Anti-Diabetic, Anti-Inammatory and Antioxidant Properties of Four Underutilized Ethnomedicinal Plants: An in Vitro Approach

Background : In the present study, antidiabetic, anti-inammatory activities and antioxidant properties in four diverse dissolvable concentrates of four lesser known ethnomedicinal plants viz. Apluda mutica, Mikania micrantha, Kyllinga nemoralis and Cleome rutidosperma were investigated. Methods: The benzene, chloroform, methanol and 70% aqueous (aq.) ethanol extract of these plants were tried for antioxidant by various established in vitro systems like total phenolic content, DPPH free radical scavenging, ABTS radical cation scavenging, metal chelating activity. Anti-diabetic and anti-inammatory activities were explored by quantifying α-amylase, α-glucosidase and protein denaturation inhibitory activities of the investigated plants. Results: Among the various solvents 70% aq. ethanol extract of M. micrantha had the highest total phenolic content (230.450 ± 0.12mg GAE/g extract), DPPH & ABTS radical scavenging activity, FRAP value (4.122± 0.004 μM Trolox equivalent/ g dry extract), anti-lipid peroxidation capacity, reducing power and metal chelating activity. The highest amount of total avonoid was detected in the 70% aq. ethanol extract of C. rutidosprema (71.050 ± 0.058 mg RE /g extract. Similarly M. micrantha also exhibited signicantly lower IC 50 values for the percentage inhibition of α-amylase (IC 50 58.44 ± 0.012 µg/ml) and α-glucosidase (IC 50 113.31 ± 0.010 µg/ml) compared to acarbose (IC 50 = 53.8 ± 0.009 µg/ml ; IC50 = 79.48 ± 0.006 µg/ml respectively) (p ≤ 0.05). Anti-inammatory activity was determined by using protein denaturation assay and M. micrantha showed signicant lower IC 50 value for the inhibition of protein denaturation (IC 50 = 89.27 ± 0.008 µg/ml) compared to other plants under investigation. Quantication of polyphenolics by HPLC showed the presence of different phenolic acids in varying amounts. Conclusion: Therefore, the results indicate that these plants were shown to contain a remarkable amount of different bio-active compounds, thus conrming their involvement in several biological activities and to serve as a potential antioxidative, anti-inammatory and anti-diabetic agent in food and pharmaceutical industries. The gradient elusion was 90 % solvent B and 10% solvent A and ow rate was settled from 1ml/min to 0.7 ml/min in 27 min, from 10 to 40 % solvent A with ow rate 0.7 ml/min for 23 min, 40% solvent A and 60% B with ow rate 0.7 ml/min primarily for 2 min and then ow rate altered from 0.7 to 0.3 ml/min in 65min, from 40 to 44% solvent A with ow rate 0.3 to 0.7ml/min in 70 min, 44% solvent A with ow rate 0.7 to 1ml/min for 10 min duration, solvent A changed from 44% to 58 % with ow rate 1ml/min for 5 min, 58 to 70% solvent A in 98 min at constant ow rate 1 ml/min. The mobile phase composition was taken to the original condition (solvent A: solvent B: 10: 90) in 101 min and allowed to run for another 4 min, before the injection of another sample. Total analysis time per sample was 105 min. The presence of phenolics were detected in HPLC chromatograms using a photo diode array UV detector at three different wavelengths (272, 280 and 310 nm) according to absorption maxima of investigated compounds. Each compound was identied by its retention time and by spiking with standards under the same conditions. The quantication of phenolic acids and avonoids in the plant extracts were analysed by the measurement of the integrated peak area and the amounts were estimated using the calibration curve of the respective standard compound. with decreasing polarity. The outcome is as per the examination by Sultana et al. on medicinal plants where aqueous ethanol displayed most extreme DPPH radical scavenging activity over absolute ethanol (Sultana et al. 2009). The data suggests that polar components like phenol and avonoids of the plant studied contributed to the radical scavenging activity. The methanolic extract of these plants are also rich in their avonoid content and exhibited good scavenging activity. Hagerman et al. have reported free radicals scavenging property increases with the increase in molecular weight of the phenolic compounds and it depends on the number of aromatic rings and nature of hydroxyl group substitution (Hagerman et al. 1998).

