol

Glycosides                              Terpenoids                              Alkaloids

Fig: 5 Phyto – Chemical Test

4.3 ACUTE ORAL TOXICITY STUDIES OF EETPAE (OECD 423 GUIDELINES):

Table: 3 Acute Oral Toxicity Studies

4.4 PHARMACOLOGICAL ACTIVITY:

4.4.1 Assessment of Blood Glucose level & Thermal Hyperalgesia:

Table: 4 Assessment of Blood Glucose level & Thermal Hyperalgesia

Fig: 6 Assessment of Blood Glucose level & Thermal Hyperalgesia

4.4.2 Effect of EETPAE on Cold Plate Test & Mechanical Hyperalgesia:

Table: 5 Effect of EETPAE on Cold Plate Test & Mechanical Hyperalgesia

Fig: 7 Effect of EETPAE on Cold Plate Test & Mechanical Hyperalgesia

4.4.3 Effect of EETPAE on Formalin I & II Test:

Table: 6 Effect of EETPAE On Formalin Test- I

Fig: 8 Effect of EETPAE On Formalin Test- I

Table: 7 Effect of EETPAE On Formalin Test- II

Fig: 9 Effect of EETPAE On Formalin Test- II

4.4.4 ASSESSMENT OF BIOCHEMICAL ANALYSIS:

Table: 8 Assessment of Biochemical Analysis

Fig: 10 Assessment of Biochemical Analysis

4.4.5 HISTOPATHOLOGY:

Group I: Control Group: Normal histological architecture. The sciatic nerve tissue showed no pathological alterations. The axons, myelin sheath, and connective tissue appeared Represents the baseline healthy condition, serving as a standard for comparison with other groups.          

Group II: Diabetic Control Group: Significant degeneration of nerve fibers, axonal swelling, and vacuolization. The sciatic nerve showed marked demyelination and inflammatory infiltration, which are characteristic of diabetic neuropathy. Indicates the damaging effect of untreated diabetes on peripheral nerves.

Group III: Standard Treated Group: Improved nerve architecture compared to Group II. Reduced degeneration and partial restoration of nerve fibers. Treatment with the standard drug ameliorated the diabetic nerve damage to some extent, preserving nerve fiber integrity. Confirms the efficacy of standard treatment in reversing or preventing diabetic-induced nerve damage.

Group IV: Low Dose EETPAE Treated Group (250 mg/kg): Mild regeneration of nerve fibers with moderate reduction in vacuolization and axonal damage. The low dose of the test compound (EETPAE) shows a protective effect on the sciatic nerve, though less pronounced than the standard drug. Indicates potential neuroprotective action at a lower dose, but not completely restorative.

Group V: High Dose EETPAE Treated Group (500 mg/kg): Significant regeneration of nerve fibers, near-normal histology. The high dose of EETPAE demonstrated strong neuroprotective and regenerative effects, showing better results than the low dose. Suggests a dose-dependent effect of EETPAE in mitigating diabetic neuropathy.

GROUP - I                                                             GROUP - II

GROUP – III                                                          GROUP – IV

GROUP – V

Fig: 11 Histopathology