Abstract:

Ethnopharmacological relevance

Paederia foetida Linn. (Family: Rubiaceae) is widely used as natural remedy for diabetes mellitus by
the Nepali and Lepcha tribes of Sikkim and Darjeeling Himalayan region. The plant is administered to
a diabetic individual in the form of leaf infusion for 2-3 weeks. Therefore, we investigated the effects
of methanolic leaf extract of Paederia foetida (MEPF) on alloxan (ALX) induced diabetic renal
oxidative stress and NF-kB dependent renoinflammatory events in rat

1. Introduction

Diabetes mellitus (DM) is an increasingly global health problem today. In 2013, 382 million people
suffered from DM around the globe and type 2 accounts for 90% of the cases with no gender
disparity (Shi and Hu, 2014; Vos et al., 2013). The global diabetic population is projected to increase
to 439 million by 2030; which is equal to 7.7% of total adult population (Chen et al., 2012). DM is
characterized by altered glucose, lipid and protein metabolism. The altered metabolism results in
microvascular complications; notably including nephropathy, neuropathy and retinopathy (Giacco
and Brownlee, 2010; Kitada et al., 2010). One third of type 2 diabetics suffer from nephropathy and
have a high cardiovascular mortality and morbidity (Thomas and Karalliedde, 2015). Diabetic
nephropathy (DN) is the single most important cause of end stage renal disease (ESRD) worldwide
and contributes to 40% of the new cases in both Asian and Western countries (Keane et al., 2003).
Studies on DN, revealed that oxidative stress on kidney and chronic inflammation; both equally
contribute to its development and progression (Brownlee, 2005). NF-kB linked inflammatory cascade
is reported to have key role in the pathogenesis of DN (Sun et al., 2013).

Currently, more than 2/3rd of world’s diabetics are dependent on plant based medicine for their
successive therapy due to its lesser side effects and low coast (WHO, 2008). Paederia foetida Linn.
(Family: Rubiaceae) is a wildly grown woody climber of north-east India. The leaves of the plant are
taken as infusion to treat DM by the tribes of Sikkim and Darjeeling Himalayan region. The therapy is
given usually for a period 2-3 weeks (Chanda et al., 2013;

Chhetri et al., 2005). The vernacular names of the plant include Bhedai lata (Assamese),
Gandhabhadulya (Bengali), Gandhali (Hindi), Birilahara (Nepali), Gandhaprasarani (Sanskrit) and
Skunkvine or Chinese fever vine (English). The plant is found in central and eastern Himalayas,
Malaysia, Philippines, Malacca and India. In India, it is found mainly in Assam, Bengal, Bihar,
Meghalaya, Orissa and Sikkim (Chanda et al., 2013; De et al., 1994; Fazlin et al., 2002). The aerial
parts of the plant are very rich in paederine, paederenine, paederone, paederolone and iridoid
glycosides: asperuloside, scandoside and paederoside. An enzyme present in it, responsible for
splitting paederoside and gives its characteristics bad odour when plant is bruised (Shukla et al.,
1976; Sugumaran et al., 2005). The plant is also a rich source of friedelin, ursolic acid, ellagic acid,
vitamin C, carotene, lupeol, palmitic acid and methyl mercaptan (Soni et al., 2013). In traditional
medicine the plant is indicated for the treatment of rheumatism (Charak, 1949), difficulty in labour
(Pandey, 1952), vata disorders, piles, oedema and night blindness (Bhattacharya and Bhattacharya,
1933; Dutta, 1902; Krishna-Shastri, 1936). Leaf extract of the plant is reported to possess significant
antihyperglycaemic (Kumar et al., 2014; Morshed et al., 2012), antioxidant (Kumar et al., 2014;
Osman et al., 2009) and lipid lowering effects (Kumar et al., 2014). A very recent study revealed that
the plant effectively suppress NF-kB in animal model (Kumar et al., 2015). Based on above findings,
we hypothesized that if Paederia foetida (P. foetida) extract can inhibit NF-kB and hyperglycaemia in
vivo, then ALX induced diabetic rat with nephropathy to investigate whether alcoholic leaf extract of
the plant affect the NF-kB mediated diabetic renoinflammatory cascades.

