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Annexure –VI UNIVERSITY GRANTS COMMISSION BAHADUR SHAH ZAFAR MARG NEW DELHI – 110 002.

FINAL REPORT OF THE WORK DONE ON THE MINOR RESEARCH PROJECT

1. Project report No. : Final

2. UGC Reference No.F.: MRP-7007/16 (SERO/UGC) Dt. 30th October 2017

3. Period of report: From ___November 2017__To ___October 2019___

4. Title of research project: Anti-inflammatory efficacy of Hibiscus schizopetalus 5. (a) Name of the Principal Investigator: Dr. R. Amsaveni (b) Department : Biotechnology (c) College where work has progressed: Kongunadu Arts and Science College, Coimbatore – 641 029.

6. Effective date of starting of the project: 14-11-2017

7. Grant approved and expenditure incurred during the period of the report: a. Total amount approved Rs. ______2,20,000.00______b. Total expenditure Rs. ______2,22,654.00______

Report of the work done

1. Brief objective of the project:  To analyze the phytochemical constituents and in vitro anti-inflammatory activities of Hibiscus schizopetalus leaf and flower extracts.  To determine the in vivo anti-inflammatory activities of the H. schizopetalus leaf and flower extracts using animal models.  To evaluate the in vitro anti-haemolytic and cytotoxicity effect of the extracts.  To evaluate the gene expression profiles of COX-2 in animal models. 2. Objective of the project (For 1st year):  Extract preparation from the leaves and flowers of H. schizopetalus.  Phytochemical screening of the extracts.  In vitro anti-inflammatory activity of the extracts.  Anti-haemolytic activity of the plant extracts.  In vitro cytotoxicity analysis.

3. Work done so far and results achieved

3.1 Methods 3.1.1 Extract preparation from the leaves and flowers of H. schizopetalus Extracts were prepared by two different modes such as extraction of fresh plant material without drying and extraction after drying each plant part (leaves and flower). From each methods hot water, cold water and organic solvent (ethanol and methanol) extracts was prepared (Goyal et al., 2008) and used for further studies. 3.1.1.1 Hot water extraction

About 10 g of leaves and flowers of Hibiscus schizopetalus was boiled in 100 mL distilled water with constant stirring for 30 min. The solution was then allowed to cool to room temperature and then filtered using muslin cloth. The filtrate was centrifuged at 5000 rpm for 15 min. The supernatant was again filtered using Whattmanʼs No. 1 filter paper under strict aseptic conditions. The filtrate was collected in fresh sterilized glass tubes and stored at 4 °C until use (Goyal et al., 2008). 3.1.1.2 Cold Water Extraction

About 10 g of leaves and flowers of H. schizopetalus was macerated in mortar and pestle with 100 ml distilled water at room temperature and then filtered using muslin cloth. The filtrate was centrifuged at 5000 rpm for 15 min. The supernatant was again filtered using Whattmanʼs

No. 1 filter paper under strict aseptic conditions and the filtrate was collected in fresh sterilized glass tubes and stored at 4 °C until use (Goyal et al., 2008).

3.1.1.3 Ethanol and methanol extraction

The ethanol and methanol extract of dried plant leaves and flowers of H. schizopetalus was prepared by mixing about 5 grams of each plant material (flower and leaves) powder in 50 ml of respective solvents separately. The mixture was taken into 250 ml sterile conical flasks, plugged with sterile cotton and kept in shaking incubator with 200 rpm for 24 h. The solution was filtered through muslin cloth. This process was repeated three times after which a clear aqueous extract of the plant was taken (Zamin et al., 2014).

All the Hibiscus extracts (leaf and flower) obtained were used for phytochemical analysis, in vitro anti-inflammatory activities, in vitro antihaemolytic activity and in vitro cytotoxicity tests.

3.1.2 Phytochemical screening of the extracts All the extracts will be screened for the presence of phytochemicals such as carbohydrates, alkaloids, flavonoids, terpenoids, steroids, tannins, saponins, phenols. Test for carbohydrates

Fehling’s test

One mL of the filtrate was boiled with 1 mL each of Fehling solutions A and B on water bath. Appearance of a red or brick precipitate indicates the presence of sugar.

Test for alkaloids

The extract was mixed with 5 mL of 1% aqueous hydrochloric acid on a water bath. The filtrate is carefully tested with different alkaloidal reagents for the presence of alkaloids. Wagner’s test

About 1 mL of HCl was added to 3 mL of extract in a test tube. The mixture was heated for 20 min, cooled and filtered. Two drops of Wagner’s reagent was added to 1 mL of the filtrate and observed for reddish brown precipitate.

Test for flavonoids

Alkaline reagent test

Extracts (2 mL) was dissolved in 10% aqueous sodium hydroxide solution, it gives yellow color. A change of color from yellow to colourless on addition of dilute HCl indicates the presence of flavonoids.

Test for terpenoids

Horizon test

To 1 mL of extract, 2 mL of trichloroacetic acid (TCA) was added. The formation of yellow to red precipitate shows the presence of terpenoids.

Test for steroids

Salkowski test

A little quantity of each plant extracts was dissolved in 1 mL of chloroform and about 1 mL of concentrated sulphuric acid was added to it to form two phases. Formation of red colouration was taken as an indication for the presence of steroids.

