Investigating the Plant Growth Promoting and Biocontrol Potentiality of Endophytic Streptomyces sp. SP5 Against Early Blight in Tomato Seedling


 Background: Early blight (EB), caused by Alternaria solani, is one of the alarming diseases that restrict tomato production globally. Existing cultural practices and fungicide applications are not enough to control the early blight diseases. Therefore, the study aimed to isolate, identify, and characterize an endophytic Streptomyces strain exhibiting the potential to control early blight in tomato and also promote plant growth. Results: From a Citrus jambhiri leaf, an endophytic Streptomyces sp. SP5 isolated with antagonistic activity against Alternaria solani, Colletotrichum acutatum, Cladosporium herbarum, Alternaria brassicicola, Alternaria alternata, Fusarium oxysporum and Fusarium moniliforme. It was identified as a Streptomyces sp. designated as SP5 through 16S ribosomal DNA sequence analysis. It also produced 40μg/ml indole acetic acid which was further confirmed by TLC and HPLC analyses. Treatment of pathogen infected plants with Streptomyces sp. SP5 antagonist (culture cells/culture supernatant/solvent extract/ acetone precipitates) decreased the early blight disease incidence and significantly increased the various agronomic traits Conclusion: The present study concluded that Streptomyces sp. SP5 possessed antifungal activity against different fungal pathogens and had significant potential to control early blight disease in tomatoes and also promote plant growth by producing IAA.

The natural habitat of most Streptomyces is the soil where they may constitute from 1 to 20% of the culturable population, but Streptomyces are also abundantly present inside plant tissues as endophytes. To discover new biocontrol agents there is a need to screen new Streptomyces strains from untapped sources. Therefore, in the present study an endophytic isolate SP5, exhibiting antifungal and plant growth-promoting activities, was recovered from Citrus jambhiri leaves. The aim of this study was to characterize the isolate SP5 using a polyphasic approach and to assess its in vivo potential for controlling Alternaria solani caused early blight in tomato plants. The strain ability to promote plant growth in vivo was also assessed by observing its impact on various agronomic traits in the tomato plant.

Isolation and screening
Total of 42 different endophytic actinobacteria isolates were recovered from plant samples. Among these, 15 isolates demonstrated antifungal activity against one or more test fungi during primary screening, and 9 isolates were found to have antifungal activity in fermentation broth, inhibiting various test fungi to varying degrees. Isolate SP5 was selected for further studies because it exhibiting potent antifungal activity against all the tested phytopathogenic fungi.
Phenotypic and phylogenetic analysis of Streptomyces sp. SP5 Streptomyces sp. SP5 showed good growth on SCNA (Starch casein nitrate agar) and all ISP media except ISP2 medium. It exhibited varied cultural characteristics on different media (Table.2).
Pigmentation was not observed in any ISP medium. On SCNA medium strain showed white gray sporulation ( Fig.1a) with yellow aerial and substrate mycelia (Fig.1b). In the light microscope (100X), spore chains of SP5 were observed as exuous spore chains with a hook (Fig.1c) and it was assigned to the Streptomyces retinaculiaperti group (Li et al 2016). SEM micromorphological analysis showed chains of spores on aerial mycelium, each with 20-30 cylindrical spores (1.5-2.0 m length and 1.5 m width) and a smooth surface (Fig. 1 d). The chemotaxonomic analysis revealed the presence of LL-DAP (LLdiaminopimelic acid) in the cell wall, and no characteristic sugar was detected in whole-cell hydrolysate.
In physiological studies, Streptomyces sp. SP5 grew at temperature of 25-45 0 C (optimum at 28 0 C), pH of 5-10 (optimum at pH 7.0) and showed salt tolerance of up to 5%. In biochemical characteristics, strain SP5 hydrolyzed starch, cellulose, lipid, gelatin, and esculin but not able to hydrolyze casein. Strain SP5 showed positive results for the production of catalase, oxidase, hydrogen sulphide and citrase but gave negative results for both indole production and MRVP (Methyl Red-Voges-Proskauer) test. The strain was able to utilize most of the tested sugars as the sole carbon sources except arabinose (Additional le   Table S1). These cultural characteristics showed that SP5 belongs to the genus Streptomyces and was further con rmed by 16S rRNA sequencing.  Antifungal potential Streptomyces sp. SP5 showed antifungal activity against all the tested fungal phytopathogens. The production of active metabolites in SCN culture broth against fungal pathogens was detected after 48 h of incubation, maximum activity was reached after 120 hours, and then began to decrease as the incubation period was extended further. Additionally, during the 10-day activity pro le, there was a positive correlation between antifungal activity and biomass (Fig.3). The maximum biomass was obtained after the fth day of incubation, which coincided with the maximum activity obtained on the same day. In vitro bioassay demonstrated broad-spectrum antifungal activity of Streptomyces sp. SP5 against tested strains (Fig.4). Among the tested fungi, higher activity was observed against F. oxysporum, C. herbarum, F. moniliforme (25-28mm) as compared to A. brassicicola, A. alternata, C. acutatum, A. solani, and F. solani (20-25mm).