2. Materials and method

2.1 Drugs and Chemicals

Glibenclamide (Cat. no. D3525) and Alloxan monohydrate (Cat. no. A7413) were purchased from
Sigma Aldrich, St. Louis, MO, USA; and Methanol (Cat. no. 106009) from Merck, Darmstadt,
Germany. Carboxymethylcellulose Sodium (Cat.no. C9481) Sodium citrate (Cat. no. PHR1416), Citric
acid (Cat. no. C2219000), Phosphate buffer (Cat. no. P5244), Sodium chloride (Cat. no. 746398),
Ellman’s reagent (Cat. no. D8130), Thiobarbituric acid (T5500), Lowry reagent (Cat.no. L3540), SOD
estimation kit (Cat. no. 19160), Catalase estimation kit (Cat. no. CAT100) and Glutathione (Cat. no.
Y0000517) were procured from Sigma Aldrich Corporation. Vettest strips were purchased from IDEXX
laboratories, Westbrook, USA. ELISA kits for estimation of pro-inflammatory cytokines and NF-kB
were purchased from Life technologies, CA, USA, (TNF-α, catalog no. KRC3011) and Elabscience
Biotechnology, Wuhan, China (IL-1β; Cat. no. E-EL-R0012, IL-6; Cat. no. E-EL-R0015 and NF-kB, Cat.no.
E-EL-R0674).

2.2 Plant materials

Paederia foetida Linn. was collected from Sivasagar district, Assam, during the month of August-
September and authenticated in Govt. Ayurvedic college, Guwahati, India. A voucher specimen (no.
NIPER/PCT/PTH/01) of the same was deposited in the herbarium of Botanical Survey of India.

2.3 Method of extraction

The leaves were separated from the other plant materials and dried under shade. The dried leaves
were grounded into coarse powder using a mechanical grinder. Then the powders were soaked in
methanol for 7-days and filtered through a muslin cloth. The filtrate obtained was evaporated in a
rotatory evaporator (IKA RV10, Malaysia) to get methanolic leaf extract of Paederia foetida (MEPF).
The recovery was 17.83% w/w (calculated for dried leaves). Further, the extract was freeze dried to
remove the residual water content. Then it was suspended in 0.2% w/v carboxymethylcellulose
(CMC) to make a uniform suspension and stored in 2-8οC.

2.4 Animals and induction of diabetes

Albino rats (wistar strain), sex male, weighing around 160-180gm were procured from College of
Veterinary Science (CVSc.), Khanapara, Guwahati and acclimatized to laboratory conditions
maintained at (25 ± 2)οC and 12-hours light-dark cycle for two weeks. The rats were provided with
normal pellet diet and tap water ad libitum. After two weeks, rats were grouped in such a way that
the variations in weight among the experimental groups are minimized. The overnight fasted
animals, except the normal control group, were administered with single intraperitoneal (i.p.) dose of
alloxan (ALX), 150 mg/kg body weight (b.w.), in 0.1M citrate buffer, pH 4 (Abdul-Hamid and
Moustafa, 2014; Ezejiofor et al., 2014). ALX administered rats were allowed to drink 5%w/v glucose
water for 24-hours to prevent acute hypoglycaemia. Fasting blood sugar (FBS) level was measured
after 7-days using an Accucheck glucometer from Roche diagnostic, India (Belhekar et al., 2013). Rats
with blood sugar level ≥ 200 mg/dL were considered diabetic and included for further study. Before
starting the experiment, ethical clearance was taken from Institutional Animal Ethical Committee
(IAEC), Gauhati Medical College & Hospital, Guwahati, Assam (approval no. MC/05/2015/55).

2.5 Experimental design

Group I: Non-diabetic rats receiving 0.2% (w/v) aq. solution of CMC/day for 28-days (p.o.);

Group II: Diabetic rats receiving no treatment;

Group III: Diabetic rats receiving MEPF at 250 mg/kg b.w./day for 28-days (p.o.);

Group IV: Diabetic rats receiving MEPF at 500 mg/kg b.w./day for 28-days (p.o.) and

Group V: Diabetic rats receiving Glibenclamide at 10 mg/kg b.w./day for 28-days (p.o.)