Test for tannins

Ferric chloride test

The extracts were boiled for 5 min in water bath and added 2 drops of 5% FeCl3 to it. Production of greenish precipitate was an indication for the presence of tannins.

Test for saponins

Frothing test The extracts were shaken with a drop of sodium bicarbonate in test tubes.

Formation of honey comb like froth which persisted for 15 min indicates the presence of saponins. Test for phenols

Ferric chloride test

The extract was dissolved in 5 mL of distilled water. To this few drops of neutral 5% ferric chloride solution was added. A dark green colour indicates the presence of phenolic compounds.

In vitro anti-inflammatory activity of the extracts The tests such as inhibition of albumin denaturation, Membrane stabilization and Proteinase inhibitory action will be carried out to study the in vitro anti-inflammatory activities of the extracts. 3.1.3 In vitro anti-inflammatory activity

3.1.3.1 Inhibition of albumin denaturation

The reaction mixture consisted of test extracts and 1% aqueous solution of bovine albumin fraction. The pH of the reaction mixture was adjusted using small amount of 1N HCl.

The samples were incubated at 37 °C for 20 min and then heated at 57 °C for 20 min. After cooling the samples, the turbidity was measured spectrophotometrically at 660 nm. The experiment was performed in triplicates (Sakat et al., 2010). Percent inhibition of protein denaturation was calculated.

3.1.3.2 Membrane stabilization test

3.1.3.2.1 Heat induced haemolysis

Fresh whole human blood (5 mL) was collected and transferred to the heparinized centrifuged tubes. The tubes were centrifuged at 3000 rpm for 10 min and were washed three times with equal volume of normal saline (0.9% NaCl). The volume of the blood was measured and reconstituted as 10% v/v suspension with normal saline (Sadique et al., 1989). The reaction mixture (2 mL) consisted of 1 mL of plant extracts and 1 mL of 10% RBC suspension. In case of control tube instead of plant extract only saline was added. Aspirin was taken as a standard drug.

All the centrifuge tubes containing the reaction mixture were incubated in a water bath at 56 °C for 30 min. At the end of the incubation, the tubes were cooled under running tap water. The reaction mixture was centrifuged at 2500 rpm for 5 min and the absorbance of the supernatants was taken at 560 nm. The experiment was performed in triplicates (Shinde et al., 1999). Percent membrane stabilization activity was calculated.

3.1.3.3 Proteinase inhibitory action

The reaction mixture (2 mL) was containing 0.06 mg trypsin, 1 mL 20 mM Tris HCl buffer (pH 7.4) and 1 mL test sample. The mixture was incubated at 37 °C for 5 min and then 1 mL of 0.8% (w/v) casein was added. The mixture was incubated for an additional 20 min. About

2 mL of 70% perchloric acid was added to terminate the reaction. Cloudy suspension was centrifuged and the absorbance of the supernatant was read at 210 nm against buffer as blank.

The experiment was performed in triplicates. The percentage inhibition of proteinase inhibitory activity was calculated (Sakat et al., 2010).

3.1.4 In vitro antihaemolytic activity

The human venous blood samples were collected in EDTA tubes from well nourished healthy adults (25-30 years of age) and the tubes were centrifuged at 1,500 rpm for 10 min.

Further the plasma was discarded from the tubes and the settled RBCs were washed three times with saline (0.9% NaCl). Then the RBCs were diluted with saline phosphate buffer to give 4%

(v/v) suspension. Leaf and flower extracts were dissolved in 1 mL of saline was added to 2.0 mL of RBC suspension and the volume was made up to 5.0 mL with saline. This mixture was pre- incubated for 5 min at room temperature and then 0.5 mL of H2O2 solution in buffered saline was added to induce oxidative degradation of the membrane lipids. The tubes were incubated for one hour and further the reaction mixture was centrifuged at 1500 rpm for 10 min. Then the extent of haemolysis was measured spectrophotometrically at 540 nm (Saradha et al., 2013). The percentage of haemolysis inhibition was calculated.

3.1.5 In vitro cytotoxicity analysis The in vitro cytotoxic effect of H. schizopetalus leaf and flower extracts on peripheral blood mononuclear cells (PBMC) as well as in Vero cell lines was carried out. To collect PBMC about 5 mL of peripheral blood was collected in a tube containing EDTA. Then this defibrinated blood was diluted with 2.5 mL of tissue culture medium (DMEM). The diluted blood was layered carefully with pipette onto 2.4 mL ficoll paque (polymer of sucrose). The ficoll and blood should not mix while layering. Further the tubes were centrifuged at 2500 rpm for 25 min at 20 °C. Mononuclear lymphocytes and monocytes present at the ficoll-plasma interphase forms a band. All the other cells sediment at the bottom as a pellet. The PBMC layer was carefully removed with the minimal underlying histopaque and it transferred to a sterile 50 mL centrifuge tube. The PBMC is washed by adding equal volume of tissue culture medium, and gently mixed by re-suspending the cells. The cells were centrifuged at 2500 rpm for 15 min at 20 °C. The supernatant was removed and the cells were re-suspended in the tissue culture medium. The final pellet was re-suspended in complete medium containing 10% serum.

