Analysis of Phytochemicals, Antibacterial and Antioxidant Activities of certain Medicinal Plants of

International Journal of Bio-Technology and Research (IJBTR) ISSN (P):2249-6858; ISSN (E):2249-796X Vol. 10, Issue 1, Jun 2020, 49-62 Š TJPRC Pvt. Ltd.

ANALYSIS OF PHYTOCHEMICALS, ANTIBACTERIAL AND ANTIOXIDANT ACTIVITIES OF CERTAIN MEDICINAL PLANTS OF GARHWAL AGAINST HUMAN PATHOGENS Dr. JYOTI MEHTA* & IFFET AARA Department of Microbiology, Uttaranchal College of Science and Technology, Uttarakhand, India ABSTRACT Background and purpose:Medicinal plants are a source of invaluable bioactive constituents, which form the backbone of traditional medicines. The use of plant extracts and phytochemical compounds, both with known antimicrobial potential, can be of great significance in therapeutic purposes. The present study was designed to assess the phytochemical composition, antibacterial and antioxidant activities of medicinal plant extracts. Material and Methods: Five medicinal herbs (Ficus religiosa, Ficus benghalensis, Cannabis sativa, Hibiscus rosasinensis, and Terminalia chebula) were collected from Kulhan area of Dehradun (Uttarakhand) and their ethnomedicinal data were gathered from traditional healers and elders of the study area. Plant extracts were made by using solvents methanol, ethanol and chloroform in an orbital shaker. Extraction yield and phytochemical analysis was performed using standard

Gram-negative bacteria (E. coli and Shigella spp) and3 Gram-positive bacteria (S. epidermidis, B. cereus and S. aureus) by well diffusion technique. In most of the cases, inhibition of bacterial growth was proportional to extract concentrations. The free radical scavenging activity of the extracts was screened by 1, 1-diphenyl-2-picrylhydrazyl (DPPH) assay. Results:Lowest amount of extraction yield was obtained in methanol extract of H. rosasinensis (7.18%) and maximum yield was found in F. religiosa methanol extract (37.36%). All plants exhibited antimicrobial properties

Original Article

phytochemical procedures. Antibacterial activities of plant extractswere evaluated at 100, 150 and 200 mg/ml against 2

against one or more of the tested pathogens but T.chebula extracts were found most active against test pathogens and were further tested for antioxidant property. The antioxidant capacity showed maximum activity in the methanolic extract of T.chebulaat concentrations 100, 200, 300, 400 and 500 Âľg/ml and the percent inhibition of writhing response by the extract was 47.04%, 57.61%, 65.28%, 78.13%, and 92.43% respectively. T. chebula fruit could be potential sources of new antimicrobial and antioxidant agents.

CONCLUSIONS These findings confirmed that the methanol extract of T.chebula fruit is rich in bioactive phytochemical compounds and might be a promising source of antimicrobial compounds as well as antioxidant activity.T. chebula may act as a possible antioxidant compound for biological systems susceptible to free radical-mediated reactions.

KEYWORDS: Antibacterial Agents, DPPH, Free Radical Scavenger & Plant Extracts

Received: Oct 22, 2019; Accepted: Nov 12, 2019; Published: Jan 31, 2020; Paper Id.: IJBTRJUN20205

1. INTRODUCTION Garhwal Himalayas are a rich in a variety of medicinal plants. Biodiversity is blessed with a perennial source of medicinal plants, which contributed in thedevelopment of drug discovery[1] for treating human and animal disease that depends mainly on natural products obtained from plants, animals, minerals and microorganisms. According to the previous estimations, out of 20% plant species studied scientifically worldwide, only about 6% are screened

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for its pharmaceutical potential[2,3]. Screening of various active phytochemical constituents and antioxidant potential, including heavy metal is a major part of pharmaceutical drug discovery. Although NorthEast India is full of medicinal plants, yet very few studies have been carried out to explore its phytochemical composition and antioxidant properties[4-6].

Indeed, plants are a rich source of valuable secondary metabolites, including quinones, tannins, terpenoids, alkaloids, flavonoids and polyphenols that are used by plants as defense mechanisms against microorganisms, insects and herbivores.