On 28th day of the treatment, the animals were individually isolated in metabolic cages to measure
the urine output for 24-hours. After completion of the study, individual blood glucose levels and
body weights of the rats were recorded. Overnight fasted rats were anaesthetized and sacrificed by
cervical dislocation after collection of blood samples. Serum was separated from blood samples and
stored at – 80οC. Both kidneys were collected and processed with ice-cold saline. A part of the
kidneys from each animal was stored in neutral

buffered formalin (10% v/v) for histological examination. The rest of the kidneys were then
homogenized in ice-cold 0.1M phosphate buffer solution (pH 7.4). The tissue homogenates were
centrifuged at 12000g using a refrigerated centrifuge (Thermo Scientific Fresco 21), at 4οC, for 30
min. Resultant supernatants were collected and stored at – 80οC for estimation of various
parameters.

2.5 Assessment of glomerular function and biochemical changes

Glomerular function was assessed by measuring serum and urine creatinine levels using a semi auto
analyzer (IDEXX Vet Test Analyzer, 8008). The creatinine clearance (CrCl) was calculated using
formula; CrCl= [Urine creatinine (µmol L-1)/Serum creatinine (µmol L-1) X Volume of urine (ml/min.)]
(Robertshaw et al., 1989). Moreover, blood urea nitrogen (BUN) and albumin levels were measured
by an automated analyzer to assess renal dysfunction. Serum bilirubin, aspartate aminotransferase
(AST),alanine aminotransferase (ALT), triglycerides (TRIGs) and total cholesterol (TCHOL) were
estimated by same method to evaluate the effects of MEPF on biochemical markers.

2.6 Assessment of oxidative stress

MDA (Melondialdehyde) contents in the tissue homogenates were estimated by reacting with
thiobarbituric acid, as an index of lipid peroxidation (Okhawa et al., 1979). Superoxide dismutase
(SOD) and catalase (CAT) activities in the tissue samples were measured by using commercially
available colorimetric assay kits (Sigma Aldrich, St. Louis, MO, USA). Manufacturer’s instructions were
followed while carrying out the assays. Quantitation of kidney tissue total protein contents were
done by standard Lowry’s method (Lowry et al., 1951). Ellman’s reagent based assay was carried out
to determine the activity of GSH in the renal tissue (Ellman, 1959).

2.7 Assessment of diabetic renal inflammation

ELISA was carried out to ascertain the effect of MEPF on NF-kB dependent diabetic renal
inflammatory pathway. NF-kB p65 ELISA kit was purchased from Elabscience Biotechnology, Wuhan,
China. Manufacturer’s protocols were strictly followed while carrying out the assay. Commercially
available TNF-α ELISA kit was purchased from Life technologies, CA, USA; and IL-1β and IL-6 ELISA kits
were purchased from Elabscience Biotechnology, Wuhan, China. Serum TNF-α, IL-1β, IL-6 and tissue
NF-kB levels were estimated out from the standard plots constructed from the absorbance readings
of serially diluted standards.

2.8 Histopathological assessment

After 10 days, formalin stored kidneys were processed consecutively for dehydration and clearing
using acetone and xylene. The dehydrated tissues were impregnated into paraffin blocks and cut into
thin (5μm) sections, using a rotary microtome (Thermo Scientific, HM 325). The sections were
stained with eosin and haematoxylin. Pathological abnormalities in stained sections were identified
by a pathologist under 40X magnification without prior information about the groups.

2.9 Statistical analysis

One-way analysis of variance (ANOVA) was carried out using Graph-pad Prism software followed by
Tukey’s multiple comparison test. Values were expressed in Mean ± SEM; where n=6. Data were
considered statistically significant while p<0.05.

3. Results

3.1 Effects of MEPF body weight and blood glucose level

Diabetic rats showed significant reduction in their body weight when compared with control rats
receiving 0.2% w/v of CMC only. But this was not significantly affected by any of the used doses of
MEPF. Diabetic group receiving only ALX, exhibited marked elevation of blood glucose level. MEPF
treatment at 500 mg/kg b.w., remarkably restored blood sugar levels in the treated diabetic rats and
the effect was comparable to standard drug GLB (Table 1).

3.2 Effects of MEPF on biochemical parameters and renal function

Creatinine clearance, an index of GFR, significantly reduced in the diabetic untreated rats. A
significant improvement was observed while treated with MEPF; especially at the dose of 500 mg/kg
b.w. Diabetic control rats exhibited marked elevation of serum bilirubin, BUN, AST and ALT; whereas
serum albumin content significantly decreased. Although, MEPF oral administration reversed these
changes, the effects were more pronounced only at the dose of 500 mg/kg b.w. On the other hand,
with respect to plasma lipid contents (TRIGs & TCHOL), MEPF in both 250 and 500 mg/kg b.w. doses;
significantly normalized its level. The standard drug GLB showed overall significant results (Table 1).