Active metabolite extraction
The active metabolites from culture supernatant were extracted in diethyl ether. After the concentration of the organic phase using rotavapor, a light-yellow colored crude extract was obtained, which was redissolved in chloroform. Diethyl ether solvent extract showed pronounced activity against phytopathogen A. brassicicola, A. solani, F. oxysporum, F. solani (15-20mm). However, the activity was also observed in the aqueous phase which indicated that the bioactive compounds were not fully recovered by solvent extraction. Therefore, another technique i.e., Acetone precipitation was employed to recover the bioactive metabolites from the culture supernatant. The active metabolites through precipitation showed antifungal activity against A. brassicicola, A. solani, C. acutatum, F. oxysporum, F. solani, and C. herbarum (20-30mm).

Stability of active metabolites in the culture supernatant
The stability of metabolites present in the culture supernatant was investigated under various physical stresses. Active metabolites remained completely stable at 37 0 C after 1h exposure. However, a loss of 20, 48, and 68 % in the residual activity was observed at 70ºC, 100ºC, and autoclaving (121 °C for 20 min), respectively. The metabolites remained active at extreme pH values (from 2 to 14) with 92 and 84% residual activity, respectively. The metabolites were photostable in both UV and sunlight (Table.3). Plant growth-promoting potential Streptomyces sp. SP5 produced 40μg/ml indole acetic acid in an initial screening for indole acetic acid production. TLC and HPLC analysis demonstrated the ability of strain Streptomyces sp. SP5 to produce IAA. The ethyl acetate crude extract displayed a clear pink-colored spot on the TLC plate after being treated with salkowski regent at Rf value corresponding to standard IAA (0.9). After the detection of IAA in TLC, partial puri cation of crude extract containing IAA was done using silica gel chromatography. HPLC analysis was used to precisely classify and quantify the IAA production. The partially puri ed extract and corresponding reference authentic standard both displayed peaks at the same retention time (18.6 min) as shown in Fig.5. Experiments were carried out with different values of the incubation period, temperature, pH, and L-tryptophan concentration for the highest IAA production by Streptomyces SP5. The effect of the incubation period, as shown in (Fig.6a), showed that IAA production began within 24 hours, increased steadily over time, reached a maximum of 58.43μg/ml after 6 days, then began to decline with additional incubation, reaching 5.45 μg/ml after 10 days. Within the pH range 4 to 10, the impact of pH on IAA production was investigated. The data in Fig.6b show the effect of pH on IAA production. Streptomyces SP5 produced the maximum IAA of 65.32 µg/ml at an initial pH of 7.0. Acidity and high alkalinity of the medium led to a marked decrease in growth and IAA production. Incubation temperature had a signi cant impact on IAA production. At 28ºC, the maximum production of 58.63 µg/ml IAA was achieved (Fig.6c). The effect of L-tryptophan concentration on IAA production were also investigated, and it was discovered that in the absence of L-tryptophan i.e. IAA precursor, the strain was unable to produce IAA. With the increase in concentrations of L-tryptophan from 0 to 5 mg/ml, there was an increase in the production, and the maximum biosynthesis of 99.65 µg/ml IAA was achieved at a concentration of 5 mg/ml L-tryptophan (Fig.6d). IAA production decreased slightly with higher Ltryptophan concentrations. Additionally, a positive correlation between IAA production and biomass was observed with optimization parameters.
Streptomyces sp. SP5 In vivo biocontrol of early blight diseases by and its impact on tomato plant growth promotion   Additional le Table S2, Fig: 8, 9). During the period of an experimental study, early owering and fruiting were also observed in plants treated with antagonists (culture cells/protein precipitates) both in the presence and absence of pathogen stress whereas no such stages were observed in control and pathogen infested plants. The data indicated the biocontrol and plant growth-promoting potential of Streptomyces SP5 and its metabolites.

