{"id":51732,"date":"2023-09-30T10:30:15","date_gmt":"2023-09-30T10:30:15","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=51732"},"modified":"2023-10-07T11:38:01","modified_gmt":"2023-10-07T11:38:01","slug":"assessment-of-efficacy-and-safety-of-methanolic-leaf-extract-of-prosopis-juliflora-in-lipopolysaccharide-induced-systemic-inflammatory-response-syndrome-sirs-in-wistar-albino-rats","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol16no3\/assessment-of-efficacy-and-safety-of-methanolic-leaf-extract-of-prosopis-juliflora-in-lipopolysaccharide-induced-systemic-inflammatory-response-syndrome-sirs-in-wistar-albino-rats\/","title":{"rendered":"Assessment of Efficacy and Safety of Methanolic Leaf Extract of Prosopis Juliflora in Lipopolysaccharide Induced Systemic Inflammatory Response Syndrome (SIRS) in Wistar Albino Rats"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Systemic Inflammatory Response Syndrome (SIRS) is an exaggerated defense response exerted by the body as a result of noxious or harmful stimuli such as infection, trauma, acute inflammation, ischemia or reperfusion and surgery, in order to find and get rid of the source of insult or stimuli. SIRS involves the release of certain inflammatory mediators and acute phase reactants that modulate hematological, autonomic, endocrine and immunological alterations in the body. Though the surge in acute phase reactants is defensive, a dysregulated cytokine storm would result in a severe inflammation causing reversible or irreversible end organ dysfunction and death.1<\/sup><\/p>\n\n\n\n

\u201cThe American\nCollege of Chest Physicians\/Society of Critical Care Medicine-sponsored sepsis\ndefinitions consensus conference\u201d held in 1991 in Chicago, Illinois established\na set of clinical parameters to identify those subjects with SIRS easily in any\nclinical setting. SIRS is objectively defined by satisfaction of any two\nof the following criteria: \u201c1. Body temperature over 38 or\nunder 36 degrees Celsius, 2. Heart rate greater than 90 beats\/minute, 3.\nRespiratory rate greater than 20 breaths\/minute or partial pressure of CO2<\/sub>\nless than 32 mmHg, 4. Leukocyte count greater than 12000 or less than 4000\n\/microliters or over 10% immature forms or bands\u201d.2<\/sup><\/p>\n\n\n\n

Whenever an inflammation is induced by an\ninfectious or noninfectious stimuli, complex immune responses set in, involving\nthe humoral and cell mediated immunity, complement system and cytokines. The\npro inflammatory cascade is mediated by the cytokines IL-1and TNF-alpha, that\ninduce NF-kB leading to the release of IL-6, IL-8 and INF gamma and the\ncompensatory anti-inflammatory drive is mediated by IL-4 and IL-10. If the\nbalance between the pro inflammatory and anti-inflammatory pathways shifts in\nfavor of pro-inflammatory, SIRS sets in. (1)<\/sup> If not treated, the\nongoing inflammation could result in multiorgan failure and sometimes death.\nHence, early diagnosis of SIRS, identification of its etiology and prompt\nintervention is very crucial to prevent multi organ dysfunction.<\/p>\n\n\n\n

The mortality due\nto SIRS and sepsis is more than 35%. In addition to hemodynamic support,\nantibiotics and steroids are used in the management of SIRS.1<\/sup> In\ndiseases like COVID- 19 (Corona Virus Disease-19), SIRS can be a life-\nthreatening complication. Although, steroids can help in this situation, there\nis no specific treatment for SIRS of varying etiologies including COVID-19.\nThere is always a need for potential therapeutic options that could combat the\ninflammation in SIRS and prevent organ dysfunction.<\/p>\n\n\n\n

The World Health\nOrganization (WHO) reports that 88% of countries in the world use traditional\nmedicines for various ailments and it has been an integral resource for years\nand mainstay for a few countries where they do not have proper access to the\nconventional medicine (3). Traditional\nmedicines have gained popularity during the pandemic in reducing the symptoms\nassociated with COVID-19 infection and inflammation. Few plants such as Nigella\nsativa<\/em> (black cumin), Gymnanthemum amygdalinium<\/em> and Azadirachta\nindica<\/em> were evaluated for their efficacy in COVID-19 associated\ninflammation due to their anti-inflammatory, antiviral and immune modulatory\nproperties.4<\/sup> Similarly, Kabasurakudineer (KSK), a herbal concoction\nwith several ingredients have gained popularity in India during COVID-19 and\nthe Government of India promoted the use of KSK for prophylaxis and treatment\nof COVID-19, as it has antipyretic, anti-inflammatory, antioxidant and\nimmunomodulatory properties.5-9<\/sup><\/p>\n\n\n\n

Prosopis\njuliflora<\/em> (PJ), an invasive plant that belongs\nto the family Fabaceae, can grow in variety of soils and varied climatic\nconditions. The plant is available abundantly in Tamil Nadu. The\nphytoconstituents present in P.juliflora <\/em>include phenols, tannins,\nsteroids, terpenoids, flavonoids and alkaloids and various parts of the plant\nincluding seeds, fruits and leaves have been reported to have potent\nanti-inflammatory, antioxidant, anti-bacterial, anti-ulcer, anti-fungal and\nanticancer properties.10<\/sup> There\nare no adequate data available in the literature evaluating the effectiveness\nof PJ in severe forms of inflammation. With this background, we wanted to\nevaluate the efficacy of PJ leaf extract in LPS induced SIRS in rats in\ncomparison to the steroid, Hydrocortisone. <\/p>\n\n\n\n

Objectives<\/strong><\/p>\n\n\n\n

The primary\nobjective was to evaluate the efficacy of methanolic leaves extract of P.juliflora\n<\/em>in LPS induced SIRS and the secondary objective was to evaluate the safety\nof methanolic leaves extract of P.juliflora in <\/em>LPS induced SIRS in\nWistar albino rats.<\/p>\n\n\n\n

Outcomes<\/strong><\/p>\n\n\n\n

Primary outcomes<\/strong><\/p>\n\n\n\n

To evaluate the efficacy of methanolic leaves extract of P.juliflora <\/em>in LPS induced SIRS with the following:<\/p>\n\n\n\n

Body temperature<\/p>\n\n\n\n

Total WBC count<\/p>\n\n\n\n

Inflammatory markers- Interleukin-6 (IL-6), interleukin-10 (IL-10), Tumor necrosis factor (TNF-\u03b1), Interferon gamma (IFN-\u03b3), Ferritin and C-reactive protein (CRP)<\/p>\n\n\n\n

Histopathological assessment of Liver, Kidney and Brain <\/p>\n\n\n\n

Secondary outcomes<\/strong><\/p>\n\n\n\n

To evaluate the safety of methanolic leaves extract of P.juliflora in <\/em>LPS induced SIRS with the following:<\/p>\n\n\n\n

