INTRODUCTION:

Systemic bacterial infections caused by Extensive Drug Resistant (XDR) gram-negative bacilli bacterial pathogens are major risk factors for out breaking of hospital-acquired and community infections¹.

Multiple drug-resistant Enterobacteriaceae of different E.coli pathogenic strains such as O157:H7, O104:H4, O104:21, 018:K1 lethal toxins caused for bloody diarrhea, hemolytic uremic syndrome (HUS)². In September 2006, E coli O157:H7 strain was an endemic outbreak in the farm of San Benito County, California, North America from organic uncooked spinach and caused foodborne illnesses, 199 people, including 31 people were severely suffered from kidney failures with HUS and among three (3) of them died³. In the month of May 2011 a novel strain E coli O104:H4 strain was an outbreak in an organic farm in Bienenbüttel, Lower Saxony in the Northern Germany from contaminated vegetables and slowly it became epidemic spread all over Europe and some parts of USA causing infection in a total 2989 people among them 855 people suffered from HUS with chronic kidney failure and among fifty-three (53) people have died^4,5.

Some of the pathogenic E coli strains are producing extended-spectrum β lactamases (ESBLs), showed highly resistant to a wide range of antibiotics such as penicillin, cephalosporin, ceftazidime, ceftriaxone, cefepime by hydrolyzing or modifying the beta-lactam and oxyiminobeta- lactam ring structures have emerged within the community setting as an important cause of gastroenteritis, urinary tract infections, neonatal meningitis, hemolytic uremic syndrome, peritonitis, septicemia, and pneumonia^6,7. Abuse use and the long term administration these chemotherapeutic antibiotics in the treatment of Entero bacteriaceae mediated bacterial infections can cause the suppression of both innate and adaptive immune response against bacterial infections⁸, adverse effect on normal flora⁹ and induce upregulation of inflammatory mediators such as Interleukin-1β, Interleukin-6, Tumor necrosis factor-α and Nitric oxide of the host results in susceptibility of systemic infections causes the pathogenesis of peritonitis, cholecystitis, septic wounds and bedsores¹⁰. Peritosis is the most common bacterial sepsis causing a high rate of morbidity and mortality. Among the different bacterial pathogenic strains, E.coli remains the most common cause of intraperitoneal infections¹¹.

Traditionally some of the medicinal plants used for the treatment of inflammatory related infectious diseases such as sepsis and abdominal infection such as bacterial induced peritonitis¹². Medicinal plants contained diversified secondary metabolites showing significant antimicrobial activity against infectious bacterial, fungal pathogens and showed Immunomodulatory activities by stimulating phagocytosis, complement activation and suppression of Lipopolysaccharide induced production of proinflammatory mediators and suppression of Ig-E antibody production by B-cells¹³.

Sida schimperiana Hochst. A. Rich, belongs to the family Malvaceae, widely distributed in Ethiopia, Eritrea, Uganda, Kenya, North Tanzania, and India¹⁴. In Ethiopia it is usually well known as Chifrig in Amharic and has been used as traditional medicine for the treatment of wound healing, gastrointestinal and skin related infection¹⁵.

In Tigray region of Ethiopia, the fresh leaf powder of S.schimperiana used as a topical application on external injury for blood clotting. Solomon Yeabyo et.al.,¹⁶ reported that the S. schimperiana root chloroform (SSRCH) extract showed promising broad spectrum of in vitro antibacterial activity against tested multiple drug resistant gram negative bacteria such as E. coli (0.625μg/ml ), V. cholera (24.87μg) and gram positive bacteria E. faecalis (11.24μg) and respectively with less MIC values due to presence of secondary metabolites such as tannins and phenolic compounds, terpenoids, flavonoids, alkaloids and cardiac glycosides. In the present study was evaluate the in vivo immuno protective evidences of chloroform root extract of S. schimperiana on E.coli induced peritonitis in albino Wistar rats in relation to immuno stimulant properties by exploring the neutrophil function.

MATERIALS AND METHODS:

Plant Material Collection and Authentication:

The fresh roots of S. schimperiana were collected from around Adwa town, Central Zone of Tigray region, Northern Ethiopia in February 2018. The plant material was authenticated by National Herbarium, Department of Biology, Addis Ababa University, Ethiopia. A herbarium of the selected plant was submitted to the Department of Biology and given the voucher specimen number was SY 001.