Discussion
Fungal phytopathogens pose a signi cant threat to current agricultural systems around the world, since they are responsible for signi cant drop agricultural yield globally. Despite attempts to tackle them through the use of resistant crop varieties, chemical pesticides, and various other agricultural practices, there is widespread concern about public health and environment protection as a result of the harmful effect of agrochemicals (30,31). Thus, greater emphasis must be laid on controlling the phytopathogens by natural means. Therefore, there has been an increasing interest in the isolation of agriculturally important microbes from rare and scanty sources for their exploitation in biological control and plant growth promotion (32). Among microbes, actinobacteria, especially Streptomyces produce major bioactive metabolites which exert positively antagonistic effects on a wide range of plant pathogenic fungi (33)(34)(35)(36). Their lamentous and sporulating properties enable them to thrive in threatening environments (37). Agriculturally important antibiotics such as blasticidin S, kasugamycin, polyoxins, and validamycin A have been explored as commercial biological control agents from Streptomyces spp. (38). As actinobacteria are well known excellent source of bioactive compounds, it is predicted that such organisms from unique habitats may prove useful for this purpose, and one such habitat could be the plant endosphere. Researcher isolated endophytic actinobacteria from different plants and are thought to be new sources of various bioactive compounds that can be used to ght pathogenic microorganisms (39)(40)(41)(42). Multiple reports have described the isolation of Streptomyces from plant roots followed by stems and leave exhibits antifungal and plant growth-promoting activities (43,44).
In this research study, a Streptomyces sp., designated as Streptomyces sp. SP5, was isolated from leaves of Citrus jambhiri, and characterized through a polyphasic approach. shelf-life is dependent on environmental conditions such as soil temperature, soil pH, nutrient availability and water status, one of the major issues in the commercialization of biocontrol agents is their loss of viability over time (50). However, antifungal metabolites produced by Streptomyces sp.SP5 was found to be photo-thermostable and pH stable. These characteristics of metabolites make them suitable for the development of effective biocontrol agents for varied climate conditions throughout the globe.
The Streptomyces sp. SP5 also exhibited plant growth-promoting activity by synthesizing indole acetic acid (IAA). It is one of the most physiologically active auxins which stimulate and facilitate plant growth (51)(52)(53). Although several studies have reported IAA production in soil-dwelling Streptomyces spp. (52,54,55) and biosynthesis of IAA by endophytic Streptomyces (56-57). Streptomyces sp. SP5 has been found to produce a signi cant amount (40µg ml -1 ) of phytohormone IAA when grow n in YMB broth supplemented with tryptophan. TLC analysis revealed identical Rf value with IAA standards which is inconsistent with previous studies (52). HPLC is a reliable method for the identi cation of auxins thus characterization of IAA was performed by using HPLC. The retention times of partially puri ed peak of the extract was comparable to those of authentic IAA standard, con rming IAA production by Streptomyces sp. SP5. Results of the classical optimization experiment showed that the incubation time, temperature, and pH played an important role in IAA production which is in agreement with other reports (58,59). Plantassociated microbes synthesized IAA via tryptophan (trp)-dependent and -independent pathway (60). IAA production from Streptomyces sp. SP5 increased two-folds in the presence of L-tryptophan which strongly indicated that Streptomyces sp. SP5 synthesized IAA via the tryptophan-dependent pathway. It was also observed that beyond optimized concentrations of tryptophan IAA production were adversely affected in Streptomyces sp. SP5. Myo (52) also reported that higher tryptophan concentration affects the capability of strain to produce IAA. Manulis  control early blight, anthracnose and bacterial spot of eld tomatoes. Both BCAs strongly suppressed the growth of A. solani in dual culture agar diffusion bioassay. S. griseoviridis successfully controlled anthracnose and early blight diseases whether applied before pathogen or simultaneously with the pathogen but S. lydicus provided protection only when applied 48h before the exposer pathogen.
Moreover, during the pot trial Streptomyces sp. SP5 also signi cantly increased various growth parameters of the tomato plants such as shoot and root length in the absence of pathogen stress. These ndings support previous research that found Streptomyces spp. not only protected plants from pathogens, but also enhanced plant growth and physiology (63, 70-72). Dias (71) observed a signi cant increase in shoot length (1.7%-31.89%) and root length (49.15% -88.13%) of tomato plants treated with different Streptomyces isolates. In our previous studies, we reported Streptomyces sp. MR14, the cells and metabolites of which signi cantly enhanced various plant growth traits i.e. shoot length (32.36%), root length (59.88), shoot fresh weight (67.65), root fresh weight (73.97), shoot dry weight (50%) and root dry weight (116.88) in tomato plants (63). However, Streptomyces sp. SP5 reported in the present study showed highly pronounced effect on various plant growth parameters and increased shoot length, root length, shoot fresh weight, root fresh weight, shoot dry weight and root dry weight by 108.59%, 131.70%, 115.63%, 210.95%, 264.23% and 538.63%, respectively in tomato plants.