Complete Blood Count (CBC)<\/p>\n\n\n\n

Renal Function Tests (RFT) \u2013 Urea, creatinine<\/p>\n\n\n\n

Liver Function Tests (LFT) \u2013 Aspartate Transaminase (AST), Alanine Transaminase (ALT), Alkaline Phosphatase (ALP), Total bilirubin and Direct bilirubin<\/p>\n\n\n\n

Mortality<\/p>\n\n\n\n

Material and methods<\/strong><\/p>\n\n\n\n

Drugs and chemicals<\/strong><\/p>\n\n\n\n

Lipopolysaccharide (Escherichia coli 0111: B4) was obtained from Sigma aldrich for inducing SIRS<\/p>\n\n\n\n

P.juliflora <\/em>was identified and certified by authorized botanist <\/p>\n\n\n\n

Hydrocortisone was commercially obtained from nearby Chemist shop<\/p>\n\n\n\n

Kits<\/strong><\/p>\n\n\n\n

Estimation of rat plasma\nIL-6, IL-10, TNF-\u03b1 and INF-\u03b3 was done using commercially available ELISA kits\nas per the standard operating procedures.<\/p>\n\n\n\n

Dose selection<\/strong><\/p>\n\n\n\n

The doses of Hydrocortisone and PJ were selected\nbased on the previous literature reports that evaluated the anti-inflammatory\nactivity of these interventions.11, 12<\/sup>\n<\/p>\n\n\n\n

Experimental design<\/strong><\/p>\n\n\n\n

The study was initiated after\nobtaining approval from the Institutional Animal Ethics Committee (Ref no:\nIAEC1\/Proposal:59\/A.Lr\/Dt:05.03.2021). 60 male Wistar albino rats, 6 to 8 weeks\nold, weighing 200-250 grams were used in this experiment.\nThe animals were housed for about 14 days out of which the duration of\nexperiment was 7 days. The rats were given standard pellet feed and kept in\npolypropylene cages (3 rats in each cage) with 12 hours light-dark cycle.\nBedding, temperature and humidity were maintained as per the CPCSEA (Committee\nfor the Purpose of Control and Supervision of Experiments on Animals)\nguidelines. <\/p>\n\n\n\n

The rats were\ndivided into 4 groups with 18 rats in groups 1, 2 & 3 and 6 rats in group\n4. Groups 1-3 were further divided into 3 subgroups- A, B and C. Rats in group\n1 were not given any treatment and served as inflammation (SIRS) control while\nin groups 2 and 3, the animals received Hydrocortisone 5 mg\/kg IV 12th<\/sup>\nhourly and methanolic leaf extract of Prosopis juliflora <\/em>2 mg\/kg 12th<\/sup>\nhourly respectively from day 1 to day 3. <\/p>\n\n\n\n

SIRS was induced\nin all the rats of groups 1 to 3 on day 4 with single intraperitoneal injection\nof LPS (procedure explained in detail in next section). The rats in the subgroups A, B and C of groups 1,2 and 3 were\nsacrificed on day 5, 6 and 7, ie., 24, 48 and 72 hours after LPS injection\nrespectively. <\/p>\n\n\n\n

Group 4 animals\nserved as normal control as SIRS was not induced and no treatment was given.\nThey were sacrificed on day 4 to get control data for all parameters (presumptive\nbaseline data indicating the pre induction levels).\nThe treatment was continued with hydrocortisone and PJ leaf extract in groups 2\nand 3 till the animals were sacrificed. The detailed treatment schedule from\nday 1 to day 7 in all the groups is provided in table 1. <\/p>\n\n\n\n

Table 1: Treatment Schedule<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Groups<\/strong><\/p>\n<\/td>\n

\n

Subgroups<\/strong><\/p>\n<\/td>\n

\n

Treatment<\/strong><\/p>\n<\/td>\n

\n

Sacrifice<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Prior to LPS injection (day 1-3)<\/strong><\/p>\n<\/td>\n

\n

LPS injection (day 4)<\/strong><\/p>\n<\/td>\n

\n

After LPS injection (day 5-7)<\/strong><\/p>\n<\/td>\n

\n

(After LPS injection)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Group 1 (Inflammation control)<\/strong><\/p>\n

(n=18)<\/p>\n<\/td>\n

\n

Subgroup A (n=6)<\/p>\n<\/td>\n

\n

No treatment<\/p>\n<\/td>\n

\n

No treatment<\/p>\n<\/td>\n

\n

No treatment<\/p>\n<\/td>\n

\n

At 24hrs<\/p>\n

(Day 5)<\/p>\n<\/td>\n<\/tr>\n

\n

Subgroup B (n=6)<\/p>\n<\/td>\n

\n

No treatment<\/p>\n<\/td>\n

\n

No treatment<\/p>\n<\/td>\n

\n

No treatment<\/p>\n<\/td>\n

\n

At 48hrs<\/p>\n

(Day 6)<\/p>\n<\/td>\n<\/tr>\n

\n

Subgroup C (n=6)<\/p>\n<\/td>\n

\n

No treatment<\/p>\n<\/td>\n

\n

No treatment<\/p>\n<\/td>\n

\n

No treatment<\/p>\n<\/td>\n

\n

At 72hrs<\/p>\n

(Day 7)<\/p>\n<\/td>\n<\/tr>\n

\n

Group 2 (standard treatment)<\/strong><\/p>\n

(n=18)<\/p>\n<\/td>\n

\n

Subgroup A (n=6)<\/p>\n<\/td>\n

\n

Hydrocortisone 5mg\/kg, 12th<\/sup> hourly, IV<\/p>\n<\/td>\n

\n

Hydrocortisone 5mg\/kg, 12th<\/sup> hourly, IV<\/p>\n<\/td>\n

\n

Hydrocortisone 5mg\/kg, 12th<\/sup> hourly for 24hrs, IV<\/p>\n<\/td>\n

\n

At 24hrs<\/p>\n

(Day 5)<\/p>\n<\/td>\n<\/tr>\n

\n

Subgroup B (n=6)<\/p>\n<\/td>\n

\n

Hydrocortisone 5mg\/kg, 12th<\/sup> hourly, IV<\/p>\n<\/td>\n

\n

Hydrocortisone 5mg\/kg, 12th<\/sup> hourly, IV<\/p>\n<\/td>\n

\n

Hydrocortisone 5mg\/kg, 12th<\/sup> hourly for 48hrs, IV<\/p>\n<\/td>\n

\n

At 48hrs<\/p>\n

(Day 6)<\/p>\n<\/td>\n<\/tr>\n

\n

Subgroup C (n=6)<\/p>\n<\/td>\n

\n

Hydrocortisone 5mg\/kg, 12th<\/sup> hourly, IV<\/p>\n<\/td>\n

\n

Hydrocortisone 5mg\/kg, 12th<\/sup> hourly, IV<\/p>\n<\/td>\n

\n

Hydrocortisone 5mg\/kg, 12th<\/sup> hourly for 72hrs, IV<\/p>\n<\/td>\n

\n

At 72hrs<\/p>\n

(Day 7)<\/p>\n<\/td>\n<\/tr>\n

\n

Group 3<\/strong><\/p>\n

(P.juliflora<\/em>)<\/strong><\/p>\n

(n=18)<\/p>\n<\/td>\n

\n

Subgroup A (n=6)<\/p>\n<\/td>\n

\n

P.juliflora <\/em>leaf extract 2mg\/kg, 12th<\/sup> hourly, oral<\/p>\n<\/td>\n

\n

P.juliflora <\/em>leaf extract 2mg\/kg, 12th<\/sup> hourly, oral<\/p>\n<\/td>\n

\n

P.juliflora <\/em>leaf extract 2mg\/kg, 12th<\/sup> hourly for 24hrs, oral<\/p>\n<\/td>\n