3.1.5.1 Evaluation of cell viability by MTT assay

The cells were harvested from 25 cm2 T-flask. Appropriate dilution was made with the medium and serum. About 5000 to 10000 cells were seeded per well in the 96 well plates. Before seeding the cells, the cells were counted by using haemocytometer and according appropriate dilutions were made. The dilutions were prepared which gives 5000-10000 cells per 100 µL.

After seeding the cells, the plate was incubated for 24 h and then H. schizopetalus leaf and flower extracts were at added to the wells. Further the plates were incubated for 24 h, and then

10 µL of MTT (5 mg/mL) was added to each well and it was incubated for an hour. Then the medium was removed without disturbing the cells. Added 100 µL of isopropanol to each well in order to extract the visible blue color precipitate formed at the bottom of the wells. The plate was read at 594 nm in ELISA plate reader. The absorbance seen in control was taken as 100% cell viability. Survival rate was calculated using the equation, % Survival = [S/C] × 100, where “C” is the net absorbance of the control and “S” is the net absorbance of the sample.

3.2 RESULTS

3.2.1 Phytochemical analysis

Qualitative analysis was carried out to screen the phytochemicals present in the extracts of H. schizopetalus. The results of the phytochemical analysis are presented in table 1 and table 2 carbohydrates, alkaloids, flavonoids, terpenoids, steroids, tannins, saponins, phenols

Table 1 Phytochemical analysis of leaf and flower extracts of H. schizopetalus (Fresh

plant material without drying)

S.No. Tests Cold water Cold water Hot water Hot water leaf flower leaf flower 1 Alkaloids - + - + (Wagnerʼs test) 2 Carbohydrate - + - + (Fehlings test) 3 Phenol - - - - (Ferric chloride test) 4 Saponins - + - + (Frothing test) 5 Steroids - + - + (Salkowski) 6 Flavanoids - + - + (Alkaline reagent test) 7 Tannins + - + - (Ferric chloride test) 8 Terpenoids - - - - (Horizon test)

Table 2 Phytochemical analysis of leaf and flower extracts of H. schizopetalus (Plant

material after drying)

S.No. Tests Cold Cold Hot Hot Ethanol Ethanol Methanol Methanol water water water water extract extract extract extract leaf flower leaf flower leaf flower leaf flower 1 Alkaloids - + - + + - - - (Wagnerʼs test) 2 Carbohydrate - + - + - - - - (Fehlings test) 3 Phenol ------(Ferric chloride test) 4 Saponins - + - + + - - - (Frothing test) 5 Steroids - + - + - - - - (Salkowski) 6 Flavanoids ------(Alkaline reagent test) 7 Tannins - - + - + - - - (Ferric chloride test) 8 Terpenoids ------(Horizon test)

In vitro Anti-inflammatory activity

3.2.2 Inhibition of albumin denaturation

To investigate on the mechanism of anti‐inflammatory activity, the ability of extract to inhibit protein denaturation was studied. It was found that the H. schizopetalus fresh hot water flower extract of about 73±0.14% and dry hot water flower extract showed 70±0.18% was effective in inhibiting heat induced albumin denaturation (Table 3).

Table 3 Inhibition of albumin denaturation by H. schizopetalus leaf and flower

extracts

Fresh plant material without drying Plant material after drying

Inhibition of albumin denaturation (%) Inhibition of albumin denaturation (%) Cold water Hot water Cold water Hot water Ethanol Methanol extract extract Leaf 54±0.07 62±0.06 50±0.19 59±0.27 49±0.13 56±0.04

Flower 56±0.11 73±0.14 52±0.17 70±0.18 45±0.21 54±0.06

3.2.3 Membrane stabilization test

Stabilization of the RBC membrane was studied to further establish the mechanism of

anti‐inflammatory action of H. schizopetalus. The percentage of inhibition of H. schizopetalus

fresh hot water flower extracts and dry hot water flower extracts was found to be 82±0.07% and

79±0.16%, respectively (Table 4, Figure 1).

Table 4 Membrane stabilization activity of H. schizopetalus leaf and flower extracts

Fresh plant material without drying Plant material after drying

Inhibition of albumin denaturation (%) Inhibition of albumin denaturation (%) Cold water Hot water Cold water Hot water Ethanol Methanol extract extract Leaf 55±0.12 78±0.08 52±0.11 69±0.22 60±0.22 63±0.06

Flower 59±0.16 82±0.07 56±0.19 79±0.16 53±0.07 67±0.08

3.2.4 Proteinase inhibitory action

Aqueous extracts of H. schizopetalus exhibited significant antiproteinase activity. The

percentage of inhibition of H. schizopetalus H. schizopetalus fresh hot water flower extracts and

dry hot water flower extracts was found to be 72±0.18% and 68±0.17%, respectively (Table 5). Table 5 Proteinase inhibitory activity of H. schizopetalus leaf and flower extracts

Fresh plant material without drying Plant material after drying

Inhibition of albumin denaturation (%) Inhibition of albumin denaturation (%) Cold water Hot water Cold water Hot water Ethanol Methanol extract extract Leaf 56±0.17 65±0.12 51±0.09 66±0.11 55±0.24 44±0.12

Flower 55±0.07 72±0.18 53±0.13 68±0.17 62±0.06 51±0.08

3.2.5 In vitro antihaemolytic activity

Antihaemolytic avtivity of 74±0.19% and 71±0.18% was shown by H. schizopetalus

fresh hot water flower extracts and dry hot water flower extracts, respectively (Table 6).