The plant materials viz. Mikania micrantha , Apluda mutica , Kyllinga nemoralis and Cleome rutidosperma were collected from various locations of Kolkata and Howrah, India and identi cations were authenticated at Botanical Survey of India, Howrah. The voucher specimens of each plant materials were preserved at the Plant Chemistry department of our o ce under registry no. BSI/Chem/SD 006, BSI/Chem/SD 007, BSI/Chem/SD 008, and BSI/Chem/SD 009 separately. Plant materials were shade dried, pulverized and stored in an airtight container for further study.

Extraction of plant material
For preparation of plant extracts, one gram dry sample was extracted separately with 20 ml of methanol, 70% aq. ethanol, chloroform and benzene with continuous stirring, for 18-24 h at ambient temperature. The extracts were subsequently ltered with Whatmanno.1 lter paper and diluted to 25 ml with corresponding extracting solutions. These extracts were analyzed for their antioxidant pro ling, anti-diabetic and anti-in ammatory study.

Antioxidant activity
Estimation of total phenolic content Folin-Ciocalteu procedure was followed to estimate total phenolic content of the plant extracts (Datta et al. 2019), and was expressed as mg/g gallic acid equivalent (GAE). To 100 μl of different plant extract, 1.0 ml of Folin-Ciocalteu reagent and 0.8 ml of sodium carbonate (7.5%) were added and incubated for 30 mins after proper mixing. Absorption was measured at 765 nm (UV-visible spectrophotometer Shimadzu UV1800).
Estimation of total avonoid content Estimation of total avonoid content was achieved following the standard method (Datta et al. 2019) and was expressed as mg/ g rutin equivalent (RE). To 0.5 ml of extract, 0.5ml of ethanolic aluminium chloride (2%) solution was added.Theabsorbance was measured at 420 nm(UV-visible spectrophotometer Shimadzu UV 1800) after 1 hr incubation.

Ferric Reducing Antioxidant Power (FRAP) Assay
Ferric Reducing Antioxidant Power (FRAP) was measured and expressed as trolox equivalent (TE) mg/ g dry extract (Datta et al. 2019). The FRAP working solution was prepared by mixing 300 mM sodium acetate buffer (pH 3.6), 10 mM TPTZ (2, 4, 6-tripyridyl triazine) solution in 40 mM HCl and 20 mM ferric chloride solution in a ratio of 10:1:1 (v/v/v). 1 ml of plant extract was then added to 2.85 ml of FRAP working solution and incubated at 37 °C for 30 min in dark. The increase in absorbance was measured at 593 nm in a UV-visible spectrophotometer (Shimadzu UV 1800).

Measurement of reducing power
The reducing power of the extracts wascompletedand calculated as ascorbic acid equivalent (AAE) in mg/ g of dry material (Datta et al. 2019). 100 μl of plant extracts were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and 2.5 ml potassium ferricyanide (1%). The mixture was incubated at 50°C for 20 min.2.5 ml aliquots was taken from mixture and added to the equal volume of 10% trichloroacetic acid, which was then centrifuged at 3000 rpm for 10 min. The upper layer of the resulting (2.5 ml) was added to the equal amount of distilled water and 0.5 ml freshly prepared ferric chloride solution (0.1%) and kept in dark for 5 min. The absorbance of resulting solution was estimated at 700 nm in a UV-visible spectrophotometer (Shimadzu UV 1800). DPPH (2,2-Diphenyl-1-picryl-hydrazyl) assay Free radical scavenging ability of the extracts was determined using the stable radical DPPH (1, 1-diphenyl-2-picrylhydrazyl) following the process of Dutta et al (Datta et al. 2019). 100 μl of the tested sample were mixed with 3.9 ml of methanolic DPPH solution (25 mg/L). After 30 mins incubation in dark, the absorbance was measured at 517 nm in a UV-visible spectrophotometer (Shimadzu UV1800). The capability of test sample to scavenge the DPPH radical was determined.

