Radical Scavenging , Proteases Activities , and Phenolics Composition of Bark Extracts from 21 Medicinal Plants

Stem barks derived from twenty-one medicinal plants were extracted in methanol (100%) and acetone-water (70 : 30 v/v) and at room as well as at reflux temperature conditions. Total phenolic contents, determined using FC (Folin Ciocalteu) reagent, ranged from 528 to 715mgGAE/g of crude extract. 15 out of 21 plants showedDPPH activity more than 90% and the rest of plants exhibited the activity in the range of 87–89%. The methanolic extract of P. granatum obtained at room temperature showed the highest antiradical activity (96%). The extracts with similar % radical scavenging of DPPH showed significant variation in EC


Introduction
The human body may produce oxygen-centered free radicals and other reactive oxygen species as by-products during physiological and biochemical processes.Overproduction of such free radicals causes oxidative damage to biomolecule (e.g., lipid, protein, and DNA) leading to chronic diseases, for example, cancer, arthritis, atherosclerosis, Alzheimer's disease, and diabetes [1,2].On the other hand, lipid peroxidation is one of the main reasons for deterioration in food products.
Synthetic antioxidants such as BHT, BHA, TBHQ, and PG are usually used to overcome this problem.Most of these synthetic antioxidants are found to have toxic and carcinogenic effects on human health [3].Several reports [4][5][6] reveal that natural compounds are capable of protecting against reactive oxygen species (ROS) mediated damage.Therefore, natural compounds may have potential application in prevention and curing of diseases [7].
The antioxidant constituents of plants are gaining the interest of scientists and food manufactures because the future demand is functional food with specific health effects.The antioxidant property of plant extracts has been attributed to their polyphenol contents.They improve the defensive system of plants against infection and injury.They are usually found as glycosylated derivatives or as salt with inorganic sulfates or organic acids [8].They play a key role as antioxidants due to the presence of hydroxyl constituents and their aromatic nature which enable them to act as reducing agents, hydrogen donors, and singlet oxygen quenchers.In addition, they have a metal chelation potential [9].
To assess radicals scavenging ability, different in vitro methods have been used such as ABTS [10], DPPH [11], and DMPA [12].Although these assays do not reproduce in vivo conditions but are useful to rank antioxidant activity of substances.In this paper, DPPH method has been applied to the bark extracts of the selected medicinal plants.UV-Vis spectrum of 1,1-diphenyl-2-picryl hydrazyl free radical (DPPH • ) shows a characteristic absorbance close to 515 nm in methanol.This method presents the advantage of using stable and commercially available free radical and has been 2 Journal of Chemistry extensively applied on plant extracts and on various food materials [13,14].
In the field of pharmaceutical research, enzyme inhibition is an important area which has resulted in the discovery of a number of useful drugs.Specific inhibitors interact with enzymes and block their activity towards their corresponding physiological substrates.The importance of enzyme inhibitors as drugs is enormous, since these molecules have been used for treating a large number of physiological conditions.Plant derived protease inhibitors serve in the defense mechanisms of plants against pests and plant pathogens.These inhibitors can be classified into a number of families on the basis of their specificities to inhibit the cleavage of specific peptide sequences within the proteins.
