Phytochemical and Pharmacological evaluation of hibiscus hispidissimus griff

To assess phytochemical with pharmacological studies of Hibiscus hispidissimus griff belong to family malavaceae. Preliminary phytochemical analysis reveals the presence of steroids, triterpenes, saponins, steroidal saponins and phenols. Evaluation of anti-inflammatory, anti-microbial with antioxidant action were performed on aerial parts of methanolic extract of Hibiscus hispidissimus. Invitro antioxidant activity was performed by 2, 2 -diphenyl- 1- picrylhydrazyl (DPPH) assay, hydroxy radical scavenging method and superoxide radical scavenging activity.The results of invitro antioxidant study reveal that % inhibition of H. hispidissimus  was higher compared to ascorbic acid. Anti-inflammatory studies were performed using carrageenan-induced rat paw oedema animal model, for anti-inflammatory studies, the extracts were compared with standards like indomethacin, and it shows a remarkable zone of inhibition ranging from 58.97 to 71.73 respectively. The anti-bacterial and antifungal activity of plant extracts were studied for the occurrence of inhibition zones. The activity was performed by the cup plate method. Ethanolic extract of H. Hispidissimus  shows significant anti-bacterial effect against S. Aureus, B. Subtilis, P. Vulgaris and E. coli using ciprofloxacin (50µg/ml) as standard.The extracts show remarkable inhibition of zone of inhibition, and results were compared with that of standard drugs against the organism tested. In conclusion, the ethanolic extract of H. hispidissimus  shows  significant antioxidant, anti-inflammatory and anti-bacterial properties.

Anti-bacterial, Antifungal, Anti-in lammatory, Phytochemical studies A To assess phytochemical with pharmacological studies of Hibiscus hispidissimus griff belong to family malavaceae. Preliminary phytochemical analysis reveals the presence of steroids, triterpenes, saponins, steroidal saponins and phenols. Evaluation of anti-in lammatory, anti-microbial with antioxidant action were performed on aerial parts of methanolic extract of Hibiscus hispidissimus. Invitro antioxidant activity was performed by 2, 2 -diphenyl-1-picrylhydrazyl (DPPH) assay, hydroxy radical scavenging method and superoxide radical scavenging activity.The results of invitro antioxidant study reveal that % inhibition of H. hispidissimus was higher compared to ascorbic acid. Anti-in lammatory studies were performed using carrageenan-induced rat paw oedema animal model, for anti-in lammatory studies, the extracts were compared with standards like indomethacin, and it shows a remarkable zone of inhibition ranging from 58.97 to 71.73 respectively. The antibacterial and antifungal activity of plant extracts were studied for the occurrence of inhibition zones. The activity was performed by the cup plate method. Ethanolic extract of H. Hispidissimus shows signi icant anti-bacterial effect against S. Aureus, B. Subtilis, P. Vulgaris and E. coli using cipro loxacin (50µg/ml) as standard.The extracts show remarkable inhibition of zone of inhibition, and results were compared with that of standard drugs against the organism tested. In conclusion, the ethanolic extract of H. hispidissimus shows signi icant antioxidant, anti-in lammatory and anti-bacterial properties.

INTRODUCTION
Plants have been used for medicinal purpose long before prehistoric period. Traditional system of medicine continues to be widely precised on many accounts. Drugs obtained from natural sources are used as a reservoir for many biochemical products which are used as extractions for development of many formulations which are non-reactive, nontoxic and free from side effects. Drug resistance for transmittable ailments have directed to enlarged importance in the use of natural product as medicine for diverse human illnesses (Das, 2016). Hibiscus hispidissimus Grif ith (synonym Hibiscus furcatus DC. non wild., Hibiscus aculcatus Roxb.non walter), locally as 'coffort root' or 'Big thicket Hibiscus' or 'Pine Hibiscus' in English and "Uppancham" in Malayalam. Has another name wild hibiscus (Hibiscus hispidissimus, 1854) belong to the family malavaceae shown in Figure 1 (India biodiversity portal, 2005). The plant is widely distributed in southwestern parts of India, South Africa, Sri Lanka, Myanmar and Thailand (Harborne, 1994).
Hibiscus species are medicinally important and many of the species are evaluated for their antioxidant and antibacterial properties.
The selected species had greater tribal importance as medicine for many human ailments like digestion, as anthelminthic, cooling drink, remedy for poisons, swellings and for eye disorders and also in treating liver disorders especially in southern parts of India. Hence, the present work was to investigate phytochemical and pharmacological activity for Hibiscus hispidissimus.

