Journal of Pharmacognosy and Phytochemistry 2020; 9(5): 1349-1352

E-ISSN: 2278-4136
P-ISSN: 2349-8234
www.phytojournal.com Protective effect of ENervin on diabetic
JPP 2020; 9(5): 1349-1352
Received: 15-07-2020 neuropathic condition in Schwann cells
Accepted: 22-02-2020

Subashri Beulahpriyadarsini B Subashri Beulahpriyadarsini B and Mallika Jainu

NRight Nutrition Private
Limited, Guindy, Tamil Nadu,
India
Abstract
Treatment of Diabetic Neuropathy depends on three main approaches: risk factor management and
Mallika Jainu intensive glycaemic control and symptomatic pain management. It’s a type of nerve damage caused by
Bright Care Research Centre Pvt long-term high blood sugar levels. ENervin is a polyherbal formulation very effective to rejuvenate
Ltd., Cennai, Tamil Nadu, India nervous system and used for the therapy of Diabetic Neuropathy. Thus, a preliminary study has been
planned to observe and evaluate the effect of ENervin on diabetic neuropathy. The ENervin tablet shows
the presence of five important compounds like ar-tumerone, stigmasterol, gingerol, lupeol and trilaurin
which was confirmed by HPLC analysis. In this experiment, different concentrations of ENervin
decreased the Schwann cells in diabetic condition and its beneficial action was compared with
Pregabalin, an oral capsule used to treat neuropathic pain. Future preclinical and clinical research of
ENervin may help us to strongly support the usage of this polyherbal medicine for the treatment of pain
in diabetic neuropathy.

Keywords: ENervin, pregabalin, diabetic neuropathy, phytochemicals

Introduction
Diabetic Neuropathy (DN) is the damage to automatic, motor and/or sensory nerves that result
from metabolic or vascular derangement in patients with long standing Diabetes mellitus [1].
The relationships among Schwann cells, axons, and the perineurial barrier emphasize the key
role Schwann cells plays normal functions of the nerve. Schwann cells are responsible for
action potential velocity through insulation of axons, maintenance of axonal calibre, and
correct localization of Na+ channels; immunological and functional integrity of the nerve
through the perineurial blood-nerve-barrier; and effective nerve regeneration. In diabetic
neuropathy, many of these facets of nerve function are defective. The Schwann cells were
selected for experimental DN model, this may be due to the apparent localization of aldose
reductase enzyme in Schwann cells. Thus, measurements derived from whole nerve were
largely assumed to relate to Schwann cells [2]. Although increasing knowledge of the
complexity of the etiology of diabetic neuropathy has highlighted the need for in vitro studies,
much of this work has yet to be done. The present study attempts to find the role of ENervin
on Schwann cells in diabetic neuropathy condition. The most common form of neuropathy in
diabetes is known as distal symmetrical polyneuropathy: it can act on all types of neurons, but
affects those with longest axons first. Distally innervating neurons must maintain a larger
cellular volume (and hence higher metabolic efficiency) [3]. This also implies a greater
dependence on Schwann cells and target tissues for mechanical and neurotrophic support.
Therefore, these neurons are susceptible to defects in support cells [4]. Patients present with
numbness, tingling, and/or pain that typically started in their toes and slowly spreads
proximally. Painful diabetic neuropathy (PDN) is a debilitating consequence of diabetes that
may be present in as many as one in five patients with diabetes. The objective assessment of
PDN is difficult, making it challenging to diagnose and assess in both clinical practice and
clinical trials. No single treatment exists to prevent or reverse neuropathic changes or to
provide total pain relief [5].
There’s no cure for diabetic neuropathy, but studies showed that treatment can slow its
progression. Keeping your blood sugar levels within a healthy range is the best way to
decrease the likelihood of developing diabetic neuropathy or slow its progression. There is a
growing need for studies to evaluate the most potent drugs or combinations for the cure of DN
to improve the pain relief and quality of life. Herbal formulations are found to be very
Corresponding Author: effective to rejuvenate nervous system and in the management of diabetic neuropathy [6]. Thus,
Subashri Beulahpriyadarsini B the present study was conducted to find the protective effect of ENervin on diabetic
NRight Nutrition Private
Limited, Guindy, Tamil Nadu,
neuropathic conditions in Schwann cells.
India
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Materials and Methods Cell viability by MTT assay