Proteases refer to a group of enzymes, whose catalytic function is to hydrolyze peptide bond of protein.They play a key role in a variety of biological processes, both at the physiological level and in infection.Proteases are classified into serine, threonine, cysteine, aspartate, and glutamic acid proteases.Most widely studied group of proteases is serine proteases and many pathological disorders are caused by deficiencies in the normally exquisite regulation of the activity of proteolytic enzymes, resulting in abnormal tissue destruction and the aberrant processing of other proteins.Elastase, chymotrypsin, and trypsin are the subclass of serine proteases based on the type of substrate.Trypsin cleaves polypeptide chains on the C-terminal side of a positively charged side-chain containing arginine or lysine.
Acacia nilotica (Fabaceae) is used for cold, bronchitis, diarrhoea, dysentery, biliousness, bleeding piles, and leucoderma [15].Acacia modesta commonly known as "Phulai" is chewed and sucked for gastric pain.Albizzia lebbeck (Mimosaceae) is used against cataract, asthma, ophthalmopathy, leprosy, diarrhea, and all types of poisoning [16].Antiallergic [17], anti-inflammatory [18], and anticonvulsant [19] activities of this plant have also been documented.Cassia fistula (Fabaceae) has a high therapeutic value and is used to cure anorexia, rheumatism, inflammatory diseases, and skin disorders and as an analgesic.Besides, it also finds use in the treatment of haematemesis, pruritus, leucoderma, and diabetes.The hepatoprotective activity [20] and the hypoglycemic activity [21] have also been reported.Cinnamomum zeylanicum (Lauraceae) showed many biological properties as analgesic, antiseptic, antispasmodic, aphrodisiac, astringent, carminative, insecticide, and parasiticide.It has mildly astringent and aromatic properties and is used in European medicine.It has a place for the treatment of colic and diarrhea [22].Different parts of Eucalyptus rostrata (Myrtaceae) are used in traditional medicine for the treatment of diarrhea, relaxed throats, chronic dysentery, malaria, infection of the upper respiratory tract, and certain skin diseases, and as an astringent in dentistry and for cuts [23].According to Ayurveda and Unani system of medication, Eugenia jambolana (Myrtaceae) is sweet, digestive, astringent, used to cure bronchitis, asthma, dysentery, and ulcers.Its external use shows good wound healing properties [24].
Ficus (Moraceae) are being used traditionally in a wide variety of ethnomedical remedies all over the world [25].
Mangifera indica (Anacardiaceae) possesses various pharmacological activities, including anti-inflammatory, antimicrobial, antioxidant, hepatoprotective, and hypoglycemic activities.Mangiferin isolated from M. indica is effective in against herpes simplex virus type 2 in vitro.It also possesses immunomodulatory action [26].Michelia champaca (Magnoliaceae) is used in the treatment of brain disorders fever, colic, leprosy, and postpartum protection, in childbirth, and as febrifuge.It is also used as anti-inflammatory and antipyretic agent [27].Various parts of Mimusops elengi (Sapotaceae) have been used as a febrifuge, astringent, purgative, and stimulant [28].The pounded seeds pasted with oil are used for the treatment of obstinate constipation [29].Punica granatum (Punicaceae) is used to treat stomach ache and laxative and respiratory pathologies in traditional medicine [30].Psidium guajava (Myrtaceae) is used as antidiarrhoeal, gastroenteritis, dysentery, antibacterial, colic, and pathogenic germs of the intestine [31].Polyalthia longifolia (Annonaceae) is used as a febrifuge and depressed the heart, lowered blood pressure, and stimulated respiration [32].The fungicidal effect of P. longifolia has also been reported by many workers [33].Zizyphus jujuba (Rhamnaceae) possess multiple medicinal properties such as antifertility, analgesic, and antidiabetes [34].
The aim of this study was to screen the bark extracts with respect to their total phenolic contents, radical scavenging, and proteases activity in order to find new potential drugs from natural sources.