Chemical and Regents:

All the chemicals used in this study were obtained from HiMedia Laboratories Pvt. Ltd. (Mumbai, India), Sigma-Aldrich Chemical Co. (Milwaukee, WI, USA). All the chemicals used in this study were of analytical grade.

Preparation of Plant Extract

The roots of S. schimperiana were washed 3-4 times with running tap water followed by sterile distilled water. Then, roots were cut into small pieces, spread out on paper sheets and it was dried in a shaded area at room temperature of 25°C for 15 days. The roots were coarsely powdered and used for extraction.

About (100 gm) of the powdered plant root material was packed into a thimble and extracted with using 500ml of chloroform by using Soxhlet’s apparatus. The Soxhlet’s extraction processes were carried out until the solvent in the siphon tube of Soxhlet’s apparatus become colorless. The extracts were filtered and concentrated using a rotary evaporator under reduced pressure, the extracts obtained were dried and stored at 40⁰C¹⁷. Then the yield value was calculated as

Extraction obtained

Percentage of yield = –––––––––––––––––––––– ×100

Total amount of material

Phytochemical Analysis:

The preliminary phytochemical analysis was carried out for the S. schimperiana root chloroform extract which shown the in vivo immunoprotective against E.coli 018:K1 induced peritonitis in albino Wistar rats to identify different phytoconstituents present in the plants¹⁸.

Acute Toxicity Assay (OECD 423, 2001):

The acute toxicity assay used to determine the lethal dose (LD₅₀) of S. schimperiana root chloroform extract, according to the methods described by the Organization for Economic Co-operation and Development (OECD) guidelines. The six weeks old male albino Wistar rats of weighing 200 ± 25gm were obtained from the Pinnacle Biomedical Research Institute, Bhopal (M.P). The animal experiment was approved by the Institutional Animal Ethics Committee (IAEC) of Pinnacle Biomedical Research Institute Bhopal (Reg. No.1824/PO/ERe/S/15/CPCSEA). Protocol Approval Reference No. PBRI/IAEC/17059. Toxicological studies help to decide whether a new drug should be adopted for clinical use or not. Each group contains three animals (n=3) the animals were kept fasting for overnight.

The determination of acute toxicity was performed by observation of the Wistar rats that were given S. schimperiana root chloroform extract for 14 days¹⁹. A total of fifteen (15) healthy Wistar rats were divided into five groups, each group contains three rats. Group-I was used as a control group and four other groups (II- V) were treated with S. schimperiana root chloroform extract with each dose of 5mg/kg, 50mg/kg, 300mg/kg, and 2000mg/kg, body weight administered by oral. The Wistar rats in all groups were observed daily for 14 days to determine the symptoms of toxicity, including death.

The mortality and/or the moribund status of the animal during this period were recorded. The determination of the acute toxicity by adopting fixed-dose as per the guidelines of CPCSEA and 1/10th, 1/5th of LD₅₀, cut-off values of the plant extract was selected as screening doses for E. coli induced in vivo model of peritonitis in albino Wistar rats.

E. Coli Induced Peritonitis in Albino Wistar Rats.

Preparation for Bacterial Inoculum:

In brief, E. coli strain 018:K1 were grown on Nutrient broth Medium (3g of beef extract, 5g of peptone and 5g of NaCl, pH 7, sterilized by autoclaving at 121°C temperature and 15 lbs pressure for 30 min.) from a single colony and incubated for 16–18 hrs at 37°C to obtain mid lag phase cultures. Then the bacteria were centrifuged at 200rpm for 10 min at 4°C and the pellets were resuspended in PBS to an OD of 0.1 at 660nm, with a spectrophotometer, corresponding to 2X10⁴ CFU/ml²⁰.

Systemic Infection of Peritonitis by E. Coli²¹

E. coli induced in vivo model of peritonitis used for the evaluation of the antimicrobial and protective potential of S. schimperiana root chloroform extract. The animals were divided into four groups each group containing five animals (n=5).