Metal chelating property
For the estimation of metal chelating property of experimental plant extract, the process of Lin et al. was followed with slight modi cations (Datta et al. 2019). 1 ml of plant extracts was added to a 200 µl ferrous chloride (2 mM) and 400µl ferrozine (5 mM). The mixture was incubated for 10 mins and absorbance was taken at 562 nm.
Anti-lipid peroxidation in linoleic acid system Anti-lipid peroxidation was assayed as described by Dutta et al, with modi cations (Datta et al. 2019). 1ml of plant extract was added 130 µl of linoleic acid solution followed by addition of 99.8% ethanol (10 ml) and 10 ml sodium phosphate buffer (pH 7, 0.2M). The mixture was made upto25 ml and incubated at 40 0 C upto 360 hours. Extent of oxidation was measured by thiocyanate method. 75% ethanol (10ml), 30% aq. Solution of ammonium thiocyanate (0.2 ml), sample solution (0.2 ml) and ferrous chloride (20mM in 3.5% HCl, 0.2 ml) added sequentially. The absorbance was measured at 500 nm after 3 mins incubation.
Quantitative pro ling of phenolic acids and avonoids by RP-HPLC

Preparation of standard solutions
Stock solution of 1mg/ml concentration of different phenolic acids and avonoids viz. protocatechuic acid, gentisic acid, chlorogenic acid, p-hydroxy benzoic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid, salicylic acid, gallic acid and ellagic acid, catechin, rutin, myricetin, quercetin, naringin, apigenin and kaempferol were prepared by dissolving 10 mg of the respective phenolic acids in 1 ml HPLC-grade methanol and the and the resulting volume was made up to 10 ml with the solvent for the Mobile phase (methanol and 0.5% aq. acetic acid 1:9). A standard curve was obtained by further dilution at 20, 40, 60, 80 and 100 μg/ml with the mobile phase solvent system. The standard and working solutions were ltered through 0.45 μm PVDF-syringe lter prior injection.
Preparation of sample solution 1 g of each coarsely powdered plant samples were extracted using 5 ml 70% aq. ethanol with constant stirring for 24 h. The process was repeated for three times and the nal volume was made upto 10 ml. The extracts were ltered through 0.45 μm PVDF-syringe lter prior injection.

Chromatograph analysis
Quanti cation of phenolic acids and avonoids were performed following the method of Dutta et al (Datta et al. 2019). Separation was achieved by a reversed phase Acclaim C18 column (5 micron particle size, 250 x 4.6 mm). The mobile phase contains methanol (Solvent A) and 0.5% aq. acetic acid solution (Solvent B) and the column was thermostatically controlled at 28 0 C and the injection volume was kept at 20 μl. The gradient elusion was 90 % solvent B and 10% solvent A and ow rate was settled from 1ml/min to 0.7 ml/min in 27 min, from 10 to 40 % solvent A with ow rate 0.7 ml/min for 23 min, 40% solvent A and 60% B with ow rate 0.7 ml/min primarily for 2 min and then ow rate altered from 0.7 to 0.3 ml/min in 65min, from 40 to 44% solvent A with ow rate 0.3 to 0.7ml/min in 70 min, 44% solvent A with ow rate 0.7 to 1ml/min for 10 min duration, solvent A changed from 44% to 58 % with ow rate 1ml/min for 5 min, 58 to 70% solvent A in 98 min at constant ow rate 1 ml/min. The mobile phase composition was taken to the original condition (solvent A: solvent B: 10: 90) in 101 min and allowed to run for another 4 min, before the injection of another sample. Total analysis time per sample was 105 min. The presence of phenolics were detected in HPLC chromatograms using a photo diode array UV detector at three different wavelengths (272, 280 and 310 nm) according to absorption maxima of investigated compounds. Each compound was identi ed by its retention time and by spiking with standards under the same conditions. The quanti cation of phenolic acids and avonoids in the plant extracts were analysed by the measurement of the integrated peak area and the amounts were estimated using the calibration curve of the respective standard compound.