Conclusion
The results of in vitro and in vivo studies are highly signi cant and data reveals that Streptomyces sp. SP5 is superior to the previously reported Streptomyces spp. and might be used as a potential biological agent to control fungal phytopathogens and as an effective biofertilizer to promote plant growth.

Sample collection
Leaves and roots of healthy plants (Cinnamon basil, Ricinus communis, Epipremunum aureum, Citrus jambhiri, Hibiscus rosasinensis were collected from "Botanical Garden" of Guru Nanak Dev University, Amritsar, Punjab, India (31°37′45″N and 74°49′36″N). Plant samples were wrapped aseptically in plastic bags and taken to the laboratory where they were processed within 4 hours.

Isolation of endophytic strains from plant samples
The collected plant samples were washed under running tap water for 1-2 minutes to remove soil particles. The samples were then surface sterilized by 70% ethanol for 10 min followed by treatment with 1% sodium hypochlorite solution for 15 min (21). Plant samples were then repeatedly washed with sterilized water, air-dried in a laminar air ow hood, and cut into small pieces using a sterile razor blade.
The small pieces of sterile samples were placed on SCNA medium (starch casein nitrate agar). To inhibit the growth of fungi and other non-Streptomyces bacteria, cycloheximide (50g/ml) and nalidixic acid (50g/ml) were added to the medium. The plates were then incubated at 28°C for 7-21 days. Isolated actinobacteria colonies were subcultured and puri ed on SCNA plates. Isolate spores were reserved as a stock in 20% glycerol at -20 °C for potential use.
Screening and selection of the isolate Primary screening determines the ability of the microorganisms to produce an antifungal metabolite without providing a signi cant idea about the production potential of the organism. Modi ed Kirby Bauer antibiotic susceptibility test was used for primary screening (22). The actinobacterial isolates were cultured for seven days on the SCNA medium at 28 ºC. Six mm agar discs from well-grown culture of actinobacterial isolates were placed on PDA plates which were already seeded with the test phytopathogenic fungi (100 µl of 10 6 spore ml -1 ). The plates were then kept at 4ºC for 30 min for the diffusion and then incubated at 28ºC. The zone of inhibition was determined after 48 h.
Following primary screening, the active isolates were subjected to secondary screening. Three discs of actinobacterial culture with a diameter of 6 mm were transferred to 250 ml Erlenmeyer asks containing 50 ml of starch casein nitrate broth and incubated for 10 days at 28 ºC in a rotary shaker at 180 rpm.
Mycelia were extracted by centrifuging at 10,000 g for 20 minutes at 4 ºC and the supernatant was used for bioassay. The PDA plates inoculated with test fungi (100 µl of 10 6 spore ml -1 ) were punctured with sterile cork borer to create wells (6 mm in diameter) and 200µl of culture supernatant was transferred to each well under aseptic conditions. The plates were incubated at 28 ºC for three days and observed for antifungal activity of the isolates as clear zones of inhibition (in mm) around well. Out of 9 isolates from secondary screening, isolate SP5 was selected for further studies based on its broad-spectrum antifungal activity.
Morphological, physiological, and biochemical characterization of Streptomyces sp. SP5 Cultural characteristics were determined as per methods described by Shirling and Gottlieb (23) in the International Streptomyces Project (ISP). Bright light microscopy and electron microscopy were used to examine the strain morphological characteristic. Physiological and biochemical characterization such as growth at different temperatures (20-50°C), pH (5.0-12.0), salt concentration (0-20% w/v), and capacity to generate different hydrolytic enzymes were carried out according standard protocols (24). According to Shirling and Gottlieb (1966), sugars assimilation as carbon source was investigated. The major diagnostic features of Streptomyces, a sugar pattern in whole-cell hydrolysate and an isomer of diaminopimelic acid (DAP) in the cell wall, were determined using Lechevalier and Lechevalier (1980).
Ampli cation was done for 40 cycles in an PCR machine (Eppendorf-gradient). Each cycle included a denaturation stage of 1 minute at 94 0 C, an annealing step of 1 minute at 50 0 C, and an extension step of 2 minutes at 72 0 C and a nal extension of 10 minutes at 72 0 C. The QIA quick gel extraction kit (Qiagen, Germany) was used to purify the obtained PCR product. The Institute of Microbial Technology (IMTECH), Chandigarh, India, sequenced the 16S rRNA gene of Streptomyces sp. SP5. For the detection of phylogenetic neighbors and calculation of pairwise 16S rRNA gene sequence similarities, the EzTaxon server (http://www.ezbiocloud.net) was used (27). The Clustal W program was used to align the nearly complete sequence (1400 bp). Phylogenetic trees based on bootstrap values (1000 replications with MEGA6 software) were constructed using neighbor-joining and maximum-parsimony methods (28-29).
Time course experiment related to antifungal metabolites production and growth For antifungal pro ling determination, seed culture was prepared, three discs (6mm diameter) of 7 days old Streptomyces sp. SP5 culture were inoculated into Erlenmeyer asks (250 ml) containing 50 ml of starch casein nitrate broth and incubated at 28°C at 180 rpm for a week. Inoculating the production medium (SCN broth) with seed culture (2%) and incubating at 28 °C for 10 days with agitation at 180 rpm was used for fermentation. After every 24 hours, the asks were harvested, and the culture broth was centrifuged at 10,000g for 10 minutes to remove the biomass. The biomass was dried for 2 days at 60 °C and measured (mg). The activity against test fungal phytopathogens was determined using the remaining cell-free culture supernatant. The test fungi (100 µl of 10 6 spore ml -1 ) were seeded on PDA, which were punctured with sterile cork borer to create 6 mm wells. The plates were held in the refrigerator for 1 hour after adding 200 µl of culture supernatant to allow active metabolites to diffuse. After diffusion, the plates were incubated at 28 °C for 4-5 days. The antifungal activity of the isolate was measured in millimetres as zones of inhibition around wells.