3.3 Effects of MEPF on antioxidant status

MEPF oral administration significantly lowered lipid peroxidation in the treated ALX induced diabetic
rat kidney. Renal tissue SOD and CAT of diabetic control group showed significant decline in their
activity compared to control group. On MEPF supplementation at a dose of 500 mg/kg b.w.,
significantly improved activity of these antioxidant enzymes (SOD & CAT). Non-enzymatic
antioxidant, GSH also showed significant drop in its activity in the diabetic kidney without treatment.
A marked augmented activity of GSH was found in MEPF treated groups. The antioxidant effects of
MEPF observed at a dose of 500 mg/kg b.w. on the diabetic rats were comparable to GLB (Table 2).

3.4 Effect of MEPF on diabetic renal inflammation

Fig. 2 explains the effects of MEPF on serum pro-inflammatory cytokines (TNF-α, IL-6 & IL-1β) and
the renal NF-kB p65 concentrations. The ALX treated diabetic control rat serum showed high
concentrations of pro-inflammatory cytokines [Fig. (i) TNF-α, (ii) IL-6 & (iii) IL-1β], whereas a higher
expression of NF-kB p65 fraction was detected in the kidney tissue [Fig. (iv)]. MEPF treatment
significantly supressed these serum pro-inflammatory cytokines; but at a dose of 250 mg/kg, failed
to produce significant alteration of serum IL-6 level. Renal tissue NF-kB p65 fraction was dose
dependently down regulated by 28-days oral MEPF treatment.

3.5 Effect of MEPF on renal histopathology

Fig. 3 depicts the comparative diabetic renal pathology in the presence or absence of MEPF and GLB;
under microscope (40X magnification). Fig. 3(i) represents renal microscopic view of a nondiabetic
control rat (black arrows), showing normal glomerular architecture ( ), proximal convoluted tubules
( ) and distal convoluted tubules ( ); Fig. 3(ii) illustrates a untreated diabetic rat kidney section with
complete loss of nomal glomerular architecture showing detachment of basement membrane,
glomerular endothelial cell damages, glomerular space reduction, tubular damage with necrosis (red
arrows); Fig 3(iii) displays a diabetic kidney section on 250 mg/kg b.w. MEPF treatment with mild
improvement in glomerular appearance and tubular structure (blue arrows); Fig 3(iv) represents a
diabetic kindney on MEPF 500 mg/kg b.w. treatment with greatly improved glomerular and tubular
structure, healed nectrotised tubules, restored glomerular spaces in comparison with lone ALX
treated diabetic kidney (blue arrows). Fig. 3(v) shows diabetic kidney section on 10 mg/kg GLB
treatment with almost restored normal renal architecture (green arrows).

4. Discussion

The pathogenesis of DN has been related to both oxidative stress and chronic renal inflammation.
However, source of oxidative stress producing oxygen derivatives (i.e. reactive oxygen species, ROS)
in the kidney is not clearly defined. Studies on human DN have linked ROS in renal cells to high blood
glucose concentrations in diabetic individuals (Brownlee, 2005; Susztak, 2006). On the other hand,
lipophilic derivatives of ALX can also cause selective toxicity to GLUT2 expressing renal tubular cells.
However, the toxicity is severe only on systemic administration (Lenzen, 2008). Likewise, renal
inflammation in human DN is reported to be NF-kB linked and pro-inflammatory cytokines can
initiate the event (Balakumar et al., 2009; Mezzano et al., 2004).

Plant originated drugs, in terms of safety profile, always find advantages over existing synthetic drugs
for the management of diabetes and its complications. Here, we investigated the effect of indigenous
plant P. foetida extract, a folk antidiabetic medicine (Chhetri et al., 2005), recently reported to have
inhibitory action against transcription factor, NF-kB (Kumar et al., 2015), on various oxidative stress
markers and NF-kB dependent renal inflammatory pathway of DN.