\n

At 24hrs<\/p>\n

(Day 5)<\/p>\n<\/td>\n<\/tr>\n

\n

Subgroup B (n=6)<\/p>\n<\/td>\n

\n

P.juliflora <\/em>leaf extract 2mg\/kg, 12th<\/sup> hourly, oral<\/p>\n<\/td>\n

\n

P.juliflora <\/em>leaf extract 2mg\/kg, 12th<\/sup> hourly, oral<\/p>\n<\/td>\n

\n

P.juliflora <\/em>leaf extract 2mg\/kg, 12th<\/sup> hourly for 48hrs, oral<\/p>\n<\/td>\n

\n

At 48hrs<\/p>\n

(Day 6)<\/p>\n<\/td>\n<\/tr>\n

\n

Subgroup C (n=6)<\/p>\n<\/td>\n

\n

P.juliflora <\/em>leaf extract 2mg\/kg, 12th<\/sup> hourly, oral<\/p>\n<\/td>\n

\n

P.juliflora <\/em>leaf extract 2mg\/kg, 12th<\/sup> hourly, oral<\/p>\n<\/td>\n

\n

P.juliflora <\/em>leaf extract 2mg\/kg, 12th<\/sup> hourly for 72hrs, oral<\/p>\n<\/td>\n

\n

At 72hrs<\/p>\n

(Day 7)<\/p>\n<\/td>\n<\/tr>\n

\n

Group 4 (normal control)<\/strong><\/p>\n

(n=6)<\/p>\n<\/td>\n

\n

—<\/p>\n<\/td>\n

\n

No treatment<\/p>\n<\/td>\n

\n

No treatment<\/p>\n<\/td>\n

\n

No treatment<\/p>\n<\/td>\n

\n

On day 4, ie when SIRS was induced in other groups, these animals were sacrificed to get control data<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Note: n=number of rats<\/p>\n\n\n

Induction of systemic inflammatory response syndrome<\/strong><\/p>\n\n\n\n

Among the various animal models\nfor systemic inflammatory response syndrome in rats, we used LPS for inducing\nSIRS. Lipopolysaccharide (Escherichia coli 0111: B4) 5mg\/kg was dissolved in\n0.1 ml\/20gm body weight PBS (Phosphate buffered saline), and given intraperitoneally\nto the rats of groups 1, 2 and 3.13<\/sup>\n<\/p>\n\n\n\n

Extract preparation <\/strong><\/p>\n\n\n\n

P.juliflora<\/em>\nplant was identified and certified by the authorized botanist before starting\nthe experiment. The leaves of P.juliflora<\/em> (3kg) were washed, shade dried\nfor 3 days and grounded to fine powder, mixed with 80% methanol at 20% (w\/v)\nconcentration with continuous mixing in rotary evaporator for 24 hours at room\ntemperature. The extract was then filtered through Whatman no:4 filter paper,\nand the excess solvent were removed using rotary evaporator at 55o<\/sup>C.\nThe extract was stored at 4\u00baC for the experiment.14<\/sup><\/p>\n\n\n\n

Study assessments <\/strong><\/p>\n\n\n\n

After sacrificing the animals at their respective time points as detailed in Table 1, the following parameters were assessed:<\/p>\n\n\n\n

CBC (complete Blood Count)<\/p>\n\n\n\n

Renal Function Tests (RFT) \u2013 Urea, creatinine<\/p>\n\n\n\n

Liver Function Tests (LFT) \u2013 Aspartate Transaminase (AST), Alanine Transaminase (ALT), Alkaline Phosphatase (ALP), Total bilirubin and Direct bilirubin<\/p>\n\n\n\n

Inflammatory markers- Interleukin (IL)-6, Interleukin (IL)-10, Tumor necrosis factor (TNF)-\u03b1, Interferon gamma (IFN)-\u03b3, Ferritin and C-reactive protein (CRP)<\/p>\n\n\n\n

Histopathological examination of liver, kidney and brain <\/p>\n\n\n\n

Statistical analysis<\/strong><\/p>\n\n\n\n

The\ndata were analyzed using GraphPad Instat software (version 3.1). Quantitative data\nwere summarized as mean \u00b1 standard deviation. Inferential statistics was done\nusing repeated measures ANOVA (Analysis of Variance) for within group\ncomparison and one-way ANOVA for between group comparison, followed by Post hoc\nanalysis using Tukey\u2019s multiple comparison test. P value <0.05 was\nconsidered significant.<\/p>\n\n\n\n

Results <\/strong><\/p>\n\n\n\n

All the rats in groups 1-3\ndeveloped SIRS, as evidenced by increased body temperature and total WBC count,\nas per the SIRS criteria.1<\/sup>\nThere was no mortality in any of the groups. The clinical, haematological,\nbiochemical and inflammatory markers observed in the study were statistically\nanalysed within the groups and between the groups and the results are discussed\nparameter wise in the following section. Statistically significant p values are\ndenoted by * in the tables and graphs.<\/p>\n\n\n\n

Body temperature<\/strong><\/p>\n\n\n\n

Within group analysis of the\nbody temperature was carried out only in subgroup C of groups 1, 2 and 3 as it\nhad the data at all time points. Between group analysis was carried out by\ncomparing the subgroup C of groups 1, 2 and 3.<\/p>\n\n\n\n

In contrast to inflammation\ncontrol (group 1), where the rats had elevated body temperature till the end\n(72 hours), hydrocortisone and PJ extract treated rats showed significant\ndecrease in the body temperature. The body temperature of hydrocortisone\ntreated rats was significantly low at 24 hours after LPS injection when\ncompared to inflammation control, but it was not significantly reduced with PJ\nin comparison to group 1 (inflammation control). At 48 hours and 72 hours, the\nreduction in body temperature was similar in both hydrocortisone and PJ treated\nrats (Table 2). <\/p>\n\n\n\n