Group-I Control (Normal Saline+ E. coli (i.p.)

Group-II Standard Drug (Ofloxacin 5 mg/kg body weight (p.o.)+ E. coli (i.p.)

Group-III S. schimperiana root chloroform extract (200 mg/kg body weight (p.o.)+ E. coli (i.p.)

Group-IV S. schimperiana root chloroform extract (400 mg/kg body weight (p.o.)+ E. coli (i.p.)

The experimental design was involved administration of each of the three testing samples (200mg and 400mg/kg body weight) of selected plant extract i.e, S. schimperiana root chloroform extract and Standard antibiotic Ofloxacin 5mg/Kg body weight by daily oral dose (p.o.) for a period of 7 days, dosing regimens was started on day -1,-2, -3, 0, 1, 2 and 3 relative to the day of challenge (day 0) with 2×10⁴CFU of E. coli CFU/ml (i.p.).

Monitoring of Mortality:

The survival studies of 6 rats per treatment group were inoculated with E. coli and the mortality occurs primarily between 24 and 48 hrs after infection. The mortality was assessed every 2 h in this period, thereafter mortality was monitored every 6 hrs. The Survival was monitored for all experimental groups until 14 days²².

Test of Bacterial Clearance from Peritoneal Cavity:

To assess the possible effect of S. schimperiana root chloroform extract on E. coli induced peritonitis, the animals (n=5per group) were orally pre-treated with the extract and intra-peritoneal administrated with 2×10⁴ CFU/ml of E. coli in 200μl of sterile PBS after 48 hours of oral treatment²¹.

Sample Harvesting:

After the monitoring of the mortality, the treated (Groups I-IV) rats were sacrificed, and assess the in vivo antibacterial activity of S. schimperiana root chloroform extract by determination of CFU/ml. All the four groups of treated rats were anesthetized by inhalation of diethyl ether and the peritoneal cavity was washed with 5ml of sterile PBS saline by using an 18-gauge needle and peritoneal lavage fluid was collected in sterile tubes for determination of CFU²².

Determination of CFU:

Fifty microliters (50µl) of peritoneal lavage fluid from all tested Groups I-IV were placed on ice and serially diluted with sterile saline. Twelve (12µl) microliters of each dilution were spread on sterile nutrient agar plates and incubated overnight at 37°C after which the number of colonies was counted²⁰.

Neutrophils Function Test¹⁹:

Adult male Wister rats were weighing about 200 ± 25g were divided into four groups of 5 animals each. The dosages of S. schimperiana root chloroform extract administered to the different groups were as follows:

Group-I received normal saline for 14 days

Group-II received Standard Drug (ofloxacin 5mg/kg body weight (p.o.) for 14 days

Group-III received (SSRCH – 200mg) S. schimperiana root chloroform extract (200mg/kg body weight (p.o.) for 14 days

Group-IV received (SSRCH-400mg) S. schimperiana root chloroform extract (400mg/kg body weight (p.o.) for 14 days

The rats were treated orally with Standard Drug (ofloxacin 5mg/kg, S. schimperiana root chloroform extract at the doses of 200 and 400mg/kg/day for 14 days .On the 14th day the blood samples were collected from the retro-orbital plexus in heparinized vials and analysed for total leukocyte count (TLC) using an automatic cell counter analyser. The differential leukocyte count (DLC) was performed by fixing the blood smear and staining with Leishman’s stain, and percentage neutrophils in each sample were determined.

After the initial counts, blood samples were incubated with 80mg nylon fibres/ml for 15 min at 37^oC. The incubated blood samples were again analysed for TLC.

The Product of TLC and Percentage Neutrophils Gave the Neutrophil Index of Blood Sample.

The percent neutrophil adhesion was calculated as follows:

Neutrophil adhesion (%) = (NIu- NIt) /NIu X 100

NIu = Neutrophils index of the untreated blood sample

NIt = Neutrophil index of treated blood sample

Statistical Analysis:

The results obtained in this study were expressed as a mean inhibition zone (mm) ± S.D of three replicates. The statistical difference between the test and control groups were evaluated by one-way analysis of variance (ANOVA) by Graph pad prism 6.0 software and followed by Turkey multiple comparison t-tests .*p≤0.05, **p≤0.01, ***p≤0.001 represents a significant difference between the control with the test group.