During the experimental period, the ALX treated diabetic rats exhibited hyperglycaemia, polydipsia
and polyuria; the typical characteristics of DM (American Diabetes Association, 2010). At end of the
experiment, on serum analysis, we found that the serum bilirubin, BUN and creatinine levels in the
diabetic control rats were considerably higher than that of nondiabetic control rats. On the other
hand, serum albumin and GFR decreased to a greater extent. These findings confirmed glomerular
injury and renal dysfunction in the ALX treated groups (Thomas and Karalliedde, 2015). We observed
a significant reversal of altered serum markers and GFR in the MEPF treated groups, after 28-days
oral administration. However, the effects were more pronounced at a dose of 500 mg/kg b.w. Also, at
500 mg/kg b.w. dose, MEPF remarkably normalized the elevated blood sugar level in the treated
diabetic rats. Our findings were consistent with the previous reports of a reduction in blood glucose
level in rats with DM by P. foetida extract (Ahmed et al., 2014; Morshed et al., 2012). Blood glucose
lowering property of the extract could be due to the presence of iridodial glycoside contents (i.e.
scandoside) and ursolic acid in it (Miura et al., 1996; Zhang et al., 2006). Ursolic acid facilitates
phosphorylation of insulin receptors and stimulates glucose uptake through
10

inhibition of protein tyrosine phosphatase 1B (Zhang et al., 2006). Moreover, it can potentiate the
beta cell function in a partially damaged pancreatic islet (Jang et al., 2009). In DM, metabolic and
regulatory processes get altered due to lack of insulin availability precipitating hyperlipidemic state.
Lipids such as TRIGs and TCHOL start accumulating in various body parts. The veins and arteries are
more prone to such attacks leading to vascular complications of DM; related to lipid deposition
(Goldberg, 1981). In the present study, we observed that serum TRIGs and TCHOL levels in
hyperglycaemic, diabetic rats were abnormally high. Oral treatment with MEPF significantly
normalized this elevated serum lipids. Three bioactive principles viz. asperuloside, ellagic acid and
ursolic acid, present in it, could be responsible for the observed antidyslipidemic action (Hirata et al.,
2011; Kannan and Quine, 2013; Somova et al., 2003). A study on asperuloside clearly demonstrated
that this glycoside, in hyperlidemic state, can stimulate metabolic activities across several organ
systems (Fujikawa et al., 2012). Alike, hyperlipidemia reducing ability of ellagic acid has been linked
to HMGCoA reductase, the key enzyme responsible for cholesterol biosynthesis, inhibiting property
(Kannan and Quine, 2013).

In DN, lipid peroxidation increases with the substantial failure of antioxidant machineries (SOD, CAT
and GSH) in the kidney tissues leading to oxidative stress. Decreased activity of SOD, CAT and GSH in
diabetes; protecting cell membrane and its constituents from oxidative damage, is due to over
production of ROS (Giacco and Brownlee, 2010). In the present study, the decreased activity of SOD,
CAT and GSH, in the kidneys of the diabetic rats with no treatment, indicated oxidative stress. Oral
treatment with MEPF revitalized the diminished activity of these enzymatic (i.e. SOD and CAT) and
non-enzymatic (i.e. GSH) free radical scavengers. Recently, potentiating of bodily free radical
scavenging system activity by P.foetida leaf extract has been reported, supporting the present
findings (Kumar et al., 2014). Moreover, we observed a simultaneous increase in the level of
thiobarbituric acid reactive substances (MDA) along with the decrease in activity of free radical
scavenging system in the kidney of control diabetic rats. This confirmed a high degree of lipid
peroxidation in the ALX treated diabetic rats receiving no treatment (Chaudhry et al., 2007; Prince et
al., 1998). MEPF administration greatly reversed this increased renal tissue MDA level. This reversal
could be due to the presence of bodily free radical scavenger sparing principles like ellagic acid,
friedelin, ursolic acid etc in the extract (Somova et al., 2003; Sunil et al., 2013; Yüce et al., 2008). The
free radical scavenging activity and anti-lipid

11

peroxidation property of ellagic acid has been attributed to its metal chelating action (Yüce et al.,
2008).