Recovery of antifungal metabolites
For the production of antifungal active metabolites, fermentation was carried out by inoculating the strain (at a concentration of 2.5% from seed culture) in a production medium and incubating for ve days at 28°C under shaking (180 rpm). Fermentation was terminated when the maximum antifungal activity was observed (5th day) and culture broth was centrifuged at 10,000g at 4 ºC for 10 min to separate the mycelium.
The active metabolites from culture supernatant were recovered by solvent extraction. Different organic solvents (hexane, diethyl ether, chloroform, ethyl acetate, butanol) having a wide range of polarity were screened to work out the best extractant. The supernatant of Streptomyces sp. SP5 was extracted twice in the ratio of 1:1 (supernatant: solvent). Rotavapor (BUCHI Rotavapor R-210) was used to concentrate the obtained organic phase to dryness. The obtained crude extracts were redissolved in methanol (1ml) and tested for antifungal activity against test strains. Acetone precipitation was also employed to extract out the antifungal metabolites from the culture supernatant. For acetone precipitation, two volumes of 100% chilled acetone were added to the culture supernatant and kept for 1 hour. at -20 o C. The precipitates were recovered by centrifugation at 10,000 rpm for 10 minutes at 4 o C, and were redissolved in distilled water.
The antifungal activity of precipitates was determined against different test phytopathogenic fungi using the agar well diffusion method.