RESULTS:

Table -1: Colour, Consistency and Percentage yield (w/w) of S. schimperiana root chloroform extract

Extract	Colour	Consistency	Percentage of yield (g/100g)
Chloroform extract	Dark green	Non – Sticky	2.9

As showed in table-1, the dried root chloroform extract of S. schimperiana reported a percentage yield of 2.9 g/100g and shown dark green color appearance with non-sticky consistency.

Qualitative Phytochemical analysis of S. schimperiana root chloroform extract:

The qualitative phytochemical analysis of chloroform root extracts of S. schimperiana as shown in table-2, presence of secondary metabolites such as alkaloids, flavonoids, Tannin and Phenolic Compounds, terpenoid, Saponins, Steroids.

Table-2: Preliminary phytochemical screening of S. schimperiana root chloroform extract

S. No	Phyto-constituents	Test	Result
1	Alkaloids	Dragenderoff’s Test	+
2	Flavonoids	Lead acetate test	+++
3	Phenolic Compounds	Ferric Chloride Test	+++
4	Tannins	Gelatin Test	+++
5	Terpenoids	Horizon Test	++
6	Saponins	Frothing Test	++
7	Steroids	Liebermann-Burchards Test	++

(+) score indicate slight positive reaction for Secondary metabolites

(++) score indicate definitive positive reaction for Secondary metabolites

(+++) indicate significant reactions were obtained for Secondary metabolites

Acute Toxicity Studies of S. schimperiana Root Chloroform (SSRCH) Extract:

Toxicology is an important aspect of ethnopharmacology, that deals with evaluate the safety potential of bioactive substances on living organisms before the development and used as a drug. An acute toxicity study was carried out according to the Organization for Economic Co-operation and Development (OECD) guidelines. In acute toxicity studies, no mortality was observed for 24-48 hours in mice for different doses (5-2000 mg/kg bw) of S. schimperiana root chloroform (SSRCH) extract. The lethal dose (LD50) cut-off doses obtained S. schimperiana root chloroform (SSRCH) extracts were shown in table -3. Based on these LD50 values, two doses (200 and 400mg/kg body weight) of S. schimperiana root chloroform were selected for effective doses for E. coli induced in-vivo model of peritonitis in male albino Wistar rats.

Table-3: Acute oral toxicity study of S. schimperiana root chloroform (SSRCH) extract

S. schimperiana root extract

LD₅₀Cut off Dose along with 1/10^th and 1/5^th of Extracts

ED₅₀Effective Dose

LD₅₀Cut Off Dose

1/10^thof LD₅₀

1/5^thof LD₅₀

SSRCH

2500 mg/kg body weight

250mg/kg

body weight

500mg/kg

body weight

250mg/kg body weight

The effect of S. schimperiana root chloroform (SSRCH) extract on percentage of mortality rate on E. coli induced peritonitis in albino Wistar rats:

The in vivo antibacterial effect of S. schimperiana root chloroform (SSRCH) extract on E. coli induced peritonitis in albino Wistar rats were shown in the table-4. SSRCH-200mg and 400mg/kg bw shows the maximum protection of Wistar rats against E. coli induced peritonitis through by exhibiting the in vivo antibacterial activity and maintain the survivability of Wistar rats between 20% and 60% respectively shown in table-5.

Table-4: The effect of S. schimperiana root chloroform (SSRCH) extract on percentage of mortality rate (protective nature) in hours (1hr – 24 hr) and days (2^nd day – 10^th day) on E. coli induced peritonitis in albino Wistar rats.

Treatment Extracts	Mortality rate in Hours									Mortality rate in Days									Total Death
Treatment Extracts	0	1	2	4	8	12	16	20	24	2	3	4	5	6	7	8	9	10
Group-I	0	0	0	2	0	0	2	1	0	0	0	0	0	0	0	0	0	0	5
Group-II	0	0	0	0	0	0	0	0	0	0	0	0	1	0	0	0	0	0	1
Group-III	0	0	0	0	0	0	0	0	0	0	0	1	1	0	1	0	1	0	4
Group-IV	0	0	0	0	0	0	0	0	0	0	0	1	0	0	0	1	0	0	2

Group-I Control (Normal Saline+ E. coli (i.p.)