Nowadays, indirect roles of oxygen-containing free radicals in biological systems as causative agents in various chronic disorders are no longer hidden[7]. Antioxidant plays a role in free radical scavenging activity in biological systems and reduce the widespreadrisk of various chronic diseases and their prolong side effects[8]. There are many synthetic antioxidants, but they have harmful effects, hence, there is a need for more potent and less toxic antioxidants[9,10]. Plants have been found to possess polyphenolic compounds, such as flavonoids thatcontain antioxidant property[11]. Some, such as terpenoids, give plants their odors property; quinones and tannins control plant pigmentation. These bioactive ingredients are said to be responsible for the antimicrobial potential of plant extracts in vitro[12,13]. Medicinal plantslike Ficus religiosa, Ficus benghalensis, Cannabis sativa, Hibiscus rosasinensis, and Terminalia chebula are easily available and have useful extraction from different parts like leaves, seeds, roots etc. C. sativa (Cannabaceae) is an angiosperm[14] and known by the name Marijuana in America and bhang, ganja and charas in India. The plant and its preparations have been used for its sedative, narcotic, antispasmodic, analgesic, antimicrobial and many other properties including its use for photophobia, asthma and piles[15, 16]. H. rosasinensis (Malvaceae) is commonly known as China rose or shoe flower. It is the national flower ofMalaysia. Traditionally, the flowers can be used to treatasthma[17,18] and they have been found to possess chemical ingredients such as cyanidin, quercetin, hentriacontane, calcium oxalate, thiamine, riboflavin, niacin and ascorbic acids.

There are many studies which confirmthe presence of such compounds with antimicrobial activity in various plant parts[19,20]. F. religiosa Linn (Moraceae) commonly known as ‘Peepal tree’, considered as a sacred tree to both Hindus as well as Buddhists. It has got mythological, religious and medicinal importance in Indian culture since ancient times[21-23].

F. religiosa is used as an ayurvedic medicine whichbelongs to a class of drugs called rasayana. Rasayana arerejuvenators, antioxidants and relieve body stress[24,25], ailments of the central nervous system, gastrointestinal tract, endocrine system, reproductive system, respiratory system and infectious disorders[26]. Another plant, F. bengalensis L. (Moraceae) is commonly known as Banyan tree. The plant has been extensively used in Ayurvedic and Unani medicines to treat health problems. Its aerial roots are antiemetic and also used topically to pimples. Latex from aerial plant parts mixed with honey which is used orally to control hyperglycemia in Pakistan[27]. T. chebula, commonly known as ‘King of Medicine’ in Tibet and is always listed at the top of thelist in Ayurvedic Materia Medica due to its extra ordinarypower of healing[28]. T. chebula contains tannins, chebulic acid, glycosides, sugar, triterpenoids, steroids and good quantity of phosphoricacid. It is commonly used to controldiseases like constipation, diarrhea, ulcer, gastroenteritis, asthma, cough, dyspepsia, urinary discharge, tumours, skin disease, anorexia[29]. It is also reported to possess antibacterial, antifungal, antiviral, carcinogenic, antioxidant, hypolipidemic, hepatoprotective, cardio protective and anti-diabeticpotential[30]. Due to the easy availability and pharmacological properties of Ficus religiosa, Ficus benghalensis, Cannabis sativa, Hibiscus rosasinensis, and Terminalia chebula, they were chosen to explore major biologically active phytoconstituents and antioxidant activity.

Moreover, antibacterial efficacy was tested with respect to inhibition over the growth of human pathogens viz., Gramnegative bacteria (E. coli and Shigella spp) and Gram-positive bacteria (S. epidermidis, B. cereus and S. aureus) under in vitro conditions.

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2. MATERIALS AND METHODS 2.1 Collection of Medicinal Plant Sources Roots, flower, fruit and leaves of selected medicinal plants given in table 1 were shade dried for 3-5 days in an oven at 2530℃ for few hours and finely powdered in a mixer grinder. The powder form was stored in an airtight container for further use.

S. NO. 1 2 3 4 5

Table 1: Medicinal Plants used for the Study Local name Botanical Name Part Used Peepal Ficus religiosa Leaf Bargad Ficus benghalensis Root Bhaang Cannabis sativa Leaf Gudahal Hibiscus rosasinensis Flower Harad Terminalia chebula Fruit

Family Moraceae Moraceae Cannabinaceae Lythraceae Combretaceae

2.2 Bacterial Strains S. epidermidis, B. cereus, E. coli, S. aureus, and Shigella spp. were used as test pathogens. All pathogens were obtained from the microbiology laboratory of UCST College, Dehradun.