Estimation of anti-diabetic property
Extraction of plant material 1g of powdered plant sample was extracted with 5 ml 70% ethanol on constant stirring for 24 h at the ambient temperature. The resulting extracts were ltered using Whatman No.1 lter paper and the ltrate was concentrated under vacuum at room temperature. Dried extracts were weighed and further dissolved in double distilled water to yield a stock solution concentration ranging from 5μg/ml to 50μg/ml.

α-Amylase inhibition property
This assay was carried out using a modi ed procedure of McCue and Shetty (McCue, K. Shetty 2004). Different concentration (5-50μg/ ml) of plant extract was mixed with 250 µl of sodium phosphate buffer (0.02M, pH 6.9) containing α-amylase solution (0.5 mg/ml). This solution was incubated at 25 0 C for 10 min, followed by addition of 250 µl of starch solution (1%) in 0.02M sodium phosphate buffer (pH 6.9) then further incubated at 25 0 C for 10min. The reaction was completed by addition 500 µl of dinitrosalicylic acid (DNS) reagent. The reaction mixture was incubated for 5min in boiling water. The absorbance was measured at 540 nm using spectrophotometer after cooling to room temperature and further dilution with 5ml distilled water. Acarbose (Acarbose, 25mg from Orchid Chemicals & Pharmaceuticals Ltd.) was used as standard. The α-amylase inhibitory activity was calculated as percentage inhibition (%).

α-Glucosidase inhibition property
The inhibition of α-glucosidase assay of the extracts was completed according to the method described by Kim et al. (Kim et al. 2005), using α-glucosidase from Saccharomyces cerevisiae. p-nitrophenylglucopyranoside (pNPG) solution prepared in phosphate buffer (20mM , pH 6.9) was used as substrate. 100 µl of α-glucosidase (1.0U/cc) was pre-incubated with in different concentration (5 -50μg/ml) of the extracts for 10min, followed by addition of 50 µl of pNPG (3mM) to initiate reaction. The reaction mixture was incubated at 37 0 C for 20min and terminated by adding 2ml of sodium carbonate (0.1M). The absorbance was measured at 405 nm using spectrophotometer. Acarbose was used as standard andα-glucosidase inhibitory activity was calculated as percentage inhibition (%).

Anti-in ammatory activity
The plant extract was prepared following the same procedure that used for anti-diabetic activity. The anti-in ammatory activity was studied in terms of inhibition of albumin denaturation which was studied according to Vallabh et. al. (Vallabh et. al. 2009) with minor modi cations. The reaction mixture (0.5 ml) consisted of 0.45 ml of bovine serumalbumin (5% aqueous solution) and 0.05 ml of plant extracts (50 and 200 μg/cc of nal volume) which was incubated in boiling water bath for 10 mins.After cooling the turbidity was measured at 660nm. Diclofenac (Dicloran 50 mg, JB Chemicals & Pharma Ltd.) was used as standard and protein denaturation activity was calculated as percentage inhibition (%).

Statistical analysis
All the experiments were done using triplicate samples. Results were represented as value ± standard error mean (SEM).Experimental results were subjected to univariate analysis of variance (ANOVA), followed by Tukey test (p ≤ 0.05) using the statistical package for the social sciences SPSS version17.0 (SPSS Inc., Chicago,Illinois, USA).

Results
Antioxidant activity Effect of solvent system on extractive yield The extractive yield of the different solvent extracts of the experimental plants varied according to polarity (Table 1). Maximum extractive yield was observed in methanol extract of M. micrantha (7.21±0.19%) and C. rutidosperma (7.17 ± 0.16%) and the lowest in benzene extract of C. rutidosperma (0.538 ± 0.116%). The plants showed a higher extraction yield with 70% aq.ethanol and methanol compared to their chloroform or benzene extracts.