Table 6 Antihaemolytic activity of H. schizopetalus leaf and flower extracts

Fresh plant material without drying Plant material after drying

Inhibition of albumin denaturation (%) Inhibition of albumin denaturation (%) Cold water Hot water Cold water Hot water Ethanol Methanol extract extract Leaf 49±0.16 58±0.11 45±0.24 56±0.07 53±0.13 54±0.18

Flower 53±0.06 74±0.19 50±0.17 71±0.18 51±0.21 52±0.11

3.2.6 In vitro cytotoxicity

MTT assay was carried out for PBMC and Vero cell lines in order to investigate the in vitro

cytotoxicity activity of H. schizopetalus leaf and flower extracts, in which the cells and media

served as control. The cells treated with the fresh hot water flower extract, showed prevention in

cell death and retained 100% survival rate. There is no cytotoxic effect for the extract against

both PBMC and Vero cells (Figure 2).

Figure 1 Membrane stabilization activity of H. schizopetalus flower extract

Figure 2 In vitro cytotoxicity of H. schizopetalus hot water flower extracts (without drying) on Vero cell lines

4. Objective of the project (For 2nd year):  In vivo anti-inflammatory activity of the extracts and COX2 gene expression analysis using animal models. 5. Work done so far and results achieved for the second year:

5.1 Carrageenan induced paw oedema in Swiss albino mice

In vivo anti-inflammatory activity of H. schizopetalus fresh hot water flower extract was evaluated against carrageenan induced paw oedema in Swiss albino mice. The fresh hot water flower extract was found to be the most potent after 4 hours of treatment when compared with the saline control group. Treatment with plant extract at 100 mg/kg body weight was found to highly efficient in controlling the induced inflammation in mice when compared to plant extract of 100 mg/kg body weight. In control groups the paw volume remained constant throughout the experiment. In case of carrageenan induced group the paw oedema reached a maximum volume of 1.1±0.1 cm after 120 min of induction. In case of standard diclofenac sodium treated group the paw volume decreased from1.55±0.1 cm to 0.8±0.3 cm. The 100 μL (100 mg/kg body weight) plant extract treated group showed decrease in paw volume with initial volume of 1.45±0.6 cm and decreased paw oedema after 120 min was plant extract treated group showed decrease in paw volume from 1.35±0.2 cm to 0.7±0.1 cm after 240 min of treatment. It is evident from the results that the in vivo anti-inflammatory activity of H. schizopetalus fresh hot water flower extract at 100 μL (10 mg/kg body weight) concentration is highly effective and its effect is comparable with the results of standard drug used in this study (Table 5.1 and Fig. 5.1). The maximum percentage inhibition of paw oedema around 1.35±0.2% was shown by

H. schizopetalus fresh hot water flower extract at 10 mg/kg body weight. Whereas, the standard drug diclofenac sodium showed 1.55±0.1% inhibition of paw oedema.

Table 5.1 Carrageenan induced paw oedema in Swiss albino

Groups Treatment 0 min 30 min 60 min 90 min 120 min

Paw volume (cm)

I Control 1.5±0.1 1.5±0.1 1.5±0.1 1.5±0.1 1.5±0.1

II Induced 1.4±0.6 1.9±0.2 1.7±0.1 1.6±0.2 1.6±0.1

III Extract 1.4±0.6 1.4±0.6 1.4±0.6 1.4±0.6 1.4±0.6

IV Treated 1.6±0.3 1.2±0.3 1.1±0.3 0.9±0.2 0.8±0.1

V Standard 1.5±0.1 1.5±0.4 1.2±0.3 1.0±0.3 0.8±0.3

5.2 Estimation of Myeloperoxidase (MPO) Activity

The levels of MPO showed significant increase of treated group when compared with the control (Table 5.2).

Table 5.2 Estimation of Myeloperoxidase (MPO) Activity

GROUPS µM/min/g

Control 10.9±0.24

Induced 19.5±0.14

Extract 11.8±0.69

Treated 19.21±0.25

Standard 15.05±0.58

Figure 5.1 In vivo anti-inflammatory activity

A B C

D E F

C I E T S

Fig. 5.1: Shows the in vivo anti-inflammatory activity of H. schizopetalus fresh hot water flower extract against carrageenan induced paw oedema in Swiss albino mice. Note: A- Control mice, B- Oral plant extract treatment in mice, C- Sub plantar carrageenan induction in mice after

1 h of extract treatment, D- treatment mice right hind paw, E- measuring the reading of

Inflammation induced right hind paw of mice, F-Mice after the completion of experiment,

Comparison of right hind paw of mice in all groups after 4 h of experiment. Note: C-control, I- induced, E- extract, T-treatment with 100 mg/kg body weight of plant extract, S-standard.

5.2.1 Acidic iso-MPO analysis

Acidic MPO iso enzyme of the control and treatment groups analysed from the hind paw tissue showed differential expression. Carrageenan induced paw tissue showed increased expression of acidic iso-MPO with Rf 0.66 when compared to the control. Whereas pretreatment with aqueous extracts and standard drug showed significant decreased in expression of acidic iso-MPO with Rf 0.66. This clearly shows that inflammation induced by carrageenan is reduced using the extract and standard drug (Fig 5.2).