2.3 Preparation of Organic Solvent Extract Shade-dried coarse powderwas thoroughly mixed with organic solvents, including ethanol, 70% methanol, and chloro form in the ratio of 1:10 in a conical flask. The mixture was placed at room temperature on an orbital shaker with 190-200 rpm and filters the supernatant after 48hrs through a sterilized Whatman No. 1 filter paper. The plant residue was re-extracted for 24 hrs with the addition of solvents to get final concentrated slurry. The combined filtrate thus obtained was concentrated by complete evaporation of solvent at 40℃ on a water bath to yield the crude extract. The dried extract, thus obtained was stored at 4℃ for further use[31]. The dry weight of extracts obtained by the solvent evaporation was used to determine the concentration in mg/ml[32]. A stock solution of crude extracts for each type of organic solvent was prepared by mixing the appropriate amount of crude extracts with 5% Dimethyl sulfoxide (DMSO) solvent to obtain a final concentration.

2.4 Determination of the Extraction Yield The extraction yield is a measure of the solvent efficiency to extract specific phytocompounds from the plant material. The extraction yield was calculated according to the method of Zhang et al. (2007)[33]. Briefly, the dried extracts were weighed, and then the extraction yield was calculated according to the following given formula Extraction yield (%) =

Weight of the dried extract ×100 Weight of the plant sample

2.5 Phytochemical Qualitative Analysis of Selected Plants Methanolic, chloroform and ethanolic extracts were investigated for the presence of the phytochemical by using the following standard methods given by different authors[1, 34-38].

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2.6 Detection of Glycosides Keller-Kiliani Test: 4 ml of glacial acetic acid was added to 1 drop of 2% FeCl3 andplant extract, followed by the addition of concentrated sulphuric acid (H2SO4). A brown ring formed between the layers showed the entity of cardiac steroidal glycosides.

2.7 Detection of Alkaloids Mayer’s Test: Filtrates of the extract were mixed with Mayer’s reagent (Potassium Mercuric iodide). Formation of a yellow cream precipitate gives positive result for alkaloids.

2.6 Detection of Tannins Gelatin Test: 1% gelatin solution containing sodium chloride was mixed with the extract. Formation of white precipitate confirms the presence of tannin in the extract.

2.7 Detection of Flavonoids Alkaline Reagent Test: Extracts were treated with a few drops of sodium hydroxide (NaOH) solution. Formation of intense yellow colour, which becomes colorless on the addition of dilute acid, gives positive result for flavonoids.

2.8 Detection of Proteins and Amino Acids •

Xanthoproteic Test: Extracts were treated with a few drops of concentrated Nitric acid (HNO3) solution. Yellow colour formation shows the presence of proteins.

•

Ninhydrin Test: To each extract, 0.25% ninhydrin reagent was added and boiled for few minutes. Formation of blue colourrevealed the presence of amino acid in the extract.

2.9 Detection of Phenols Ferric Chloride Test: Extracts were treated with a few drops of ferric chloride solution. Appearance of bluish black colorconfirms the presence of phenols.

2.10 Detection of Carbohydrates Fehling’s Test: Filtrates were hydrolysed with dil. HCl, neutralized with alkali (NaOH) and heated with each solution of Fehling A and B in equal amount. Formation of a red precipitate shows the presence of reducing sugars.

2.11 Detection of Saponin Foam Test: Small amount of each extract was shaken with 1-2 ml of water. If foam produced persists for 10min it indicates the presence of saponins.

2.12 Detection of Terpenoids Salkowski Test: The extract wasadded to 2ml of chloroform and 3 ml of concentrated H2SO4 carefully added to form a layer. Appearance of reddish brown colouration at the interface show positive results for the presence of terpenoids.

2.13 Detection of Steroids Salkowski Test: 2 ml of plant extract was mixed to 2ml of chloroform and 2 ml of concentrated H2SO4 and shaken well.

Chloroform layer appeared red and acid layer greenish yellow fluorescent gives positive results for steroids.

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2.14 Agar-well Diffusion Method for Evaluation of the Antimicrobial Activity A sterile cotton swab was immersed into the properly diluted inocula after adjusting the turbidity to McFarland standard.

The entire Mueller- Hinton agar plate surface was inoculated during swabbing for even growth and kept at room temperature for 15 min. The antibacterial efficacy of each plant extracts was investigated using the agar well diffusion method[39]. The concentration of plant extracts used was 100, 150 and 200mg/ml-1. Four wells (6 mm diameter) were punched at 24mm apart in the agar plates using sterile aluminum borer. The wells were filled with 100µ of different concentrations of plant extract. 10% Dimethyl sulfoxide (DMSO) and ciprofloxacin (10µg) was used as the negative control and positive control respectively. After incubation the plates were observed for the antibacterial potential and diameter of the zone of inhibition for each extract was measured.