Total phenolic and avonoid content
Total phenolic and avonoid content is expressed as mg gallic acid equivalent (GAE)/g dry extract mg rutin equivalent (RE)/ g dry extract respectively and is represented in Table 1. All the extracts showed presence of high amount of phenolics and avonoids in the 70% aq. ethanol followed by methanol extracts and it decreased with decreasing polarity. Maximum phenolic content was observed in M. micrantha (230.45 ± 0.12 mg GAE/g dry extract) and the lowest in A. mutica (141.94 ± 0.67 mg GAE/g dry extract). The total avonoid content is expressed as equivalent mg rutin equivalent (RE)/g dry extract and was observed to be comparatively lower than the phenolic content. 70% aq. ethanol extract of C. rutidosperma.(71.05 ± 0.06 RE/g dry extract) showed the highest amount of avonoid content. The avonoid content in the different solvent extracts under study also decreased with decreasing polarity of the extracting solvent.
Radical scavenging property using DPPH and ABTS radical All solvent extractsof the plants studied were subjected to radical scavenging activity assays using both DPPH and ABTS radicals and is expressed as percentage of inhibition of radicals by the extract and is depicted in Table 2. Activities varied widely, from very high to moderate. All samples demonstrated ability to scavenge DPPH and ABTS radicals, however, the plants showed more prominent activity against ABTS radical. Aq. ethanol solution of M. micrantha (87.01 ± 0.22%) and C. rutidosperma (83.85 ± 0.08%) exhibited maximum radical scavenging activity using DPPH radicals. Similar observations were made using ABTS radical with M. micrantha (93.30 ± 0.84%) and C. rutidosperma (92.67 ± 0.13 %) having the most promising activity. The scavenging activity was observed to be minimal in the benzene extract for all the plants.

Ferric ion reducing antioxidant power (FRAP)
All the four different extracts of each plant were subjected to FRAP assay which is expressed as µ mole Trolox equivalent (TE)/g dry extract and is represented in Table 2. The aq. ethanol and methanol extract showed higher FRAP activity over the chloroform and benzene extract for all the plants. Maximum activity was observed in 70 % aq.ethanol extract of M. micrantha (4.12± 0.004 µM TE) and C. rutidosperma (3.73± 0.003 µM TE). The FRAP activity of the methanol extract of these plants was also promising.
Reducing property and metal chelating activity All the previous experiments indicated that the alcoholic extract of the plants to possess better antioxidant capabilities and subsequently those had been selected for in additional studies. Reducing property is expressed as mg ascorbic acid equivalent (AAE)/g dry extract and is represented in showed the highest chelating activity expressed as percentage of inhibition of metal ions/g dry extract and represented in Table 3. Maximum chelating activity is observed in 70 % aq. ethanol extract followed by methanol extract for all the plants under study. The chelating activity of M. micrantha and C. rutidosperma in methanol extract were also noteworthy.

Anti-lipid peroxidation assay
Anti-lipid peroxidation assay is represented as percentage of inhibition of lipid peroxidation/g dry extract and is represented in Table 3. M. micrantha (63.16 ± 0.011%) showed maximum inhibition activity in 70% aq. ethanol extract. The ethanol extract showed greater e cacy in lipid peroxidation activity over other extracts for all the plants.
Antidiabetic potential by α -amylase and α-glucosidase inhibition assay Results were calculated as percentage inhibition (under experimental conditions). The inhibitory activity for the studied plants was also shown to be concentration dependent. The IC 50 of each plant extract was determined as the inhibitor concentration (estimated on basis of the dried extract) required inhibiting 50% of α-amylase activity and α-glucosidase under the described experimental conditions, represented in Table 5. Acarbose appeared to be most active as α-amylase inhibitor (IC 50