Table 2: Body temperature<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

 <\/strong><\/p>\n<\/td>\n

\n

Prior to LPS Injection<\/strong><\/p>\n<\/td>\n

\n

After LPS Injection<\/strong><\/p>\n<\/td>\n

\n

P value<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n

Groups<\/strong><\/p>\n<\/td>\n

\n

 <\/strong>Sub<\/strong><\/p>\n

groups<\/strong><\/p>\n<\/td>\n

\n

 <\/strong>Day 1<\/strong><\/p>\n

(Mean\u00b1<\/strong><\/p>\n

SD)<\/strong><\/p>\n<\/td>\n

\n

 <\/strong>Day 2<\/strong><\/p>\n

(Mean\u00b1<\/strong><\/p>\n

SD)<\/strong><\/p>\n<\/td>\n

\n

 <\/strong>Day 3<\/strong><\/p>\n

(Mean\u00b1<\/strong><\/p>\n

SD)<\/strong><\/p>\n<\/td>\n

\n

 <\/strong>At 24 hours<\/strong><\/p>\n

(Mean\u00b1<\/strong><\/p>\n

SD)<\/strong><\/p>\n<\/td>\n

\n

At 48 hours<\/strong><\/p>\n

(Mean\u00b1<\/strong><\/p>\n

SD)<\/strong><\/p>\n<\/td>\n

\n

 <\/strong>At 72 hours<\/strong><\/p>\n

(Mean\u00b1<\/strong><\/p>\n

SD)<\/strong><\/p>\n<\/td>\n

\n

Within group comparison<\/strong><\/p>\n

(Repeated measures ANOVA) <\/strong><\/p>\n<\/td>\n

\n

Inter-Group comparison<\/strong><\/p>\n

(One way ANOVA)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Group 1-<\/p>\n

Inflammation Control<\/p>\n<\/td>\n

\n

A<\/p>\n<\/td>\n

\n

35.82 \u00b1 0.19<\/p>\n<\/td>\n

\n

36.17 \u00b1 0.12<\/p>\n<\/td>\n

\n

35.12 \u00b1 0.17<\/p>\n<\/td>\n

\n

40.05 \u00b1 0.09<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

B<\/p>\n<\/td>\n

\n

36.09 \u00b1 0.22<\/p>\n<\/td>\n

\n

35.26 \u00b1 0.2<\/p>\n<\/td>\n

\n

36.19 \u00b1 0.13<\/p>\n<\/td>\n

\n

39.12 \u00b1 0.12<\/p>\n<\/td>\n

\n

38.64 \u00b1 0.18<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

C<\/p>\n<\/td>\n

\n

35.18 \u00b1 0.09<\/p>\n<\/td>\n

\n

35.94 \u00b1 0.24<\/p>\n<\/td>\n

\n

36.05 \u00b1 0.34<\/p>\n<\/td>\n

\n

39.25 \u00b1 0.14<\/p>\n<\/td>\n

\n

39.15 \u00b1 0.08<\/p>\n<\/td>\n

\n

39.12 \u00b1 0.22<\/p>\n<\/td>\n

\n

<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

Group 2-<\/p>\n

Hydrocortisone<\/p>\n<\/td>\n

\n

A<\/p>\n<\/td>\n

\n

36.22 \u00b1 0.17<\/p>\n<\/td>\n

\n

35.67 \u00b1 0.16<\/p>\n<\/td>\n

\n

35.16 \u00b1 0.17<\/p>\n<\/td>\n

\n

37.75 \u00b1 0.12<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

B<\/p>\n<\/td>\n

\n

35.01 \u00b1 0.11<\/p>\n<\/td>\n

\n

35.06 \u00b1 0.13<\/p>\n<\/td>\n

\n

36.08 \u00b1 0.19<\/p>\n<\/td>\n

\n

37.92 \u00b1 0.06<\/p>\n<\/td>\n

\n

37.44 \u00b1 0.34<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

C<\/p>\n<\/td>\n

\n

35.66 \u00b1 0.06<\/p>\n<\/td>\n

\n

35.65 \u00b1 0.18<\/p>\n<\/td>\n

\n

36.01 \u00b1 0.25<\/p>\n<\/td>\n

\n

37.93 \u00b1 0.15<\/p>\n<\/td>\n

\n

37.58 \u00b1 0.26<\/p>\n<\/td>\n

\n

35.87 \u00b1 0.23<\/p>\n<\/td>\n

\n

<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

Group 3-<\/p>\n

PJ extract<\/p>\n<\/td>\n

\n

A<\/p>\n<\/td>\n

\n

35.12 \u00b1 0.16<\/p>\n<\/td>\n

\n

35.19 \u00b1 0.18<\/p>\n<\/td>\n

\n

35.42 \u00b1 0.11<\/p>\n<\/td>\n

\n

39.98 \u00b1 0.06<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

B<\/p>\n<\/td>\n

\n

36.26 \u00b1 0.09<\/p>\n<\/td>\n

\n

36.62 \u00b1 0.28<\/p>\n<\/td>\n

\n

36.04 \u00b1 0.25<\/p>\n<\/td>\n

\n

39.02 \u00b1 0.08<\/p>\n<\/td>\n

\n

37.81 \u00b1 0.12<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

C<\/p>\n<\/td>\n

\n

36.12 \u00b1 0.14<\/p>\n<\/td>\n

\n

36.07 \u00b1 0.23<\/p>\n<\/td>\n

\n

36.08 \u00b1 0.21<\/p>\n<\/td>\n

\n

39.36 \u00b1 0.14<\/p>\n<\/td>\n

\n

37.92 \u00b1 0.07<\/p>\n<\/td>\n

\n

37.06 \u00b1 0.29<\/p>\n<\/td>\n

\n

<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

Group 4-<\/p>\n

Control (pre induction)<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

35.62 \u00b1 0.12<\/p>\n<\/td>\n

\n

36.09 \u00b1 0.13<\/p>\n<\/td>\n

\n

35.12 \u00b1 0.17<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Blood parameters<\/strong><\/p>\n\n\n\n

In this study, there is no\nbaseline data in the subgroups. Hence, the animals in group 4 were scarified at\nthe time of LPS injection in other groups and the hematological, inflammatory\nand biochemical parameters were measured. This data was considered as presumptive\nbaseline data indicating the pre induction levels for statistical purpose to\nassess whether 24 hours post LPS data satisfies induction of inflammation.\nFurther, the data of subgroups (A, B & C) in each group were comparatively\nanalyzed to check whether the parameters improve or worsen.<\/p>\n\n\n\n

At 24 hours post LPS injection<\/strong><\/p>\n\n\n\n

All the blood parameters\nshowed significant increase or decrease at 24 hours compared to presumptive\nbaseline data (group 4), in favor of inflammation in groups 1, 2 and 3, except\nferritin which did not increase significantly in hydrocortisone treated (group\n2) rats at 24 hours post LPS injection.<\/p>\n\n\n\n