Fig 5.2 Myeloperoxidase activity staining

Fig 5.2 shows the MPO activity staining with varying expressions of MPO isoenzyme of hind paw tissue in each group. The variations were confirmed either by the absence or presence of bands or by the thickness of the isoforms when visualized under white light after activity staining of MPO. Note: C-control, I-induced, E- extract, T-treatment with 100 mg/kg body weight of plant extract, S-standard.

5.3 Estimation of Catalase (CAT) Activity

The level of biomarkers of oxidative stress enzyme (CAT) in the paw tissue sample in treated group showed significant increase when compared to control and induced group (Table

5.3).

Table 5.3 Estimation of Catalase (CAT) Activity

GROUPS g -1min-1 mg

Control 80.7±0.56

Induced 86.1±0.63

Extract 89.5±1.41

Treated 94.7±1.9

Standard 96.5±0.7

5.4 Estimation of superoxide dismutase (SOD) Activity

The level of biomarkers of oxidative stress enzyme (SOD) in the paw tissue sample in treated group showed significant decrease of when compared to control and induced group

(Table 5.4).

Table 5.4 Estimation of superoxide dismutase (SOD) Activity

GROUPS (unit/ mg protein)

Control 0.48±0.11

Induced 0.65±0.01

Extract 0.38±0.04

Treated 0.38±0.03

Standard 0.44±0.27

5.5 Estimation of Malondialdehyde (MDA) Activity

The levels of MDA showed significant decrease of about in treated group when compared with the control (Table 5.5).

Table 5.5 Estimation of Malondialdehyde (MDA) Activity

GROUP (n mol/ ml)

Control 0.39±0.02

Induced 0.35±0.35

Extract 0.21±0.05

Treated 0.40±0.08

Standard 0.45±0.14

5.6 Estimation of Glutathione peroxidase (GPx) Activity

The levels of GPx showed significant decrease of about in treated group when compared with the control (Table 5.6).

Table 5.6 Estimation of Glutathione peroxidase (GPx) Activity

GROUPS m-1 cm-1

Control 71±1.3

Induced 86±1.7

Extract 80±1.3

Treated 60±1.4

Standard 50 ±1.4

5.7 In vivo anti-inflammatory activity of the extracts and COX2 gene expression analysis using animal models The mRNA from paw tissue was used for cDNA synthesis using Reverse Transcriptase - Polymerase Chain Reaction (RT-PCR) followed by COX-2 gene specific amplification compared with β-actin gene expression using respective specific primers (Using commercial kits). COX-2 expression is normally absent from most tissues and is induced by pathologic stimuli, such as bacterial lipopolysaccharide (LPS) and proinflammatory cytokines such as interleukin 1, IL-2, and TNF-α. COX-2 expressions have been found in vivo associated with inflammation, rheumatoid arthritis, seizures, ischemia, and various cancers. In the present study the control group and the extract group does not show any COX-2 gene expression. The induced group showed increased levels of COX-2 gene expression, whereas the treated and standard group showed decreased COX-2 gene expression which would be due to the protection given by the extract for the recovery of inflammation. Under basal conditions, COX-2 expression is highly restricted however; COX-2 is dramatically upregulated during inflammation.

6. Publications, if any: 1. Anusha M. R., Veluswamy Bhuvaneshwari, Manokaran Kalaiselvi, Akbar Ali Amrin, and Ramasamy Amsaveni*, In Vitro Anti-Inflammatory Activity of Hibiscus schizopetalus (Dyer) Hook. f., Vol. 12, 1–5, 2020.

7. Has the progress been according to original plan of work and towards achieving the objective. YES

8. Summary of the findings of the study: The genus Hibiscus (Malvaceae) comprises about 275 species in the tropics and sub- tropics (Dasuki, 2001). Zahid et al., (2014) reported that the methanolic extracts of H. schizopetalus (Mast) Hook flower and leaves clearly showed that the plant have powerful antioxidant activity against various antioxidant systems in vitro. With attractive and colourful flowers, plants of Hibiscus are widely planted as ornamentals and are used in traditional medicine. Of the species studied, leaves and flowers of H. mutabilis, believed to have emollient and cooling effect, are used to relieve swellings and skin infections (Dasuki, 2001). Leaves and flowers of Hibiscus rosa-sinensis L. are used as an antiseptic for boils and ulcers. Leaves of Hibiscus sabdariffa L. are used as poultice for abscesses and ulcers. Stems and roots of Hibiscus taiwanensis have been used as anti-inflammatory, antifungal, antipyretic, and antihelminthic agents (Wu et al., 2005). Flowers of Hibiscus tiliaceus L. are widely used for birth control and for treating skin infections (Rosa et al., 2006). The research into plants with alleged folkloric use as anti-inflammatory agents should therefore be viewed as a fruitful and logical research strategy in the search for new anti-inflammatory drugs. Inflammation may be potentially harmful, causing life threatening hypersensitivity reactions and progressive organ damage (Robbins, 2008). Inflammation is the reaction of living tissues to injury, infection or irritation. Lysosomal enzymes released during inflammation produce a variety of disorders which leads to the tissue injury by damaging the macromolecules and lipid peroxidation of membranes which are assumed to be responsible for certain pathological conditions as heart attacks, septic shocks and rheumatoid arthritis etc. The extra cellular activity of these enzymes is said to be related to acute or chronic inflammation. Stabilization of lysosomal membrane is important in limiting the inflammatory response by inhibiting the release of lysosomal constituents of activated neutrophil such as bactericidal enzymes and proteases, which cause further tissue inflammation and damage upon extra cellular release or by stabilizing the lysosomal membrane (Rajendran and Lakshmi, 2008). The present study focuses on the qualitative phytochemical analysis, in vitro anti- inflammatory activity of the extracts, Anti-haemolytic activity of the plant extracts, in vitro cytotoxicity analysis of H. schizopetalus. Plants are utilized extensively as raw drugs for many formulations in traditional as well as modern systems of medicine. The phytochemical screening of the plant extract confirmed the presence of several bioactive compounds like alkaloids, flavones, tannins and phenols which could be responsible for the versatile medicinal properties of this plant. H. schizopetalus fresh hot water flower extract showed 82±0.07% haemolysis inhibition. It exhibited 74±0.19% antihaemolytic activity towards RBCs. H. schizopetalus fresh hot water flower extract used in this study does not show cytotoxic effect on the PBMC and Vero cell lines, which infers that this extract screened in this study could be effectively used to cure human ailments. From the results of the study it is concluded that the H. schizopetalus, possessed considerable level of bioactive compounds