Chemicals The Schwann cells were cultured in 96-well tissue culture
HPLC grade methanol, acetonitrile, acetic acid were plates for the assay. The microplates filled with cells (100 μl)
purchased from Merck Laboratories. Ultra deionized water with a density of 3×105 were taken as negative control. The
utilized in the study was obtained from Milli-Q water cells were observed for 24 hours, and the growth medium by
purification system. The reference compounds were means of micropipette and the monolayer of cells rinsed twice
purchased from Sigma Aldrich company. Their purities were with Minimum Essential Media (MEM) devoid of Fetal
analyzed to be all above 98% by HPLC. Bovine Serum (FBS) for removing dead cells and extra FBS.
1ml of medium (without FBS) having various concentration
Chromatographic conditions of ENervin solution (1-5.0mM/ml) were added on to
The separation of phytoconstituents was implemented on a respective wells; 20 μl of MTT (5 mg/ml in PBS) were added
Shimadzu 2695 HPLC system (Shimadzu). The qualitative on to every well, and the cells kept in 5% CO2 incubator for
analysis was carried out on A Zorbax SB-C18 column (250 6-7 hrs. After removing the medium, DMSO (1ml) was added
mm×4.6 mm i.d., 5 µm). The column temperature was set at on to every well and Pregabalin (1mg/ml) was tested as a
35◦C. The mobile phase was composed of acetonitrile (A) and comparison standard drug. Propanol (50 µl) was added to the
water containing 0.4% (v/v) acetic acid (B). pellet and the plates were mixed gently to solubilize the
The gradient program was as follows: 45% A at 0–13 min, molded formazan product. The plates were kept on a shaker
45–56% A at 13–16 min, 56% A at 16–50 min, and 56–100% for 15 min and the absorbance was measured on an enzyme-
A at 50–55 min. The re-equilibration duration was 10 min linked immunosorbent assay (ELISA) (MINDRAY90) reader
between individual runs. The flow rate was kept at 1.0 at 570 nm. Each experiment was done in triplicate and the
mL/min. The injection volume was 10 µL. An online IC50 of the test samples as the percentage survival of the cells
detection wavelength was selected at wavelengths of 240 nm was calculated.
and 430 nm.
Statistical analysis
Standard solution Results were measured as mean ± S.E.M. Statistical
The ar-tumerone, stigmasterol, gingerol, lupeol and trilaurin significance was resolute by one-way analysis of variance
compounds were weighed and dissolved in methanol. The (ANOVA) and post hoc least-significant difference test by
mixed standard solution containing all reference compounds SPSS software (version 22.0). P values less than 0.05 was
was prepared in a 10 mL volumetric flask, diluting with considered significant
methanol, and stored at 4◦C. Subsequently, the stock solution
was further diluted with methanol to obtain a series of Results and discussions
concentrations of working solutions to establish calibration DN is associated with significant impairment in the nervous
curves. system of diabetic patients [7]. For the cure of DN, the
ENervin tablets can be given as a therapeutic drug for
Validation patients. So therefore, there is a need for the scientific
The HPLC method described in this article was validated justification for the usage of this drug clinically. The ENervin
interms of linearity, sensitivity, precision, stability, and tablet shows the presence of five important compounds such
accuracy. Six different concentrations of working solutions as ar-tumerone, stigmasterol, gingerol, lupeol and trilaurin
were analysed in triplicate to establish calibration curves. The along with few more compounds which was confirmed by
calibration curves of various analytes were constructed by HPLC technique (Figure 1; Table 1) and these
plotting the mean peak areas vs. the concentration of the phytoconstituents are very important for the cure of diabetic
reference compounds. The limits of detection (LOD) and neuropathic condition [8]. The early reports demonstrated that
quantification (LOQ) were determined by injecting a series of morphological changes in Schwann cells in human diabetic
dilute standard solutions until a signal-to-noise ratio (S/N) of neuropathy are now supported by an increased awareness of
3 and 10 was obtained, respectively. The precision test was molecular alterations in Schwann cells during diabetes.
performed by the measurements of intra- and inter-day Schwann cells express a wide range of receptors and, when
variability. For the intra-day precision test, the test samples they sense insults or danger signals, they increase the
were analyzed for replicates within one day, while, for the synthesis and secretion of factors that stimulate
inter-day precision test, the samples were examined in neuroprotection, regrowth and remyelination, or factors that
duplicates for three consecutive days. Quantities of the aggravate disease phenotypes [9].
analytes were calculated from their corresponding calibration
curves. The recovery was calculated with the following
equation: Recovery (%) = (amount determined−amount
original)/amount spiked×100%.

Cell culture
Schwann cells (ATCC: CRL-2765) were purchased and
cultured in T75 flasks with DMEM (containing 4 mM 1-
glutamine, 1.5 g/L sodium bicarbonate, and 5.5 mM glucose),
supplemented with 100 U/ml penicillin, 100 mg/ml
streptomycin, and 10% fetal bovine serum in an incubator.
Schwann cells were cultured for 48 h under hyperglcemic
conditions with or without ENervin treatment and then
Fig 1: HPLC analysis of ENervin
collected for further analysis.