Subgroups (24, 48 and 72 hours post LPS injection)<\/p>\n\n\n\n

Inflammation control (Group\n1):<\/p>\n\n\n\n

All the inflammatory and\nblood parameters showed significant increase from 24 hours to 72 hours, except\nHb which showed significant decline at 72 hours compared to 24 hours. These\nobservations were in favor sustained inflammation till 72 hours.<\/p>\n\n\n\n

Observations in group 2 (Hydrocortisone) and 3 (PJ):<\/p>\n\n\n\n

Hematological parameters – Significant improvement in Hb and significant reduction in WBC with hydrocortisone and PJ extract were noted at 72 hours. Though there is improvement in Hb level at 72 hours, it has not attained the preinduction levels in both treatments whereas the total WBC count returned to preinduction levels in both hydrocortisone and PJ at 72 hours (Figure 1 and Table 3).<\/p>\n\n\n\n

Renal function tests – Urea levels showed statistically significant increase after induction of inflammation (24, 48 and 72 hours) in group 2 & 3. However, there was no clinical significance as the values were within the normal range. Creatinine levels were increased after 24 hours of LPS injection in group 1, 2 & 3, but there was significant reduction in the creatinine levels at 72 hours in groups 2 & 3 and the reduction was similar in both, though the values did not reduce to the preinduction levels (Figures 2 & 3). Creatinine levels did not reduce in group 1, inflammation control. <\/p>\n\n\n\n

Liver function tests – Liver enzymes showed significant decline at 72 hours with both hydrocortisone and PJ in group 2 and 3, after an initial increase at 24 hours. AST and ALT decreased to preinduction levels at 72 hours with hydrocortisone but not with PJ. ALP returned to normal at 48 hours with hydrocortisone and at 72 hours with PJ. There was no clinically significant change noted with total and direct bilirubin (Figures 4-6, Table 4). Improvement in LFT was not noted in group 1. <\/p>\n\n\n\n

Inflammatory markers – All the inflammatory markers (CRP, ferritin, IL-6, IL-10, TNF-\u03b1 and INF-\u03b3) showed significant reduction at 72 hours with both hydrocortisone and PJ. In hydrocortisone group, ferritin was not significantly elevated at 24 hours. IL-6 & IL-10 reduced to normal (preinduction levels) at 48 hours and TNF-\u03b1 & INF-\u03b3 returned to normal at 72 hours. In PJ group, IL-10 was reduced to preinduction level at 48 hours, while Ferritin, IL-6, TNF-\u03b1 and INF-\u03b3 became normal at 72 hours. Though CRP showed significant decline at 72 hours with both treatments, it did not reduce to the preinduction levels (Figures 7-10, Table 5). <\/p>\n\n\n\n

Histopathological assessment <\/strong><\/p>\n\n\n\n

The animals in inflammation control (group 1) had inflammatory changes in liver, kidney and brain at 72 hours post LPS injection. The animals treated with hydrocortisone and PJ extract had regenerative changes in liver and kidney after 72 hours post LPS injection. But brain had better regenerative changes in Hydrocortisone group at 72 hours compared to PJ group which still had minimal inflammatory changes. The histopathological changes are presented in figure 11. <\/p>\n\n\n\n\n\n
\"\"<\/td>\n
<\/em><\/strong>

<\/p>\n

Figure <\/strong>1<\/strong>: Haemoglobin<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table 3: Total WBC count<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Groups<\/strong><\/p>\n<\/td>\n

\n

Subgroups<\/strong><\/p>\n<\/td>\n

\n

WBC (cell\/smm3<\/sup>) (Mean\u00b1SD)<\/strong><\/p>\n<\/td>\n

\n

P value<\/strong><\/p>\n

(Inter Subgroup)<\/strong><\/p>\n

(One way ANOVA)<\/strong><\/p>\n<\/td>\n

\n

P value<\/strong><\/p>\n

(Inter group)<\/strong><\/p>\n

(One way ANOVA)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Group 1-<\/p>\n

Inflammation Control<\/p>\n<\/td>\n

\n

24 hrs<\/p>\n<\/td>\n

\n

12633.33 \u00b1 1139.59<\/p>\n<\/td>\n

\n

0.0004*<\/p>\n<\/td>\n

\n

P<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

48 hrs<\/p>\n<\/td>\n

\n

14566.66 \u00b1 1216.04<\/p>\n<\/td>\n<\/tr>\n

\n

72 hrs<\/p>\n<\/td>\n

\n

15800.00 \u00b1 704.27<\/p>\n<\/td>\n<\/tr>\n

\n

Group 2-<\/p>\n

Hydrocortisone<\/p>\n<\/td>\n

\n

24 hrs<\/p>\n<\/td>\n

\n

13866.66 \u00b1 1184.34<\/p>\n<\/td>\n

\n

0.004*<\/p>\n<\/td>\n<\/tr>\n

\n

48 hrs<\/p>\n<\/td>\n

\n

13002.01 \u00b1 1300.38<\/p>\n<\/td>\n<\/tr>\n

\n

72 hrs<\/p>\n<\/td>\n

\n

11283.33 \u00b1 930.41<\/p>\n<\/td>\n<\/tr>\n

\n

Group 3-<\/p>\n

PJ extract<\/p>\n<\/td>\n

\n

24 hrs<\/p>\n<\/td>\n

\n

13916.66 \u00b1 1792.67<\/p>\n<\/td>\n

\n

0.02*<\/p>\n<\/td>\n<\/tr>\n

\n

48 hrs<\/p>\n<\/td>\n

\n

13066.66 \u00b1 628.22<\/p>\n<\/td>\n<\/tr>\n

\n

72 hrs<\/p>\n<\/td>\n

\n

11033.33 \u00b1 742.06<\/p>\n<\/td>\n<\/tr>\n

\n

Group 4-<\/p>\n

Control (Pre induction)<\/p>\n<\/td>\n

\n

NA<\/p>\n<\/td>\n

\n

9833.33 \u00b1 2593.58<\/p>\n<\/td>\n

\n

NA<\/p>\n<\/td>\n<\/tr>\n

\n

Post hoc: G1 24 vs Control: p=0.02*, G2 24 vs Control: p=0.0001*, G3 24 vs Control: p=0.0001*, G2 72 vs Control: p=0.69, G3 72 vs Control: p=0.86, G2 48 vs G3 48: p=1, G2 72 vs G3 72: p=1<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n
<\/u><\/em>

<\/p>\n

Figure <\/strong>2<\/strong>: Urea<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n
<\/u><\/em>

<\/p>\n

Figure <\/strong>3<\/strong>: Creatinine<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure <\/strong>4<\/strong>: AST<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure <\/strong>5<\/strong>: ALT<\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure <\/strong>6<\/strong>: ALP<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table 4: Bilirubin<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Groups<\/strong><\/p>\n<\/td>\n