Stability of active metabolites in the culture supernatant
The supernatant was heated at various temperatures (37 0 C, 50 0 C, 70 0 C, 100 0 C and autoclaving at 121 0 C) for 1hr, and was also exposed to a lower temperature (-20 0 C) for one hour to determine thermostability. The effect of pH on activity was determined by adjusting the pH from 2 to 14, followed by incubation for one h at 28 0 C. Photostability was tested by exposing the supernatant separately to UV Streptomyces sp. SP5 culture and then incubated at 28ºC for 7 days under shaking (180rpm). After incubation, the broth was centrifuged at 10,000g for 15 minutes to remove mycelia. After centrifugation 2ml Salkowski reagent, as modi ed by Gordon et al (1950) was added into tubes containing 1ml of cellfree supernatant and incubated for 25 min at room temperature. The developed pink color intensity measured at 530nm, which indicates indole acetic acid production. The concentration of IAA produced was estimated against the standard curve of IAA using a concentration range of 10-100 μg/ml. Extraction, puri cation, and identi cation of Indole acetic acid The culture supernatant was acidi ed to pH 2.5 with HCl and extracted twice with ethyl acetate using solvent-solvent extraction. The separated organic phase was concentrated using a rotary evaporator and redissolved in ethyl acetate (1ml). For IAA analysis, the ethyl acetate extract was separated using thinlayer chromatography (TLC) with propanol: water (8:2, v/v) as solvent system, and the chromatogram was observed after spraying the plates with Salkowski regent. IAA was partially puri ed from the crude solvent extract by using silica gel column chromatography and fractions were collected with the solvent system of ethyl acetate and hexane (20:80 v/v). Each fraction was checked for the presence of IAA by TLC using Salkowski regent. The indole containing fraction was further quanti ed by high-performance liquid chromatography (HPLC) using C-18 reversed-phase column eluting with methanol: 1 % acetic acid in water (40:60 v/v as mobile phase) at 1 ml min -1 ow rate. The pure IAA compound was spiked into the column as standard and from partially puri ed IAA extract 20 µl aliquot was analyzed. The identical retention time of the respective standard was used to assess the presence of IAA in the specimens.

Optimization of IAA production
Various factors such as incubation period, pH, temperature, and tryptophan concentration were studied using one variable at a time approach or classical approach to optimise IAA production. The effect of incubation time on IAA synthesis was investigated by growing the culture in yeast malt broth for 10 days and IAA production was checked after every 24 h. The optimal for production was determined by changing the initial pH of the medium from 2-10 using 1N HCl and 1N NaOH. Similarly, the effect temperature of incubation on IAA production was investigated by incubating Streptomyces sp. SP5 into the same medium at 20, 25, 28, 30, 35, and 40 ºC for six days. The effect of L-tryptophan concentration was calculated by adding 0, 1, 2, 3, 4, and 5 mg/ml of L-tryptophan to medium (pH 7.0) and incubating for six days. The bacterial growth rate was measured, culture broth was centrifuged at 10,000g for 10 min and the biomass was dried at 60 °C for 2 days and weighed (mg). All experimentations were performed in triplicates and average values were observed.
Streptomyces sp. SP5 In vivo biocontrol of Alternaria solani and its effect on tomato plant growth promotion E cacy of Streptomyces sp. SP5 against early blight disease was tested by conducting three independent pot trials at Guru Nanak Dev University (Amritsar) using soil drenching method. The aim was to see whether Streptomyces sp. SP5 culture cells, culture supernatant, solvent extract as well as acetone precipitates could regulate Alternaria solani (the causal fungal phytopathogen of early blight in tomato) and promote various plant growth traits. Tomato seeds susceptible to Alternaria solani (Solanum lycopersicum Mill. "Pusa Ruby") were sown in sterile soil for 3 weeks at 28±2.0 °C and after true leaf stage, a single plant was transplanted into a pot of 8cm diameter containing 100g sterile soil for each treatment. The plants were subjected to a variety of treatments by Streptomyces sp. SP5 antagonists viz. Streptomyces sp. SP5 cells, supernatant, solvent extract and Acetone precipitates, with and without pathogen (Table.1). Three replications were maintained for each treatment and the pots were kept under natural conditions (temperature 22±2.0°C, photoperiod L16: D8). Plants were watered and disease symptoms, owering, and fruiting stages were observed daily. After disease appearance in the control plants, all treated plants were uprooted carefully, and plants were washed with running tap water to remove the adhered soil, shoot & root length, and fresh & dry weights of tomato seedlings were recorded. Data obtained from in vivo were subjected to statistical analysis. Values were represented as their mean ± SE of three independent experiments and to compare the mean difference, one-way analysis of variance (ANOVA) with Tukey's post hoc test was carried out using SPSS statistical analysis software