Reducing property and metal chelating activity
Metal ions mediate lipid peroxidation to initiate a chain reaction leading to the deterioration of food (Gordon 1990). The catalysis of metal ions also correlates with etiology of cancer and arthritis (Halliwell et al. 1990). Chelating capacity of the investigated plant extracts decreased with the decreasing polarity. A similar trend is followed as recorded forreducing property. In addition, lipid peroxidation and oxidative damage of protein systems ferrous ions also catalyze the conversion of hydrogen peroxide to hydroxyl radical via Fenton reaction. Ferrous ions are the most effective pro-oxidants adequately found in food systems (Yamaguchi et al. 1998). Polyphenols with dihydroxygroups can conjugate metals, preventing metal catalyzed free radical formation (Gua et al. 1996). The metal reducing or chelating ability can be attributed to the high phenol and avonoid content in the 70% aq. ethanol extracts.

Anti-lipid peroxidation assay
Peroxidation of cell membrane lipid is associated with various pathological events such as atherosclerosis, in ammation and liver injury (Singh et al. 2012).The phenolic compounds and other chemical components may suppress lipid peroxidation through different chemical mechanisms, including free radical quenching, electron transfer, radical addition or radical recombination (Mathew and Abraham 2006). Lipid peroxidation is also associated with of rancidity of lipid foods (Gutensperger et al. 1998). The plant extracts under study have highest phenolic content in the 70% ethanol extract so their anti-lipid peroxidation activity was also higher in their 70% ethanol extract. This is in accordance with several other investigations on fruits and vegetables where a signi cant relationship between total phenolic content and peroxidation activity is evident (Velioglu et al. 1998) and in medicinal plants like Hypericum perforatum (Zou et al. 2004).