\n

Subgroup<\/strong><\/p>\n<\/td>\n

\n

Total bilirubin (mg\/dl)<\/strong><\/p>\n

(Mean\u00b1SD)<\/strong><\/p>\n<\/td>\n

\n

P value<\/strong><\/p>\n

(Inter Subgroup comparison)<\/strong><\/p>\n<\/td>\n

\n

P value<\/strong><\/p>\n

(Inter group comparison)<\/strong><\/p>\n<\/td>\n

\n

Direct bilirubin (mg\/dl)<\/strong><\/p>\n

(Mean\u00b1SD)<\/strong><\/p>\n<\/td>\n

\n

P value<\/strong><\/p>\n

(Inter Subgroup)<\/strong><\/p>\n

(One way ANOVA)<\/strong><\/p>\n<\/td>\n

\n

P value<\/strong><\/p>\n

(Inter group)<\/strong><\/p>\n

(One way ANOVA)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Group 1-<\/p>\n

Inflammation Control<\/p>\n<\/td>\n

\n

24 hrs<\/p>\n<\/td>\n

\n

0.50 \u00b1 0.09<\/p>\n<\/td>\n

\n

0.77<\/p>\n<\/td>\n

\n

0.03*<\/p>\n<\/td>\n

\n

0.12 \u00b1 0.06<\/p>\n<\/td>\n

\n

0.74<\/p>\n<\/td>\n

\n

0.46<\/p>\n<\/td>\n<\/tr>\n

\n

48 hrs<\/p>\n<\/td>\n

\n

0.51 \u00b1 0.07<\/p>\n<\/td>\n

\n

0.10 \u00b1 0.03<\/p>\n<\/td>\n<\/tr>\n

\n

72 hrs<\/p>\n<\/td>\n

\n

0.53 \u00b1 0.06<\/p>\n<\/td>\n

\n

0.11 \u00b1 0.04<\/p>\n<\/td>\n<\/tr>\n

\n

Group 2-<\/p>\n

Hydrocortisone<\/p>\n<\/td>\n

\n

24 hrs<\/p>\n<\/td>\n

\n

0.45 \u00b1 0.06<\/p>\n<\/td>\n

\n

0.63<\/p>\n<\/td>\n

\n

0.09 \u00b1 0.03<\/p>\n<\/td>\n

\n

0.65<\/p>\n<\/td>\n<\/tr>\n

\n

48 hrs<\/p>\n<\/td>\n

\n

0.42 \u00b1 0.07<\/p>\n<\/td>\n

\n

0.08 \u00b1 0.02<\/p>\n<\/td>\n<\/tr>\n

\n

72 hrs<\/p>\n<\/td>\n

\n

0.41\u00b1 0.09<\/p>\n<\/td>\n

\n

0.08 \u00b1 0.01<\/p>\n<\/td>\n<\/tr>\n

\n

Group 3-<\/p>\n

PJ extract<\/p>\n<\/td>\n

\n

24 hrs<\/p>\n<\/td>\n

\n

0.49 \u00b1 0.09<\/p>\n<\/td>\n

\n

0.38<\/p>\n<\/td>\n

\n

0.10 \u00b1 .05<\/p>\n<\/td>\n

\n

0.76<\/p>\n<\/td>\n<\/tr>\n

\n

48 hrs<\/p>\n<\/td>\n

\n

0.47 \u00b1 0.07<\/p>\n<\/td>\n

\n

0.09 \u00b1 .04<\/p>\n<\/td>\n<\/tr>\n

\n

72 hrs<\/p>\n<\/td>\n

\n

0.42 \u00b1 0.10<\/p>\n<\/td>\n

\n

0.08 \u00b1 0.05<\/p>\n<\/td>\n<\/tr>\n

\n

Group 4-<\/p>\n

Control (Pre induction)<\/p>\n<\/td>\n

\n

NA<\/p>\n<\/td>\n

\n

0.32 \u00b1 0.06<\/p>\n<\/td>\n

\n

NA<\/p>\n<\/td>\n

\n

0.06 \u00b1 0.02<\/p>\n<\/td>\n

\n

NA<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure <\/strong>7<\/strong>: C-reactive protein<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure <\/strong>8<\/strong>: Ferritin<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure <\/strong>9<\/strong>: IL-6<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure <\/strong>10<\/strong>: IL-10<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table 5: TNF- \u03b1 & INF- \u03b3<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

 <\/p>\n

Groups<\/strong><\/p>\n<\/td>\n

\n

Subgroups<\/strong><\/p>\n<\/td>\n

\n

TNF-\u03b1<\/strong><\/p>\n<\/td>\n

\n

INF-\u03b3<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

TNF-\u03b1 (pg\/ml)<\/strong><\/p>\n

(Mean\u00b1SD)<\/strong><\/p>\n<\/td>\n

\n

P value<\/strong><\/p>\n

(Inter Sub<\/strong><\/p>\n

Group)<\/strong><\/p>\n<\/td>\n

\n

P value<\/strong><\/p>\n

(Inter <\/strong><\/p>\n

group) <\/strong><\/p>\n<\/td>\n

\n

INF-\u03b3 (pg\/ml)<\/strong><\/p>\n

(Mean\u00b1SD)<\/strong><\/p>\n<\/td>\n

\n

P value<\/strong><\/p>\n

(Inter Sub Group)<\/strong><\/p>\n

(One way ANOVA)<\/strong><\/p>\n<\/td>\n

\n

P value<\/strong><\/p>\n

(Inter <\/strong><\/p>\n

group) <\/strong><\/p>\n

(One way ANOVA)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Group 1-<\/p>\n