Extraction of Phenolic Compounds.
Plant materials were collected in January 2012, from Azad Kashmir region, Pakistan, and identified at the Department of Botany, University of Gujrat, Gujrat, while the bark of C. zeylanicum and A. catechu was purchased from local market.The bark was shadedried, pulverized, grinded to powder, and extracted (50 gm) with 100% methanol and acetone-water (70 : 30) separately at room temperature for seven days and at reflux temperature for five hours (500 mL each).The crude extracts were filtered and concentrated at reduced pressure by using rotary evaporator.

Determination of Total Phenols.
Total phenols in extracts were determined using Folin-Ciocalteu reagent [35].40 L of each sample (2 mg/mL) was mixed with 0.25 mL of FC reagent and 0.8 mL of 10% sodium carbonate solution.The mixture was allowed to stand for 30 min and the absorbance was measured at 765 nm.The total phenolic contents were expressed as gallic acid equivalents (GAE) in mg/g of crude extract.Correlation studies between total phenolic contents and % scavenging of DPPH were performed.

DPP𝐻
• Radical Scavenging Assay.The radical scavenging ability of extracts was measured using the method of Majinda et al. [36].Methanolic solution (1.0 mL) of all the extracts (2 mg/mL) was added to 1.0 mL (0.2 mg/mL) methanol solution of DPPH • and absorbance was measured at 517 nm after thirty minutes.
The % scavenging of DPPH • was determined by the following formula: The amount of the sample was also determined by measuring decrease in absorbance at 517 nm to half of the initial value as EC 50 .Gallic acid and BHT were used as standard reference.

Protease Inhibition Assay.
All the extracts were subjected to protease inhibition assay according to the method of Jedinák et al. [37], with some modification.Tris buffer (100 mM) of pH 7.5 was prepared by dissolving 12.1 g of Tris (hydroxymethyl)-aminomethane in distilled water and adjusted pH 7.5 with HCl (5 M).The stock solution of trypsin was prepared by dissolving 2 mg of trypsin in 10 mL of 1.0 mM HCl. N-benzoyl-DL-arginine-paranitroanilide hydrochloride (BApNA) was dissolved in DMSO (20 mg/mL).Enzyme (0.3 mL) and inhibitor (100 L) was incubated at 37 ∘ C for 15 minutes and then 0.6 mM substrate was added and final volume was made 2.5 mL with Tris buffer.The reaction mixture was incubated at 37 ∘ C for 30 minutes.The reaction was quenched by adding 100 L of acetic acid (30%) and read the absorbance at 410 nm using UV/Vis spectrophotometer.Phenylmethanesulfonylfluoride (PMSF) was used as positive inhibitor: 2.6.High Performance Liquid Chromatography.Qualitative analysis of all plant extracts was carried out with high performance liquid chromatograph, HPLC-DAD system (Shimadzu, Kyoto, Japan) consisting of LC-20A pump, DGU-20A degasser, SPD M-20A diode array detector, a manual sample injector with a 20 L sampling loop (Rheodyne, Cottati, CA, USA), and CTO-20 A column oven.Data was integrated by using HPLC 20A software.The measurements were performed at room temperature using reverse-phase Merck C-18 column (4.6 × 250 mm, 5 m).The mobile phase was a mixture of methanol, water, and acetic acid (50 : 48 : 2) with isocratic elution.The elution flow rate was 1.0 mL/min, duration of analysis was 20.0 min, and UV detector was tuned to 254 nm.For the sample preparation, 5.0 mg of extract and for standard 0.05 mg were dissolved separately in 5 mL of mobile phase.

Statistical Analysis.
The data was analyzed using one-way analysis of variance (ANOVA) for repeated measurements.Duncan's multiple range tests were used to determine differences at each point which were considered significant at  ≤ 0.05.