A study on human DM detected higher concentrations of TNF-α than the normal, in the patient’s
serum, especially in those with DN (Moriwaki et al., 2003). The pathogenetic potential of this
pleiotropic cytokine is because of its ability to cause direct renal cell injury. Moreover, it activates
secondary messenger system, cell adhesion molecules, transcription factors, synthesis of cytokines,
acute phase proteins, MHC molecules etc. (Navarro-González et al., 2009). In this study, we detected
abnormally elevated serum level of TNF-α in the untreated hyperglycaemic diabetic rats. On MEPF
oral administration, we found a dose dependent inhibition of this cytokine in the treated diabetic
groups. We also detected an elevated serum IL-6 concentration in control diabetic rats. This was in
line with a previous report on diabetic patients where serum analysis of patients with nephropathy
detected markedly elevated IL-6 concentrations (Dalla Vestra et al., 2005). Investigations on DN,
demonstrated that IL-6 is involved in glomerular basement membrane thickening, alterations in
endothelial permeability and induction of mesangial cell proliferation (Mora and Navarro, 2006).
MEPF administration, at a dose of 500 mg/kg, led to pronounced decrease in serum IL-6 level. But at
a dose of 250 mg/kg, failed to effect significantly. A study on IL-1β involvement pattern in DN
reported that the circulating serum IL-1β level in albuminuric patients is remarkably high and its level
increases in the renal cortex of db/db mice in an age dependent fashion (Shahzad et al., 2015). We
indeed observed a high serum IL-1β level in the diabetic untreated group with reduced serum
albumin. IL-1β is thought to be associated with abnormal intraglomerular hemodynamic changes.
Additionally, it has been found to cause increase in vascular endothelial permeability and induction
mesangial cell proliferation and matrix synthesis (Mora and Navarro, 2006). In contrast to diabetic
control rats, MEPF treated rats exhibited a significant downturn in serum IL-1β level. The mechanistic
events of cytokine inhibition by MEPF are difficult to predict at present. It could be due to its
hyperglycaemia lowering property in diabetes.

Renal inflammation in DM and activation of transcription factor, NF-kB; both are positively co-related
(Balakumar et al., 2009). Pro-inflammatory cytokines can act as stimuli for NF-kB activation; notably
including IL-1β and TNF-α (Sanz, 2010). Activation leads to the degradation of inhibitory IĸB fraction
by phosphorylating p65 subunit which allows p65 subunit to translocate into the nucleus. On binding
to the target site on DNA, active NF-kB promotes transcription of several downstream genes directly
or indirectly associated DN

12

(Mezzano et al., 2004; Sanchez and Sharma, 2009). Moreover, it promotes the transcription of pro-
inflammatory cytokines; including TNF-α and IL-1β (Sanz, 2010). More advanced studies on human
diabetic nephropathy reported that NF-kB highly upregulated in diabetic kidney. (Mezzano et al.,
2004). Here we detected remarkably high concentration of p65 subunit in the untreated diabetic
kidney through ELISA while in MEPF treated diabetic groups the active NF-kB concentrations were
significantly low. Supporting our findings, a recent study demonstrated that P. foetida extract can
efficiently inhibit transcription factor NF-kB activation to suppress inflammatory response (Kumar et
al., 2015). This property MEPF may possibly be due to synergistic effect of ursolic acid, lupeol and
ellagic acid present in it (Anitha et al., 2013; Lee et al., 2016; Shishodia et al., 2003). However, MEPF
is comparable to standard antidiabetic GLB in terms of protective actions against early stage diabetic
nephropathy. The above results are in support of our hypothetical mechanism of renoprotection in
diabetes by MEPF (illustrated in Fig. 4).

Further, in renal histopathology of the diabetic control group, we observed oxidative glomerular
damages, chronic inflammatory changes and intrinsic cells undergoing necrosis. In the kidney
sections of GLB treated diabetic group, a slight distortion in renal microarchitecture was observed
with majority of changes in its tubular structures. On investigations of MEPF treated kidney sections,
we saw some minor improvements at a dose of 250 mg/kg, whilst at a dose of 500 mg/kg rats
exhibited renal microarchitectures with healed tubular cells, slightly distorted glomerulus and
preserved glomerular spaces. The observed renoprotection of the extract could be due to
suppression of hyperglycaemia mediated oxidative stress and inhibition of renal inflammatory
cascades associated with NFkB activation.

5. Conclusion

In conclusion, P. foetida methanolic leaf extract exerted prominent nephro-protection in early stage
of experimental DN in rats. Our study reports that the renoprotective action is due to attenuation of
renal oxidative stress and suppression of renoinflammatory events though inhibition of NF-kB, in
early progressive DN. Thus, it might be useful during the early stage of DN. However, our study
further necessitates in detail investigation on P. foetida to find out the active principles responsible
for its nephroprotective action in diabetes