Inflammation Control<\/p>\n<\/td>\n

\n

24 hrs<\/p>\n<\/td>\n

\n

238.23 \u00b1 16.98<\/p>\n<\/td>\n

\n

0.37<\/p>\n<\/td>\n

\n

<0.0001*<\/p>\n<\/td>\n

\n

240.52 \u00b1 15.38<\/p>\n<\/td>\n

\n

0.30<\/p>\n<\/td>\n

\n

<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

48 hrs<\/p>\n<\/td>\n

\n

249.88 \u00b1 22.54<\/p>\n<\/td>\n

\n

251.86 \u00b1 20.98<\/p>\n<\/td>\n<\/tr>\n

\n

72 hrs<\/p>\n<\/td>\n

\n

253.92 \u00b1 18.21<\/p>\n<\/td>\n

\n

256.88 \u00b1 17.64<\/p>\n<\/td>\n<\/tr>\n

\n

Group 2-<\/p>\n

Hydrocortisone<\/p>\n<\/td>\n

\n

24 hrs<\/p>\n<\/td>\n

\n

187.87 \u00b1 11.09<\/p>\n<\/td>\n

\n

<0.0001*<\/p>\n<\/td>\n

\n

188.32 \u00b111.25<\/p>\n<\/td>\n

\n

<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

48 hrs<\/p>\n<\/td>\n

\n

168.92 \u00b1 12.11<\/p>\n<\/td>\n

\n

164.56 \u00b1 12.87<\/p>\n<\/td>\n<\/tr>\n

\n

72 hrs<\/p>\n<\/td>\n

\n

138.86 \u00b1 10.99<\/p>\n<\/td>\n

\n

136.52 \u00b1 10.12<\/p>\n<\/td>\n<\/tr>\n

\n

Group 3-<\/p>\n

PJ extract<\/p>\n<\/td>\n

\n

24 hrs<\/p>\n<\/td>\n

\n

187.85 \u00b1 10.97<\/p>\n<\/td>\n

\n

0.04*<\/p>\n<\/td>\n

\n

188.36\u00b1 10.88<\/p>\n<\/td>\n

\n

0.02*<\/p>\n<\/td>\n<\/tr>\n

\n

48 hrs<\/p>\n<\/td>\n

\n

179.98 \u00b1 13.22<\/p>\n<\/td>\n

\n

178.04\u00b1 12.45<\/p>\n<\/td>\n<\/tr>\n

\n

72 hrs<\/p>\n<\/td>\n

\n

168.67 \u00b1 12.14<\/p>\n<\/td>\n

\n

166.92\u00b1 12.98<\/p>\n<\/td>\n<\/tr>\n

\n

Group 4-<\/p>\n

Control (Pre induction)<\/p>\n<\/td>\n

\n

NA<\/p>\n<\/td>\n

\n

112.87 \u00b1 10.21<\/p>\n<\/td>\n

\n

NA<\/p>\n<\/td>\n

\n

132.48 \u00b1 11.24<\/p>\n<\/td>\n

\n

NA<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Post hoc analysis<\/p>\n

TNF- <\/strong>\u03b1<\/strong><\/p>\n

G1 24 vs Control: p<0.00001*, G2 24 vs Control: p<0.00001*, G3 24 vs Control: p<0.00001*, G2 48 vs Control: p<0.00001*, G2 72 vs Control: p=0.07, G3 72 vs Control: p<0.00001*, G2 48 vs G3 48: p=0.94, G2 72 vs G3 72: p=0.02*<\/p>\n

INF- \u03b3<\/strong><\/p>\n

G1 24 vs Control: p<0.00001*, G2 24 vs Control: p<0.00001*, G3 24 vs Control: p<0.00001*, G2 48 vs Control: p=0.007*, G2 72 vs Control: p=1.00, G3 48 vs Control: p<0.00001*, G3 72 vs Control: p=0.003*, G2 48 vs G3 48: p=0.80, G2 72 vs G3 72: p=0.01*<\/p>\n\n\n\n
\"\"<\/td>\n\n

Figure <\/strong>11<\/strong>: Histopathology of vital organs (at 72 hours after LPS injection) <\/strong>Discussion:<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

Traditional medicines\nhave been used for treating variety of ailments including acute and chronic\ninflammatory conditions.15, 16<\/sup>\nIn this study, we evaluated the efficacy and safety of methanolic extract of PJ\nin comparison to Hydrocortisone. We studied a few clinical parameters,\nbiochemical &haematological assessments and inflammatory markers in all the\ngroups. The treatment was started prior to induction of SIRS and continued for\nup to 24, 48 and 72 hours. All the rats in groups 1-3 developed SIRS, as\nevident from increase in the body temperature and total WBC count at 24 hours\nafter LPS injection, both of which are important in diagnosing SIRS.<\/p>\n\n\n\n

Increase in body\ntemperature is a critical criterion in SIRS. The inflammation control group\nshowed hyperpyrexia which persisted till 72 hours (end of the study) after SIRS\ninduction. Hydrocortisone treated rats exhibited gradual reduction in the body\ntemperature at 48 and 72 hours, compared to 24 hours after LPS injection.\nThough not highly significant, PJ treated animals also showed improvement in\nfever. The ethanolic extract of PJ was evaluated for its anti-pyretic activity\nby Gopinath S.M et al., in 2013, in brewer\u2019s yeast inducted hyperthermia in\nrats and they observed that PJ extract at doses of 250 and 500 mg\/kg exhibited\npotent anti-pyretic activity, similar to the standard drug paracetamol (150\nmg\/kg).17<\/sup> Our findings in the\npresent study are consistent with Gopinath S.M et al., where PJ could be used\nin hyperpyrexia.<\/p>\n\n\n\n

Anemia is one among\nthe most significant characteristics of systemic inflammation. Hepcidin is a\nhormone that regulates iron and is produced by hepatocytes. The cytokines that\nare increased in excess amounts, as a result of systemic inflammation tend to\nincrease hepcidin levels. Hepcidin inhibits the absorption of iron in the\nduodenum and the release of iron from the macrophages in the spleen, which\ncauses anaemia.18<\/sup> Hb level was\nsignificantly reduced in inflammation control as a result of SIRS, which is\nalso associated with increased levels of inflammatory cytokines in the blood,\nbut treatment with hydrocortisone and PJ extract improved the Hb, comparable to\ncontrol. This could be due to the reduction in the levels of cytokines in the\ntreatment groups.<\/p>\n\n\n\n

It is a known fact that\nWBC count should be <4000 or >12000 in SIRS.1<\/sup> In\nour study, we observed an elevated WBC count >12000 in the inflammation\ncontrol throughout the study at 24, 48 and 72 hours, and the treatment groups\nat 24 hours. Hydrocortisone reduced the WBC count significantly at 72 hours (TC\nreduced to less than 12000). In PJ treated rats, statistically significant\ndifference was not seen, though the total count at 48 and 72 hours was\nnumerically less when compared to 24 hours. <\/p>\n\n\n\n

When the renal function\ntests (Urea and Creatinine) were analysed, both the paraments showed\nsignificant increase in inflammation control group, which could be due to\ndamage to the kidney as a result of SIRS. The treatment with hydrocortisone and\nPJ did not significantly reduce urea levels. But the creatinine levels in the\ntreatment groups showed significant reduction at 72 hours after LPS injection\nie. Hydrocortisone and PJ reduced the creatinine levels significantly, which\nindicates that PJ exerts protective effect against SIRS induced renal damage\nsimilar to Hydrocortisone, which could be due to the antioxidant and\nanti-inflammatory properties.10<\/sup><\/p>\n\n\n\n