2.15 Antioxidant Assay Diphenyl 1-1- Picrylhydrazyl Free Radical-Scavenging Capacity Measurement of the 2,2 – diphenyl- 1- picrylhydrazyl (DPPH) (sigma- Aldrich, Mexico) radical scavenging capacity was carried out according to Karamac et al (2005)[40] with little modification. Briefly, 20mg DPPH in 100ml methanol (Meyer Mexico) was mixed and 1ml of this mixture was added to prepare different concentrations (100ppm, 200ppm, 300ppm, 400ppm and 500ppm) of T. chebula selected extract (Ethanol and Methanol). After 45 min of incubation at room temperature, the absorbance was measured at 517nm with UV-visible (UV/VIS) spectrophotometer (Genesys 10 UV, thomo scientific). The percentage of free radical- scavenging capacity was calculated by the following equation.

Radical scavenging capacity (%) = (Acontrol-Asample) /Acontrol×100 Where Asample is the absorbance of DPPH mixed with T. chebula extracts and Acontrol is the absorbance of DPPH in which sample has been replaced with methanol. Ascorbic acid was used as positive control.

3. RESULTS AND DISCUSSIONS Plants synthesize hundreds of chemical compounds involved in defense against insects, fungi, diseases and herbivorous mammals. In the present study, 3 extracts (methanol, ethanol and chloroform) of the five medicinal plants namely F. religiosa, F. benghalensis, C. sativa, H. rosasinensis, and T. chebula were used. The dry extraction yield (%w/w) of the plants is shown in the table 2. In most of the cases, the amount of phytochemical extracted with methanol was higher than ethanol and chloroform. The extractive yield in methanol extracts varied from 7.18% to 37.36%, while in ethanol extracts the range was from 7.93% to 27.86%. In case of chloroform, the range varied from 8.3% to 12.27%.

S. No 1 2 3 4 5

Table 2: Extraction Yield (%) of used Medicinal Plants Extraction Yield (% w/w) Type of Extract Botanical Name Ethanol Methanol Ficus religiosa 7.93 37.36 Ficus benghalensis 11.32 7.28 Cannabis sativa 11.44 11.76 Hibiscus rosasinensis 10.34 7.18 Terminalia chebula 27.86 37.26

Chloroform 12.27 9.6 9.62 8.3 12.07

In recent years, the research on investigating phytochemical compounds having antimicrobial activities has been in demand due to their therapeutic uses inmany chronic and infectious diseases. Results obtained for qualitative analysis of phytochemical in three different solvents of collected medicinal plants are shown in Table 3. Present investigation showed www.tjprc.org

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the presence of carbohydrates and triterpenes whereas alkaloids, saponins, tannin, and amino acids are absent in all the extracts of C. sativa. Flavonoids and steroids are present in only chloroform extract and protein, triterpenes, phenols and C. glycosides are present in only methanol extract of C. sativa. Terpenes are commonly found in hop, is recognized as a potent anti- inflammatory, analgesic and anxiolytic component[41]. Some triterpenes are key contributors to the pharmacological properties of numerous medicinal herbs[42-44]. Our result are in contrary to the study of Monika et al., 2014[45] showed the presence of constituents like saponins, flavonoids, phenols, tannins and quinones in methanol extract of C. sativa. In case of ethanol extract, presence of saponins, flavonoids, phenols, tannins and quinones were reported. The presence of high quantity of phytochemicals including secondary metabolites such as phenolics and flavonoids, might contribute to the pharmacological potential possessed by certain plants[46]. Flavonoids are important secondary metabolites that exhibit medicinal properties such as antioxidant, antiinflammatory, anticancer, antibacterial and antiviral potential[47].