Results and Discussion
The bark powder of the selected medicinal plants was extracted in two different solvent systems, methanol (100%) and acetone-water (7 : 3), by using cold maceration and at reflux temperature.Total phenolic contents of the extracts were measured by F.C method [35].The results revealed that the methanolic extract of four species (A.nilotica, A. lebbeck, C. fistula, and M. indica) contained the highest amount of phenolic contents at reflux temperature.For the remaining extracts, methanol extraction at room temperature was found a useful method.No encouraging results were obtained in the acetone-water extracts.The methanolic extracts of the selected medicinal plants showed significant variation in the total phenolic contents ranging from 528 to 715 mg of gallic acid equivalent (GAE/g of crude extract).The bark extract of A. nilotica contained the highest amount of phenolic compounds (715 mg), while the extract of P. granatum, M. elengi, and M. indica showed 712, 706, and 700 mg of GAE/g of crude extract, respectively.91.6% of the selected plant extracts contained more than 600 mg of GAE/g, while 4.2% showed a range of 528-597 mg of GAE/g of crude extract (Table 1).DPPH • assay was carried out using in vitro model to assess the ability of the bark extracts to scavenge the free radical.This method was found rapid and reliable which can be applied to a large number of plant extracts [10,11].A total of 21 medicinal plant species from 11 families were collected, extracted, and tested.Their radical scavenging activity determined as % scavenging of DPPH  1).The correlation coefficients ( 2 ) of antioxidant activity with total phenolic contents of methanolic extract were found 0.8874 (room temperature) and 0.8327 (reflux temperature) and acetone-water 0.8433 (room temperature) and 0.7580 (reflux temperature).
The phenolic compounds in the methanolic extracts of E. jambolana, A. nilotica, and C. zeylanicum were identified and confirmed by high performance liquid chromatography with standard samples.The results of these plant extracts along standards are shown in Figures 2-5.Gallic acid was identified in the extract of C. zeylanicum and A. nilotica appearing at RT = 2.93 min; tannic acid at RT = 2.77 min (E.suberosa, A. catechu, P. longifolia, E. jambolana, Z. jujuba, F. infectoria, and A. nilotica); quercetin at RT = 13.7 min (C.zeylanicum); rutin   Plant polyphenols probably constitute the largest group of plant-secondary metabolites, which have an important application as defense antioxidants.They include phenols, phenolic acids, flavonoids, tannins, and lignans.They possess an aromatic ring bearing one or more hydroxyl constituents including their functional derivatives [38,39].Antioxidant activity of phenolic compounds is correlated to their chemical structure.Flavonoids have the basic skeleton of diphenyl propane with different oxidations of central pyran ring.In plants, flavonoids are found as O-glycosides with sugar or through C-glycoside, but some are present as aglycones.In general O-glycosides at C-3 of ring "C" have lower radical scavenging power than those having a free -OH at C-3 because it is thought to be related to the reduction of the free radical [40].Bark of the selected medicinal plants is associated with the cure of many ailments (Table 2).Quantitative data on the phenolic compounds in the bark extracts of the selected plants was not found in the literature but the main constituents are suggested to be phenolic acids such as gallic acid, ellagic acid, rosmerinic acid, tannic acid, fistulic acid, cinnamic acid, and ferulic acid in P. granatum, M. indica, F. racemosa, E. jambolana, C. fistula, and P. roxburghii [41][42][43] (Table 2).Myricetin glycoside, 4-O-acetyl myricetin, eujambin, and eujambolin have been identified in E. jambolana [44,45].Myricitine was isolated from M. elengi and cinnamic acid derivative from Z. jujuba [46].Epicatechin glycosides were reported from C. fistula [47], epicatechin derivatives, Psidinin A, B, and C, from P. guajava, xanthone and xanthene derivative from M. indica [48], and quercetin and kaemferol derivative from E. rostrata.Ellagic acid glycosides were identified in the bark extract of P. grantum [49]; gallic acid, protocatechuic acid, pyrocatechol, (+)-catechin, (−) Annonaceae Polyalthia longifolia Sonn Febrifuge, skin diseases, diabetes, hypertension, and bleeding piles [58] -: no reported activity.epigallocatechin-7-gallate, and (−) epigallocatechin-5, 7digallate were identified in the extract of A. nilotica [24].Statistical results of the correlation studies (average  2 = 0.8874) between antiradical scavenging activity and total phenolic contents strongly suggested that the phenolic compounds were the dominant antioxidant in the methanolic extract of the bark of selected plants (Table 2).Our results are in agreement with the reported data [6,50].The analysis of phenolic compounds and their correlation with radical scavenging activity in "E.rostrata, M. champaca, A. modesta, P. roxburghii, P. longifolia, E. suberosa, M. elengi, and F. infectoria" have been reported for the first time.Figures 3-5 display the selected HPLC profile of the medicinal plants.However, main phenolic compounds in 13 medicinal plants could not be identified in this study (Table 3).Therefore, further chemical identification will be required to reveal the possible structure activity relationship.

Table 2 :
Medicinal uses of stem bark of the selected medicinal plants.

Table 3 :
Compounds identified by HPLC in selected medicinal plants.