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Table 1: Various phytoconstituents present in the ENervin

PDA Ch1 254nm
Peak# Ret. Time Area Height Conc. Unit Mark Name
1 2.602 5.91225 381166 29.365 M Beta-sitosterol
2 2.696 2182441 315547 12.588 VM Trilaurin
3 2.982 3125403 211291 18.027 VM Lupeol
4 3.383 444366 51519 2.563 VM Gingerol
5 3.656 1068365 155033 6.162 M Difenoxin
6 3.824 187589 21625 0.630 VM Stigmasterol
7 4.021 4558567 22419 1.082 M Difenoxin
8 4.863 375251 591115 26.293 M n-hexadecanoic acid
9 5.5567 975251 47326 2.164 M ar-tumerone
10 6.520 195201 25818 1.126 M Phthalic acid esters
Total 17337647 1822860

The most recent studies have demonstrated that Schwann cells pathogenic process in DN. The vascular mechanism of the
regulate many aspects of axonal function, so that disruption of neuropathy involves impaired endoneurial blood flow to
their metabolism by diabetes results in the accumulation of peripheral nerves leading to the destruction of neuronal and
neurotoxic intermediates and compromises production of Schwann cells (SCs).
neuronal support factors, contributing to axonal degeneration, Actually the destructed Schwann cells in neuropathic
endothelial dysfunction and diabetic neuropathy. The earliest condition were reduced after ENervin treatment similar to that
descriptions of pathology in diabetic neuropathy indicated of standard control, which proves that ENervin formulation
that schwann cell neuropathy accompanied axonal showed a positive effect on injured nerve cells in DN patients
[12]
degeneration. . In this experiment, the schwann cells treated with
The majority of clinical and basic research in diabetic ENervin concentration at 10mM showed 35.6% glucose
neuropathy since then has focused on the effects on neurons. destructed nerve cells viability whereas the schwann cells
However, accumulating data from research into the cultured in glucose condition treated with pregabalin showed
development and regeneration of the PNS has identified 28.7% cell viability (Table 2, Figure 2).
Schwann cells as equally indispensable components that The diabetic injured schwann cells without treatment showed
maintain neuronal structure and function, nourish axons and 100% viability. Therefore ENervin showed 64.4% protection
promote survival and growth upon injury [10]. whereas pregabalin showed 71.3% against diabetic
Treatment which repairs nerves has yet to be found and neuropathy condition.
translated into clinical trials and eventually approved therapy
in clinical practice. Whilst a number of treatment options exist Table 2: The MTT assay results of Schwann cells treated with
and various guidelines and algorithms have been formulated, ENervin
none are satisfactory. Treatment Conc (mM) Cell viability (%)
Various symptomatic treatments have been proposed to SC untreated cells - 100.0 ± 8.4
manage neuropathic pain but few have been found to be SC + EN 1 74.1 ± 5.8
effective, with only three medications currently FDA SC + EN 2.5 60.8 ± 4.1
approved for DN [11]. The effect of ENervin on Schwann cells SC + EN 5.0 51.4 ± 4.5
(cell line for diabetic neuropathy research) was done in SC + EN 10.0 35.6 ± 2.7
comparison with the standard pharmacological drug SC + PG 1mg 28.7 ± 1.4
Pregabalin. Values are expressed as Mean ± SEM (n=3).SC-Schwann cells;
High glucose can induce oxidative stress and inflammatory SC+EN – Schwann cells +ENervin; SC+PG - Schwann cells +
responses to activate the Toll-like receptor 4 (TLR4)/nuclear Pregabalin, The IC50 of the ENervin test product against SC cells is
4.9mM.
factor-κB (NF-KB) signal pathway, which plays an important

Fig 2: Effect of various concentration of ENervin on Schwann cells in comparison with pregabalin Results were expressed as Mean ± SEM
(n=3). *p< 0.05; **p< 0.01; ***p< 0.001 statistically significant as compared with SC untreated group.

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The inhibition of destructed Schwann cells by Enervin

Tablets, one among the herbal formulation is found to be very
effective to rejuvenate nervous system and in the treatment of
Diabetic Neuropathy. This curative action may be due to the
important phytoconstituents which plays an important role for
the diabetic neuropathy cure which was already proved by the
researchers.

Conclusion
This polyherbal formulation showed a significant protection
against diabetic neuropathy condition and this effect was
comparable with that of Pregabalin. Therefore, further
preclinical and clinical studies required to support the ongoing
trial of ENervin on diabetic neuropathic patient.

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