Scanned by CamScanner Article Advanced Science, Engineering and Medicine Copyright © 2020 American Scientific Publishers All rights reserved Vol. 12, 1–5, 2020 Printed in the United States of America www.aspbs.com/asem

In Vitro Anti-Inflammatory Activity of Hibiscus Schizopetalus (Dyer) Hook. f.

M. R. Anusha1, Veluswamy Bhuvaneshwari1, Manokaran Kalaiselvi2, Akbar Ali Amrin1, and Ramasamy Amsaveni1 ∗

1PG and Research Department of Biotechnology, Kongunadu Arts and Science College, Coimbatore 641029, Tamil Nadu, India 2Department of Biochemistry, Kongunadu Arts and Science College, Coimbatore 641029, Tamil Nadu, India

Medicinal plants act as an important source of drug with potential therapeutic effects. The present study focuses on the in vitro anti-inflammatory activity of Hibiscus schizopetalus (Dyer) Hook. f. (leaves and flowers) extracts. The cold water and hot water extract was prepared for plant sample without drying and whereas cold water, hot water, methanol, ethanol extract was prepared for the plant sample after drying. The present study on extract of H. schizopetalus demonstrated in vitro anti-inflammatory properties as evidenced by inhibition of albumin denaturation, membrane stabi- lization test and proteinase inhibitory action. This justifies that the traditional use of this plant in treatment of pains and inflammation. Keywords: Hibiscus, Inflammation, Medicinal Plants.

1. INTRODUCTION types of inflammatory conditions [5]. The mechanism of Natural products have contributed enormously to the inflammation injury is attributed, in part, to release of reac- development of important therapeutic drugs that are used tive oxygen species (ROS) from activated neutrophil and in modern medicine [1]. There is a need to search new macrophages. This over production leads to tissue injury plant derived pharmacologically active compounds that by damaging the macromolecule and lipid peroxidation of contribute in the discovery of clinically useful drugs. It is membranes [6]. The use of plants for folkloric medicine as well known that about 25% of all modern drugs that are anti-inflammatory agents should be confirmed with fruit- used to treat disorders are derived directly or indirectly ful and logical research strategy for the development of form plants [2]. Nowadays people are being bombarded new anti-inflammatory drugs. Inflammation leads to life with thousands of unhealthy products, the level of sensi- threatening hypersensitivity reactions which are harmful bility in front of diseases is very high and that’s why the and progressive organ damage [7]. use of medicinal plants can represent the best solution [3]. Inflammation is due to harmful stimuli, such as The use of herbal extracts and nutritional supplements pathogens, damaged cells, or irritants and it is a part of either as alternative or complimentary medicine to the con- complex healing process to remove the injurious stimuli. ventional chemotherapy for treatment of anti-inflammatory Infection is caused by a microorganism, whereas inflam- diseases is well documented in ayurveda. This is an alter- mation is one of the healing responses against the wounds and infections caused by pathogen. The plants are one of native medicinal system that has been practiced primarily the most important sources of medicines. India is known in the Indian subcontinent about 5000 years [4] for treat- due to availability of several thousands of medicinal ing inflammatory diseases, including different rheumatic plants in the different bioclimatic zones anti-inflammatory diseases. diseases [8]. The medicinal substances packaged in a plant can be Lysosomal enzymes released during inflammation pro- safely assimilated by the body since the plants are its nat- duce a variety of disorders which leads to the tissue ural food. In India many ayurvedic practioners are using injury by damaging the macromolecules and lipid perox- various indigenous plants for the treatment of different idation of membranes which are assumed to be respon- sible for certain pathological conditions as heart attacks, ∗Author to whom correspondence should be addressed. septic shocks and rheumatoid arthritis etc. Stabilization of