In present study, F. religiosa showed the presence of carbohydrates and proteins in all the extracts whereas the chloroform extract revealed positive results for only flavonoids, carbohydrates, steroids and proteins. Methanol extract showed the presence of carbohydrate, tannin, steroids, triterpenes, proteins and glycosides whereas saponin, carbohydrates, phenols, triterpenes, proteins, and glycosides were present in ethanol extract of F. religiosa. In contrary to this, Prakash et al., 2017[48] reported alkaloids, saponin, carbohydrates, phenol, flavonoids, proteins, tannin and terpenoids in methanol extract and showed zone inhibition of 12 mm against E.coli.Screening of F. benghalensis revealed the presence of saponins, carbohydrate, phenols, and steroids in both ethanol and methanol extracts whereas protein and C. glycosides were in only ethanol and methanol extract respectively. In chloroform extract, only flavonoids and triterpenes were present. Similarly, Ogunlowo et al., 2013[49] confirmed the presence of alkaloids, glycosides, terpenoids, saponins, phenol, flavonoids and tannin in root extract of F. benghalensis. Similar analysis done by Uma et al. (2009)[50] reported the presence of carbohydrates, flavonoids, amino acids, steroids, saponins and tannins in methanol extracts of F. religiosa and F. benghalensis. Kumari et al., 2015[51] revealed tannin, flavonoids, alkaloids, terpenoids, saponin, C. glycosides, and phenols in ethanol extracts. Phytochemical investigation of H.rosasinensis revealed that carbohydrates, proteins, glycosides and amino acids were present in ethanol and methanol extract, and additionally saponin in ethanol extract and phenols in methanol extract. Flavonoids and triterpenes were only phytoconstituent present in chloroform extract. In H. rosasinensis the presence of triterpenes was seen in all the three extracts while as the presence of proteins, glycosides and amino acids was seen in both ethanol and methanol extract. Flavonoids and tripenes were seen only in chloroform extract and the saponin was only seen in ethanol extract. Similarly tannin, phlobatannins, cardiac glycosides, flavonoids, terpenoids, saponins were reported in flowers of H. rosasinensis by various authors[52-55]. Flavonoids and tannins are phenolic compounds that are a major group compounds acting as primary antioxidants. Terpenoids and saponins showed pain relieving properties as well as nervous system activities. The results are in line with Tiwari et al (2015)[56] and Udo et al (2016)[57] who reported that bioactive chemical compounds from H. rosasinensis extracts including alkaloids, tannins, saponins, flavonoids, glycosides, anthraquinones and phenols. Presence of saponin confirmed the use of H. rosasinensis as an ingredient of shampoo. The observed pharmacological properties possibly have been responsible due to the presence of flavonoids, terpenoids, saponins, and tannins. The results obtained here are in good correlation with previous reportof Gauthaman et al., 2006[55]. Present study of T. Chebula are same tothe study of Baliah and Astalakshmi, 2014[58]. Our results also agreed with the study of Harborne[38] who confirm the presence of glycosides, alkaloids, flavonoids, phenolic compounds saponin, steroids, quinone and tannin. These confirm the phytochemical constituents that are widely considered to have medicinal and pharmacological effects. The presence of phytochemicalsdiffered in different types of solvent used

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in the extracts. The presence of polyphenols and flavonoids, widely known for their antioxidant activity those confirmed multiple biological effects including anti-inflammatory and anti-cancerous properties of these plants. The presence of glycosides attributes to the wide range of biological activities like anti oxidant, antipyretic, and anti inflammation.

Table 3: Phytochemical Analysis of used Medical Plants S. NO.

Test

1

Alkaloids (Mayer’s test)

2

Saponins (Foam test)

3

Flavonoids (Alkaline reagent test)

4

Carbohydrate (Fehling test)

5

Tannin (Gelatin test)

6

Phenols (Ferric chloride)

7

Steroids/Triterpenes (Salkowski’s test)

8

Proteins (Xanthoproteic test)

9

C. glycosides (Kellar killani test)

10

Amino acids (Ninhydrin test)

Type of Extracts Ethanol Methanol Chloroform Ethanol Methanol Chloroform Ethanol Methanol Chloroform Ethanol Methanol Chloroform Ethanol Methanol Chloroform Ethanol Methanol Chloroform Ethanol Methanol Chloroform Ethanol Methanol Chloroform Ethanol Methanol Chloroform Ethanol Methanol Chloroform