Group-II Standard Drug (Ofloxicin 5 mg/kg body weight (p.o.)+ E. coli (i.p.)

Group-III Extract (200 mg/kg body weight (p.o.)+ E. coli (i.p.)

Group-IV Extract (400 mg/kg body weight (p.o.)+ E. coli (i.p.)

In Group-I, all five animals have died within 24 hrs. The first 4th hr two animals have died, next 16th hr another two animals were died and finally the remained one animal has died in 20th hr after challenge (day 0) with 2×104 CFU/ml of E. coli (i.p.). In Group-II, four animals were survived up to ten days, but only one animal was died on 5th day after challenge (day 0) with 2×104 CFU/ml of E. coli .(i.p.) In Group-III, all five animals were survived up to three days, from the 4th day onwards animals were started to die, two animals died on 4th and 5th day, another two animals have died in 7th and 9th day after challenge (day 0) with 2×104 CFU/ml of E. coli (i.p.).

In Group-IV two animals died in the 4th and 8th day and the remaining three animals were survived after challenge (day 0) with 2×104 CFU/ml E. coli. (i.p)

Table -5: The effect of S. schimperiana root chloroform (SSRCH) extract on % of mortality rate on E. coli induced peritonitis in albino Wistar rats.

Group	Total no of animals	No of dead animals	% of mortality
Group I	5	5	100
Group II	5	1	20
Group III	5	4	80
Group IV	5	2	40

S. schimperiana chloroform root (SSRCH) extract 400 mg (Group-IV) showed protection against E. coli induced peritonitis in albino rats. Hence it can be concluded that SSRCH extracts showed promising in vivo antibacterial activity against E. coli induced peritonitis compared to Standard Drug (Ofloxicin 5 mg/kg bw) and maintaining 60% survivability. The results of in vitro antibacterial activity of S. schimperiana root chloroform extracts against the Gram-negative bacteria E. coli have been correlated with in vivo E. coli induced peritonitis in Wistar rats.

The effect of S. schimperiana root chloroform (SSRCH) extract on bacterial clearance in E. coli induced peritonitis in albino Wistar rats:

The rate of bacterial clearance in the peritoneal cavity of all the tested groups (Group –I to the group –IV) were represented in table -6 and Figure-1a and-1b. At the end of 8th hr, two rats were died in Group I (Control rats) due to bacteremia and after 20th hr, all rats have died with average bacterial count 273 C.F.U/ml in the peritoneal cavity.

In Group –II, ofloxacin treated rats, the average bacterial count 73.80 C.F.U/ml in the peritoneal cavity was decreased and one rat died in the 10th day. In Group –III, 200 mg/kg bw, S. Schimperiana root chloroform extract (SSRCH) treated rats four rats

were died on 4th, 5th 7th and 9th day respectively with average bacteria count 198.80 C.F.U/ml in the peritoneal cavity by showing *p≤0.05, significance difference compared with ofloxacin

treated Group –II.

In Group –IV, 400 mg/kg bw, S. Schimperiana root chloroform extract (SSRCH) treated mice, only two rats were died on 4th and 10th day respectively with average bacteria count 114.80 C.F.U/ml in the peritoneal cavity by showing **p≤0.01 significance difference compared with ofloxacin treated Group –II.

Table-6: Bacterial clearance from E coli Induced peritoneal cavity (Determination of CFU)

Control	ofloxacin 5 mg/kg bw	200 mg/kg bw extract	400 mg/kg bw extract
273.00 ± 14.491	73.80 ± 13.664	198.80 ± 5.762	114.80 ± 14.957

Figure-1a: The in vivo antibacterial activity of S. schimperiana root chloroform (SSRCH) extracts (200 mg/kg bw and 400 mg/kg bw) and standard antibiotic (ofloxacin (5 mg/kg bw) on bacterial clearance from peritoneal cavity in E. coli induced peritonitis in albino Wistar rats.