Adv. Sci. Eng. Med. 2020, Vol. 12, No. xx 2164-6627/2020/12/001/005 doi:10.1166/asem.2020.2545 1 In Vitro Anti-Inflammatory Activity of Hibiscus Schizopetalus (Dyer) Hook. f. Anusha et al. lysosomal membrane is important in limiting the inflam- 2.4. Cold Water Extraction matory response by inhibiting the release of lysosomal About 10 g of leaves and flowers of H. schizopetalus was constituents of activated neutrophil such as bactericidal macerated in mortar and pestle with 100 ml distilled water enzymes and proteases, which cause further tissue inflam- at room temperature and then filtered using muslin cloth. mation and damage upon extra cellular release or by sta- The filtrate was centrifuged at 5000 rpm for 15 min. The bilizing the lysosomal membrane [9]. supernatant was again filtered using Whattman’s No. 1 fil- The genus Hibiscus comprises about 275 species in the ter paper under strict aseptic conditions and the filtrate was tropics and subtropics. With attractive and colorful flow- collected in fresh sterilized glass tubes and stored at 4 C ers, and are widely planted as ornamentals and are used in until use [14]. traditional medicines [10]. It is one of the least examined specie of this genus. Hibiscus schizopetalus (Mask) hook 2.5. Ethanol and Methanol Extraction (Malvaceae) is a shrub with spreading or usually drooping The ethanol and methanol extract of dried plant leaves and branches, attaining a height up to 13 feet (4 m) found in flowers of H. schizopetalus was prepared by mixing about east of tropical Africa. Leaves of H. schizopetalus are con- 5 grams of each plant material (flower and leaves) powder sumed to cure spermatorrhoea [11]. Fruits are consumed in 50 ml of respective solvents separately. The mixture to treat urinary problem arising from endocrinological dis- was taken into 250 ml sterile conical flasks, plugged with order or diabetes, and young fruits are chewed raw [12]. sterile cotton and kept in shaking incubator with 200 rpm Fruit infusion is drunk to facilitate childbirth [13]. for 24 h. The solution was filtered through muslin cloth. This process was repeated three times after which a clear 2. MATERIALS AND METHODS aqueous extract of the plant was taken [15]. All the Hibiscus extracts (leaf and flower) obtained were 2.1. Authentication of Plant Material used for phytochemical analysis, in vitro anti-inflammatory Hibiscus schizopetalus (Dyer) Hook. f.: Fresh activities, in vitro antihaemolytic activity and in vitro cyto- H. schizopetalus leaves and flowers was collected from toxicity tests. Coimbatore district, Tamil Nadu, India. The plant was authentified in Botanical Survey of India and the authenti- 2.6. In Vitro Anti-Inflammatory Activity cation number is NO.BSI/SRC/5/23/2014-15/Tech./2030. 2.6.1. Inhibition of Albumin Denaturation Freshly collected leaves and flowers of H. schizopetalus were washed in running tap water for 3 min. Then the The reaction mixture consisted of test extracts and 1% plant parts were surface sterilized using 1% mercuric aqueous solution of bovine albumin fraction. The pH of the chloride solution under strict aseptic conditions. Finally reaction mixture was adjusted using small amount of 1 N HCl. The samples were incubated at 37 C for 20 min and they were rinsed with sterile distilled water thoroughly to  remove mercuric chloride residues. Excess moisture was then heated at 57 C for 20 min. After cooling, it was mea- removed from the sterilized leaves and flower. Then they sured spectrophotometrically at 660 nm. The experiment was performed in triplicates [16]. The protein denaturation were subjected to hot water and cold water extraction. inhibition percentage was calculated using the following formula. 2.2. Extract Preparation from the Leaves and Flowers of H. Schizopetalus = × −A /A % inhibition of protein denaturation 100 1 C T Extracts were prepared by two different modes such asex- A = A = traction of fresh plant material without drying and extrac- where C absorption of the control sample, and T tion after drying each plant part (leaves and flower). From absorption of the test sample. each methods hot water, cold water and organic solvent (ethanol and methanol) extracts was prepared [14] and 2.7. Membrane Stabilization Test used for further studies. Fresh whole human blood (5 mL) was collected and trans- ferred to the heparinized centrifuged tubes. The tubes were 2.3. Hot Water Extraction centrifuged at 3000 rpm for 10 min and were washed three About 10 g of leaves and flowers of Hibiscus schizopetalus times with equal volume of normal saline (0.9% NaCl). was boiled in 100 mL distilled water with constant stir- After measuring the volume of blood, it was reconstituted ring for 30 min. The solution was then allowed to cool as 10% v/v suspension with normal saline [17]. The reac- to room temperature and then filtered using muslin cloth. tion mixture (2 mL) consisted of 1 mL of plant extracts The filtrate was centrifuged at 5000 rpm for 15 min. The and 1 mL of 10% RBC suspension. In case of control tube supernatant was again filtered using Whattman’s No. 1 fil- instead of plant extract only saline was added. Aspirin was ter paper under strict aseptic conditions. The filtrate was taken as a standard drug. All the centrifuge tubes contain- collected in fresh sterilized glass tubes and stored at 4 C ing the reaction mixture were incubated in a water bath until use [14]. at 56 C for 30 min. At the end of the incubation, the

2 Adv. Sci. Eng. Med. 12, 1–5, 2020 Anusha et al. In Vitro Anti-Inflammatory Activity of Hibiscus Schizopetalus (Dyer) Hook. f. tubes were cooled under running tap water. The reaction mixture was centrifuged at 2500 rpm for 5 min and the absorbance was read spectrophotometrically at 560 nm. The experiment was performed in triplicates [18]. Per- centage of membrane stabilization activity was calculated using the following formula. = × − A /A % of Membrane stabilization 100 1 C T A = A = where C absorption of the control sample, and T absorption of the test sample.