Collection of plant material
Plants of H. hispidissimus were collected from west Godavari, Andhra Pradesh. The plant was washed, collected and dried at room temperature. Then ground to a coarse powder, macerated with ethanol and the extract was dried by rotary vacuum. The dried residue was preserved in an airtight container and kept at 4-5 • C until further use.

DPPH assay
The antioxidant potential was assessed by measuring free radical. DPPH dissolved in methanol was prepared,1ml of this solution was added to 3 ml of test, standard and control solution at different concentration (5, 10, & 50 µg/ml).
The absorbance of resulting solutions was measured at 517 nm using UV-visible spectrophotometer. The DPPH radical scavenging was calculated by using the following formula.
DPPH radical scavenging activity = Abs control

Hydroxyl radical scavenging activity
To measure the competition between deoxyribose and test compounds for hydroxy radicals generated, hydroxy radical scavenging activity was used.
Extracts of different concentrations ranging from (0.1-1000 µg/ml) were added to the reaction mixture contains 1 ml of potassium phosphate buffer (10 mM, pH 7.4).
The time of incubation was 1 hr at 37 • c, then added every 1 ml of 1% thiobarbituric acid and 2.8% of TCA. The resulting mixture was subjected to underwater heating bath for 15 mts. The resulting solution was measured at 532 nm after cooling.
Hydroxy radical scavenging activity = Abs control
Test and standard were prepared, agar medium was prepared in a cup with sterile borer, then added 0.05 ml of test solution by using a micropipette. Cipro loxacin a potent antibiotic (50 µg/dl) was used as standard. All glass plates were incubated for 24 h at 37ºC. The diameter of the inhibition zone was recorded.

Anti-in lammatory activity
Anti-in lammatory studies were performed using carrageenan-induced rat paw oedema animal model. Samples extract was administered orally (100 & 250 mg/kg) body weight of each extract and standard (Indomethacin 20mg/kg) by rat paw oedema. Indomethacin (20 mg/kg) used as standard. Oedema developed was measured by volume displacement method (Bhatt et al., 1977;Hafeez et al., 2013).
Outcomes were stated as the percentage of inhibition of oedema calculated by using formula (1-V t /V C ) × 100, Where V t and V c are mean paw volume in treated and control group, respectively. Results were analysed using one-way ANOVA method.

Phytochemical analysis
The phytoconstituents of H. hispidissimus showed the occurrence of steroids, triterpenes, saponins, lavonoids, mucilage and phenol. The composition of Phytoconstituents in the plant has been mentioned in Table 1.

Antioxidant activity
Three in-vitro antioxidant methods were used such as DPPH assay, hydroxyl radical assay and superox-ide radical assay. Antioxidant activity results were expressed in terms of IC 50 values. The IC 50 values were calculated using the DPPH method for H. hispidissimus , and ascorbic acid was 42 & 12 µg/ml. The results are expressed in Table 2 and Figure 2. The IC 50 values using hydroxy radical scavenging method are 2.8 &1 µg/ml for ascorbic acid. Results are expressed in Figure 3. For the superoxide radical scavenging assay method, the extract and standard show IC 50 of 44 & 23 µg/ml correspondingly. The outcomes are revealed in Figure 4 and Table 3.

Anti-in lammatory activity
The extract tested at two different doses (100mg/kg & 250 mg/kg), exerted a considerable inhibitory effect on rat paw swelling after carrageenan administration with more than 50% inhibition for two doses.       Table 4 and Figure 5.

Anti-microbial activity
The

CONCLUSIONS
The present study has been taken to evaluate the phytochemical and pharmacological activity of H. hispidissimus. The phytochemical studies revealed the presence of phenolic compounds, steroids, triterpenes, saponins and lavonoids. The data indicated that extracts from leaves of H. hispidissimus had antioxidant, anti-in lammatory and antimicrobial properties. The results revealed by antioxidant parameters shows that H. hispidissimus have antioxidant activity against various free radicals. Preventing the lysosomal membrane is one of the contributions to anti-in lammatory activity. Data of anti-in lammatory activity shows that plant extract exerted a considerable inhibitory effect on rat paw swelling after carrageenan administration and thereby inducing anti-in lammatory effect. The results indicate that promising activities of H. hispidissimus, and further, it could be subjected to drug progress and treatment of various infectious diseases.