Values are mean of three replicates ± SEM. **p≤0.05, **p≤0.01, ***p≤0.001 represents a significant difference compared with the Ofloxicin (5 mg/kg bw)

Figure-1b: In vivo antibacterial activity of S. schimperiana root chloroform (SSRCH) extracts (Group III 200 mg/kg bw and Group -IV 400 mg/kg bw) and standard antibiotic (Group IV ofloxacin (5 mg/kg bw) on bacterial clearance from peritoneal cavity in E. coli induced peritonitis in albino Wistar rats.

Effect of S. schimperiana root chloroform extract (SSRCH) on neutrophil adhesion in Wistar rats.

Incubation of neutrophils with nylon fibers produced a decrease in the neutrophil counts due to adhesion of neutrophils to the fibers. As showed in the table-7, the percentage neutrophil adhesion in Group-I (control group animals) was noted to be 23.97±0.974, whereas, in Group-IV (SSRCH- 400 mg/kg) of the treated group it was found to be significantly increased **(P<0.01) levels of 36.14±5.609 as compared to their respective control group and in Group-III (SSRCH- 200 mg/kg), moderately increased *(P<0.05) in neutrophil adhesion as compared to control group i.e. (25.74±2.351) , Group II (ofloxacin 5 mg/kg ), the standard antibiotic drug slowly decreases percent neutrophil adhesion value to 18.11±3.655 (Figure-2).

Figure-2: Effect of S. schimperiana root chloroform extracts (SSRCH-200 and SSRCH-400 mg/kg bw) treatments on percentage of neutrophil adhesion. Each group consists of n=5 animals. Values are Mean ± S.E.M. P<0.05 is considered as significant. **P<0.01, *P<0.05 as compared to control group and Standard

DISCUSSION:

From the qualitative phytochemical analysis of the SSRCH, it showed the presence of plant-derived antimicrobial agents such as flavonoids, terpenoids, coumarins, and high phenolic content. The high phenolic and flavonoid content present in SSRCH accompanied by producing a significant zone of inhibition against all tested MDR bacteria¹⁶. Therefore the S. schimperiana chloroform root extract (SSRCH) was selected to evaluate protective potential against E. coli induced peritonitis in albino Wistar rats. Animal experiments (in vivo) have been extremely useful for conforming, in vitro biological activities of drugs or medicinal plants and evaluate the long term sustainability of drugs and understanding mode of action²³.

In acute toxicity studies, no mortality was observed for 24-48 hours in mice for different doses (5-2000mg/kg bw) of S. schimperiana root chloroform (SSRCH) extract. Based on these LD50 values, two doses (200 and 400mg/kg body weight) of S. schimperiana root chloroform were selected for effective doses for E. coli induced in vivo model of peritonitis in albino Wistar rats. S. schimperiana chloroform root (SSRCH) extract 400 mg (Group-IV) showed protection against E. coli induced peritonitis in albino rats by showing 60% survivability. The results of in vitro antibacterial activity of S. schimperiana root chloroform extracts against the Gram-negative bacteria E. coli have been correlated with in vivo E. coli induced peritonitis in Wistar rats. The methanolic extract of Trachy spermumammi exhibited significant protection against E. coli induced peritonitis in normal rats by reducing the viable cells of E. coli in rats²⁴.

From the bacterial clearance studies, it was demonstrated that pre-treatment with S. Schimperiana root chloroform extract 400mg/kg bw, protect the rats against E. coli induced peritonitis, comparable to standard antibiotic Ofloxicin by showing antibacterial activity, with average bacterial count 114.80 C.F.U/ml in peritoneal cavity. Several researchers reported that Synthetic antibiotics such tetracycline and streptomycin prevents the phagocytic chemotaxis, granuloma formation and inhibits the synthesis of interleukin-1 (IL-1) and tumor necrosis factor alfa (TNF-α)^25,26. A large population of the world uses plants for its healing, preventive, curative and much therapeutic property together with immunostimulatory property. The chloroform root extract of the S. schimperiana has been reported to possess both in vitro and in vivo antibacterial activity, there were no reports on immunostimulant properties is an innate defense mechanism against pathogenic and non-pathogenic agents. The phagocytic cells such as blood monocytes, neutrophils and tissue macrophages involved in phagocytosis²⁷.