Quantitative pro ling of phenolic acids and avonoids by RP-HPLC
Protocatechuic acid is widely distributed naturally occurring phenolic acid. It is also a very common compound present in human diet, present in bran and grain brown rice and also found in many fruits. This phenolic acid showed various pharmacological activities such as anti-in ammatory properties and interaction with several enzymes (Kakkar and Bais 2014). Chlorogenic acid is found in coffee and coffee beans and also found in higher plants. It has been reported to reduce blood sugar levels and potentially exert an anti-diabetic effect (Uma et al. 2010). Even though, presence of appreciable amount of chlorogenic acid in A. mutica there has been no report of its use as an anti-diabetic agent, therefore its potential can further be explored. to be bene cial for the treatment of obesity,diabetes, hypertension, and metabolic syndrome (Alam et al. 2014). M. micrantha has high amount of naringin and therefore can be considered for treatment of the above diseases. Naringenin has anticancerous activity (Kanno et al. 2005) and is being researched as a potential treatment for Alzheimer's disease (Yang et al. 2017).The presence of naringenin is detected only in C. rutidosperma and therefore the use of these plants for treatment of Alzheimer's disease needs further attention.
Antidiabetic potential by α-amylase andα-glucosidase inhibition assay Diabetes is characterized by high concentrations of blood sugar levels, which can cause serious complications, such as organ failures and/or destruction of the kidneys, eyes, and variouscardiovascular diseases. One of the therapeutic methods is to reduce the hyperglycemia by hindering the absorption of glucose, which is achieved by inhibition of carbohydrate-hydrolyzing enzymes, such as α-amylase and α-glucosidase (Bhandari et al. 2008). Synthetic drugs like miglitol, acarbose and voglibose are used in conjunction with other antidiabetic drugs, but these inhibitors exert gastrointestinal side effectslike abdominal discomfort, atulence and diarrhea on prolonged use (Shai et al. 2010). As a result of this, there isgrowing interest in discovering new and effective α-amylase and α-glucosidase inhibitors from plants with minimal or no side effects.The inhibition % for α-amylase are close to that of acarbose, and theinhibition rate for α-glucosidase was lower than that of acarbose for A. mutica, K. nemoralis and C. rutidosperma but comparable to M. micrantha. This indicated that A. mutica, K. nemoralis and C. rutidosperma are a potent inhibitor for α-amylase with mild inhibitory activity against α-glucosidase, whereas M. micrantha is a potent inhibitor for both the enzymes. There has been no previous report on the anti-diabetic effect of A.mutica and it is evident from the results that it can be considered as a promising source of anti diabetic compound. Anti-diabetic effect of the ethanol extract of the leaves of M. micrantha has been studied in alloxan induced diabetic rats (Nurdiana et al. 2013) but the present study elucidates the hypoglycaemic action through enzyme inhibitory action. Similarly for K. nemoralis, the hypoglycaemic activity has been established (Quanico et al. 2008) but not the α-amylase and α-glucosidase inhibition potential, which is elucidated here. The anti-diabetic effectof C. rutidosperma has been variously studied in animal model (Okoro et al. 2014) and its effect on glucokinase and glucose phosphatase has been presumed. In this study theexact inhibition of the above stated enzyme has been studied, which is important for thedevelopment of anti-diabetic drug from the plant sources.
Polyphenolic compounds in plants have been shown to inhibit the activities of digestive enzymes due to their ability to bind with proteins. Results from previous studies suggest that phenolic compounds isolated from medicinal plants (Nickavar et al. 2008) have potential to treat Type II diabetes mellitus. Polyphenols are widely distributed throughout the plant kingdom and thus form an integral part of the human diet.Therefore, the inhibition of α -amylase and α -glucosidase found in this study could be due to thepresence of polyphenolic compounds and may potentially provide a natural source of inhibitors. Anti-in ammatory activity by protein denaturation inhibition assay Denatured proteins are heterogeneous conformational isomers and lack in their proposed natural activities. However, the signi cance of denatured protein is expanding as it has demonstratedto be the underlying cause of many neurodegenerative and in ammatory diseases (Chang 2009). Anti-protein denaturation activity of these plant extract could be used as a potent mitigating drug in future. The anti-protein denaturation study was performed using bovine serum albumin (BSA). Antigens associated with type-III hypersensitivity reaction is expressed when protein is denatured, which is also related to diseases such as . Both M. micrantha and A. mutica contains the above compounds in substantial amount which legitimizes its activity. K. nemoralis and C. rutidosperma also possess these polyphenolic compounds but in lesser amount. Signi cant anti-in ammatoryactivity of M. micrantha was observed from ethyl acetate fraction in ear-in ammation on topical application of TPA. This study provides a preliminary idea about the e cacy of 70% ethanol extract on the anti-arthritic activity of the plant and also supports its traditional use in treatment of rheumatism. Our result is in congruence with prior study by Chakraborty and Roy (Chakraborty and Roy 2010) ,where the ethanolic extract of C. rutidosperma showed prominent antiarthritic activity in cottonpellet granuloma model. However A. mutica and K. nemoralis did not have any earlier report of anti-in ammatory activity. Studies on the capability of the 70% ethanol extract to hinder heat initiated protein denaturation demonstrate that these plants can be used for improvement of antiin ammatory drug.

Conclusion
The results of our current investigation support the potential role of four lesser known plants, traditionally used in India in folk medicineas an antioxidant, antidiabetic and anti-in ammatory agent. Both biochemical and biological tests were executed to provide a complete framework for each plant under investigation. All the tested plants demonstrated an amazing presence of secondary metabolites, involved in several biological activities. In particular, M.micrantha and C. rutidosperma has a high content of polyphenols and responsible for the signi cant antioxidant activity. Due to the abundance of bioactive metabolites, all these extracts were shown to be e cient, to inhibit α-amylase and α-glucosidase enzyme and also played an important role in the prevention of precipitation of the denatured protein aggregates involved in in ammation. In conclusion, all the data con rm thatthe investigated plants may be the potential sources of anti-diabetic and anti-in ammatory agent. Thus, the study has revealed that traditionally used easily available plants can be a low-cost source of important bioactive molecules with potential for herbal drug development.The speci c compounds responsible for biological activities need to be explored and further investigations for the most active compounds will be done in the near future. All authors were involved in the experiment, analysis, interpretation and drafting of the manuscript. All authors read and approved the nal manuscript.