2.8. Proteinase Inhibitory Action The reaction mixture (2 mL) was containing 0.06 mg trypsin, 1 mL 20 mM TrisHCl buffer (pH 7.4) and 1 mL Figure 1. Membrane stabilization activity of H. schizopetalus leaf and flower extracts. test sample. The mixture was incubated at 37 Cfor5min and then 1 mL of 0.8% (w/v) casein was added. The mix- ture was incubated for an additional 20 min. In order to dry hot water flower extract showed 70 ± 0.18% was terminate the reaction, about 2 mL of 70% perchloric acid effective in inhibiting heat induced albumin denaturation was added and the cloudy suspension was centrifuged. The absorbance of the supernatant was read at 210 nm against (Table I). buffer as blank using UV spectrophotometer. The experi- 3.1.2. Membrane Stabilization Test ment was performed in triplicates. The percentage inhibi- tion of proteinase inhibitory activity was calculated using Stabilization of the RBC membrane was studied to fur- the following formula [16]. ther establish the mechanism of anti-inflammatory action of H. schizopetalus. The percentage of inhibition of = × − A /A % of Proteinase inhibition 100 1 C T H. schizopetalus fresh hot water flower extracts and dry hot water flower extracts was found to be 82 ± where A = absorption of the control sample, and A = C T 0.07% and 79 ± 0.16%, respectively (Table II, Fig. 1). absorption of the test sample. Diclofenac sodium was used as the reference standard. The absorbance of human blood treated with the assay 3. RESULTS AND DISCUSSION mixture at 560 nm. Ethanolic extract of H. colorata 3.1. In Vitro Anti-Inflammatory Activity showed a good percentage of inhibition of haemolysis 3.1.1. Inhibition of Albumin Denaturation when compared to the other extracts. The reference drug To investigate on the mechanism of anti-inflammatory Diclofenac sodium showed 76.79% of haemolysis while activity, the ability of extract to inhibit protein denatura- it was 39.34%, 34%, 20.4%, 34% for ethanolic, acetone, tion was studied. It was found that the H. schizopetalus chloroform, and hexane extracts respectively, in the con- fresh hot water flower extract of about 73 ± 0.14% and centration of 150 g/mL.

Table I. Inhibition of albumin denaturation by H. schizopetalus leaf and flower extracts.

Inhibition of albumin denaturation (%)

Fresh plant material without drying Plant material after drying Cold water Hot water Cold water Hot water Ethanol extract Methanol extract

Leaf 54 ± 0.07 62 ± 0.06 50 ± 0.19 59 ± 0.27 49 ± 0.13 56 ± 0.04 Flower 56 ± 0.11 73 ± 0.14 52 ± 0.17 70 ± 0.18 45 ± 0.21 54 ± 0.06

Table II. Membrane stabilization activity of H. schizopetalus leaf and flower extracts.

Inhibition of albumin denaturation (%)

Fresh plant material without drying Plant material after drying Cold water Hot water Cold water Hot water Ethanol extract Methanol extract

Leaf 55 ± 0.12 78 ± 0.08 52 ± 0.11 69 ± 0.22 60 ± 0.22 63 ± 0.06 Flower 59 ± 0.16 82 ± 0.07 56 ± 0.19 79 ± 0.16 53 ± 0.07 67 ± 0.08

Adv. Sci. Eng. Med. 12, 1–5, 2020 3 In Vitro Anti-Inflammatory Activity of Hibiscus Schizopetalus (Dyer) Hook. f. Anusha et al.

Table III. Proteinase inhibitory activity of H. schizopetalus leaf and flower extracts.

Inhibition of albumin denaturation (%)

Fresh plant material without drying Plant material after drying Cold water Hot water Cold water Hot water Ethanol extract Methanol extract

Leaf 56 ± 0.17 65 ± 0.12 51 ± 0.09 66 ± 0.11 55 ± 0.24 44 ± 0.12 Flower 55 ± 0.07 72 ± 0.18 53 ± 0.13 68 ± 0.17 62 ± 0.06 51 ± 0.08

3.2. Proteinase Inhibitory Action a base for the development of novel potent drugs against Aqueous extracts of H. schizopetalus exhibited signifi- inflammations. cant antiproteinase activity. The percentage of inhibition of H. schizopetalus fresh hot water flower extracts and dry Acknowledgments: Authors are thankful to the Univer- hot water flower extracts was found to be 72 ± 0.18% and sity Grants Commission [MRP-7007/16 (SERO/UGC) Dt. 68 ± 0.17%, respectively (Table III). 30th October 2017] for providing financial assistance and In the present study it was found that the DST FIST instrumental facilities for this research work H. schizopetalus fresh hot water flower extract of about and to the Secretary, Principal and Department of Biotech- 73 ± 0.14% and dry hot water flower extract showed 70 ± nology of Kongunadu Arts and Science College for pro- 0.18% was effective in inhibiting heat induced albumin viding facilities to carry out this research work. denaturation. 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Received: xx Xxxx xxxx. Accepted: xx Xxxx xxxx.

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