Chloroform root extract (200 and 400 mg /kg ) of the S. schimperiana administered orally showed significant percentage of neutrophils adhesion. i.e 25.74±2.351 and 36.14±5.609 percentage of neutrophils adhesion compared to Group II treatment with (ofloxacin 5 mg/kg), the standard antibiotic drug slowly decreases percent neutrophil adhesion value to 18.11±3.655. Increase in adhesion of neutrophils to nylon fibres which correlates to the process of margination of cells in blood vessels. The neutrophil adhesion was significantly increased with the dose of 400 mg/kg/day when compared to untreated control and standard admistration of saponins of M. cymbalaria (50mg/kg per day/ and 100mg/kg per day 14days) significantly (p<0.001) increased neutrophils adhesion index values 20.82 and 25.17²⁸. The Neutrophils from untreated control mice shown 34.33 + /3.44% phagocytosis of E.coli, 3.5 hours after challenge as compared to this, phagocytic function of neutrophils of the T. cordifolia treated group was 53.66 +/5.68 % (p < 0.001), stimulated, as compared to control and that in the gentamicin treated group shown 19 +/2.75 as compared to control²⁹.

CONCLUSION:

The in vivo anti-bacterial activity studies (E. coli induced peritonitis in albino Wistar rats) revealed that the S. schimperiana chloroform root (SSRCH) extracts 200mg /kg bw (Group – III) and 400mg/kg bw (Group-IV) showed protection against E. coli induced peritonitis in albino Wistar rats by showing 20% and 60% survivability. It can be concluded that the S. schimperiana chloroform root (SSRCH) extracts 400 mg kg bw (Group-IV) showed profound in vivo antibacterial activity against E. coli induced peritonitis in Wistar rats, with average bacteria count 114.80 C.F.U/ml in the peritoneal cavity, comparable to standard antibiotic ofloxacin. Neutrophil adhesion in vivo experimental studies revealed that the chloroform root extract of the S. schimperiana (400mg/kg bw) showed a significant increase (**P<0.01) in adhesion of neutrophils to nylon fibers compare to standard. S. schimperiana (400mg/kg bw) chloroform root extract exhibiting significant in vivo antibacterial activity against E. coli induced peritonitis in albino Wistar rats, by showing increasing neutrophil adhesion.

Therefore, the in vivo studies demonstrated that the S. schimperiana chloroform root extract shown promising in vivo antibacterial activity by enhancing the immunostimuation effect. The results of the present study support the traditional use of S. schimperiana root in the treatment of various infectious diseases and also suggest that chloroform root extracts of S. schimperiana possess a broad spectrum of bioactive antibacterial agents.

ACKNOWLEDGMENTS:

The authors sincerely thanks to the Department of Chemistry, Aksum University, and Axum, Ethiopia for providing laboratory facilities, Medicinal plant was authenticated by National herbarium, Department of Biology, Addis Ababa University, Ethiopia.

FINANCIAL SUPPORT:

This project work was funded by Ministry of Education, Ethiopia in the form Student Project work.

CONFLICT OF INTEREST:

The authors confirm that this article content has no conflict of interest.

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Received on 22.04.2020 Modified on 28.05.2020

Research J. Pharm. and Tech. 2021; 14(4):2262-2269.

DOI: 10.52711/0974-360X.2021.00400

Animals	TLC (A)		Neutrophil % (B)		Neutrophil index (A×B)		Neutrophil Adhesion %
Animals	UB	NFTB	UB	NFTB	UB	NFTB	Neutrophil Adhesion %
Control	8.92±0.702	7.96±0.632	38.56±0.934	32.85±0.866	343.95±13.811	261.48±12.710	23.97±0.974
Standard	7.94±0.577	7.10 ±0.583	30.14±4.467	27.60±4.008	239.31±47.420	195.96±24.162	18.11±3.655
Extract 200 mg/kg	9.02±0.566	6.91±0.329	24.53±1.010	23.75±0.884	221.07±13.639	163.94±5.863	25.74±2.351
Extract 400 mg/kg	8.97±0.957	7.49±0.537	26.60±1.293	20.25±1.474	238.70±27.706	152.32±21.813	36.14±5.609