Derangement of liver\nenzymes is common in sepsis and SIRS, as liver has two opposite roles. It acts\nnot only as a source of inflammatory mediators but also a target organ for\ninflammatory cytokines mediated damage. Progressive liver damage can occur in\nSIRS as a part of MODS, sometimes leading to fulminant hepatic failure.\nIncrease in bilirubin levels is not usually proportionate to the enzyme levels\nand isolated hyperbilirubinemia is a rare finding in SIRS.19<\/sup> LPS induced SIRS in mice models showed\nelevated alanine transaminase enzymes starting from 2 hours and peaking in 24\nhours.13<\/sup> In our study, we could observe an increase in total\nbilirubin after inducing SIRS. Though the values are statistically significant,\nit is not having much of clinical relevance as the values fall within the\nacceptable range. The liver enzymes (AST, ALT and ALP) were elevated in the\ninflammation control but in hydrocortisone and PJ treated groups, there was a\nsignificant reduction at 72 hours compared to 24 hours after LPS injection. PJ\nleaf extract, in addition to anti-inflammatory activity, has free radical\nscavenging action and protects against liver damage by reducing the expression\nof pro-inflammatory cytokines in the liver.11<\/sup>\n<\/p>\n\n\n\n

Serum ferritin is an\nacute phase reactant, and the levels correlate with the degree of acute and chronic\ninflammation. It may be used as biomarker of uncontrolled inflammation and to\nassess the effectiveness of an intervention in inflammation.20<\/sup> In our study, we observed elevation in\nthe ferritin levels after injecting LPS in inflammation control and PJ groups.\nFerritin level was similar to control in hydrocortisone treated rats.\nHydrocortisone and PJ treated groups showed reduced ferritin levels at 72 hours\ncompared to 24 hours post LPS injection. Though PJ group did not have similar\nferritin levels at 24, 48 and 72 hours as that of Hydrocortisone, within group\nanalysis showed that the levels were significantly reduced at 72 hours compared\nto 24 hours.<\/p>\n\n\n\n

Serum CRP can be used as\nan early marker of inflammation in SIRS patients, as it increases within 24\nhours of onset of SIRS.21<\/sup>\nSimilar to ferritin, CRP levels elevated after LPS injection in all the groups\nat 24 hours, which gradually decreased at 48 hours and 72 hours. Hydrocortisone\nstarted reducing the CRP levels at 48 hours as compared to PJ, where the\nreduction was highly significant at 72 hours after induction of SIRS. <\/p>\n\n\n\n

With regard to the\ninflammatory cytokines analysed in our study (IL-6, IL-10, TNF-\u03b1 and INF-\u03b3),\nall the inflammatory markers were elevated after SIRS induction. However,\ntreatment with hydrocortisone and PJ reduced the levels of these\npro-inflammatory cytokines.  The\nanti-inflammatory effect of PJ was proven in carrageenan paw edema model in\nrats, where the PJ leaf extract given in the doses of 100, 200 and 400 mg\/kg\nshowed potent anti-inflammatory activity in terms of reduction in the paw\nvolume.22<\/sup> The findings of\nChoudhary PK et al., are supported by our current research, where PJ leaf\nextract has shown favourable effect on the pro-inflammatory markers, in SIRS.\nThus, PJ leaf extract may be considered as a potential therapeutic option in\nacute inflammation. <\/p>\n\n\n\n

The in-hospital mortality\nrate in sepsis is about 36.9% and the duration of SIRS before the onset of\norgan failure is found to be an important prognostic factor in sepsis.23<\/sup> Hence, prompt intervention and early\ntreatment of SIRS is essential in reducing the morbidity and mortality in\nsepsis. In addition to ensuring hemodynamic stability with IV fluids,\nvasopressors and inotropes may be used in some cases. If SIRS is due to sepsis,\nbroad spectrum antibiotics would be used. Steroids in low doses such as 200 to\n300 mg of hydrocortisone or other equivalent improve the survival rate in SIRS\nand sepsis. The cytokines including IL-1 and TNF-alpha are the early mediators\nof SIRS and their role is of paramount importance in shifting towards a\npro-inflammatory drive. These two mediators in turn stimulate NF-KB which will\ninduce massive release of other cytokines like IL-6, IL-8 and interferon gamma.\nIn order to antagonize the effect of these pro-inflammatory cytokines, a\ncompensatory anti-inflammatory response syndrome (CARS) is activated, which is\nmediated by the release of anti-inflammatory cytokines such as IL-4 and IL-10.\nThe balance between SIRS and CARS decides the rate of inflammation and organ\ndamage.1<\/sup> Hence, any treatment option that is able to combat the\nactive inflammation in SIRS by inhibiting the pro inflammatory cytokines could\nbe used in addition to other supportive treatment in SIRS.<\/p>\n\n\n\n

In this study, we were\nable to get potential information with regard to the efficacy of PJ towards\nreducing the clinical symptoms and inflammatory markers in SIRS. With the\nadvent of COVID-19, focus has been shifted towards the traditional medicine and\nAYUSH treatments for ameliorating the symptoms associated with infection and\ninflammation. <\/p>\n\n\n\n

PJ leaf extract exhibited\nfavourable effect on the clinical symptoms, liver enzymes, kidney function and\ninflammatory markers in SIRS. It was safe at the dose used and hence could be\nconsidered for further evaluation in severe acute and chronic inflammatory\nconditions. The major limitation of this study is that the biochemical,\nhaematological and inflammatory markers were analyzed only once at different\ntime points of sacrifice ie., 24, 48 and 72 hours in the sub-groups. Baseline\nand end of study comparison in the same animal was not done, as we need more\nthan 5 ml of blood to do all these assessments, which is not possible with tail\nvein or retro orbital puncture when the animal is alive. The required amount of\nblood was collected through cardiac puncture after sacrificing the animals.\nMore precise data could have been obtained if before-after comparisons were done.\nBut still, this study was able to generate data to show the efficacy of PJ in\nSIRS as we had inflammation control and normal control rats, which we used for\ncomparing the data.<\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

Based on the results observed in this study,\nit was noted that PJ exhibited anti-inflammatory activity at a dose of\n4mg\/kg\/day in terms of improvement in body temperature, total WBC count and\ninflammatory markers. In addition, PJ was found to have a protective role\nagainst SIRS induced renal and liver damage, as indicated by the improvement in\ncreatinine and liver enzymes. No mortality was seen in any of the groups. The\neffect of PJ on most of the parameters assessed was comparable to\nhydrocortisone. Hence, it can be concluded that PJ has a potential therapeutic\nrole in severe acute inflammatory conditions like SIRS. <\/p>\n\n\n\n

Acknowledgement<\/strong><\/p>\n\n\n\n

The authors thankfully acknowledge the Indian Council of Medical Research (ICMR), India for providing financial assistance to this research study. The authors also thank Chettinad Academy of Research and Education for supporting them in conducting this research study.<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

There is no conflict of interest.<\/p>\n\n\n\n

Funding Source<\/strong>s<\/p>\n\n\n\n

The research was partially funded by ICMR\nunder \u201cFinancial support for MD\/MS\/DM\/MCh\/DNB\/DrNB\/MDS thesis\u201d scheme [Ref no:\n3\/2\/Dec-2020\/PG-Thesis-HRD (30)].<\/p>\n\n\n\n

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