C. sativa + + + + + -/+ -/+ +/+ + + -

F. religiosa + + + + + + + -/+ +/+ +/+ + + + + -

F. benghalensis + + + + + + + +/+/-/+ + + -

H. rosasinensis + + + + + -/+ -/+ -/+ + + + + + + -

T. chebula + + + + + + + + + + + +/+ +/+/+ + + + -

(+) = Positive; (-) = Negative The results of antimicrobial investigation of ethanol, methanol and chloroform extracts of five plantsare presented in Table 4. The antimicrobial effects of all the plant extracts against 5 bacterial strains examined were assessed by the presence or absence of inhibition zones. Antibacterial activity of C. sativa showed that methanol extract showed best antibacterial activity as compared to ethanol and chloroform extract. In contrary to the present study Tandon et al., 2017[59] reported that ethanolic extract was found effective against S. aureus, E. coli and P. aeruginosa. The activity of C. sativa is due the presence of alkaloids or phenyl moiety of cannabinoids which act as a good antimicrobial agent[60]. Naveed et al., 2014[61] reported 24.1 mm against S.aureus, and 22.2 mm against E.coli. Methanol extract showed 21 mm zone of inhibition for both S. aureus and E.coli and ethanol extract measured 17 mm and 13 mm inhibition zone for S. aureus and E. coli, respectively. Hemaiswarya et al., 2009

[62]

reported that chloroform extracts of F. religiosa showed a strong

inhibitory activity against S. typhi and S. typhimurium. Finding of Jiwala et al., 2008[63]suggested the rich phytochemicalcontent of F. religiosa have good antioxidant activity. Aqueous and ethanolic extracts of F. religiosa leaves showed antibacterial effect against S. paratyphi, S. dysenteriae, S. typhimurium, S. aureus, and E. coli etc[64-66]. Our study was different from the previous study of Uma et al. (2009)[67] who reported antibacterial activity of F. religiosa against enterotoxigenic E. coli(ETEC) in methanol extracts as good activity in comparison with chloroform and aqueous extracts.

Similar to our study, Aswar et al., 2008[68] confirmed thatmethanolic extract was comparably more effective to inhibit the growth of microbes thanaqueous and chloroform extracts. www.tjprc.org

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The present report showed that ethanol and chloroform extracts of F. benghalensis exhibits good activity as compared to methanol extract. In one of the previous studies, similarly ethanolic extract showed antibacterial activity against S. aureus and E. coli[69,51]. Our results are in agreement with Subba et al., 2016[53] and Bhaskar et al. (2011)[52] who stated that ethanolic fraction of H. rosasinensis flower had saponins but not with Afify and Hassan (2016)[70]. The leaf extracts are high enough in essential nutrients required for optimal physiological performance and the maintenance of good health. Among all the above medicinal plants good antibacterial activity was shown only in ethanol and methanol extract of T. chebula as compared to ciprofloxacin (positive control), it showed zone of inhibition of 22mm and 21mm respectively, against B. cereus while against E. coli, 19 mm and 24 mm was measured.

Table 4: Evaluation of Antibacterial Activity of Extracts Against Test Pathogens

(-) = No inhibition zone; Eth = Ethanol; Met = Methanol; Chl = Chloroform. Results were expressed in terms of meanÂąstandard deviation (SD)

Figure 1: Antibacterial Activity of Methanolic Extract of T. chebula at different Concentration.

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Methanolic extract of T. chebula showed the best antibacterial effect against E. coli, S. epidermidis, Shigella spp., and S. aureus at different concentration (Figure 1).

1 The presence of phytochemicals such as phenolics, alkaloids, flavonoids, steroids and saponins in T.chebula provide a reason why the plant possessed biological activities that are of pharmacological importance.. The presence of high phenolic, flavonoid and vitamin C contents could be attributed to its pharmacological activity associated with free radical scavenging activity. The model of scavenging the stable DPPH radicals has been widely accepted as a tool to evaluate the free radical-scavenging radical scavenging activities of sample[71]. Out of five medicinal plants only methanolic nolic and ethanolic extracts of T. chebula was as tested for antioxidant assay. Methanol extract of T. chebula exhibited good DPPH radical scavenging (92.43%) (92.43 followed llowed by ethanol extract (84.24%) (84.24 was represented in figure 2. The high content of phytochemicals appeared to have been responsible for the high antioxidant ant capacity of the plant. Similar to the present investigation, a large number of reports also showed an increasing trend of reducing power activity with increase the gradual increment of plant concentration[72]. Data from previous studies as well as our results reveal the great potential of used plants for therapeutic treatment, in spite of the fact thatthey have not been completely investigated. Therefore, more studies need to be conducted conducted tosearch for new compounds responsible for their antibacterial and antioxidant activities. Once isolated, isolated and beforebeing used in new medical treatments, they should have their toxicity tested in vivo.

Figure 2: Concentration-Response Concentration of T. chebula Fruit Extract xtract in DPPH assay. assay REFERENCES 1.

Sofowara A. Medicinal plants and traditional medicine in Africa. Spectrum Books Ltd. Ibadan: Nigeria.1993; Nigeria 289–300.

2.

Lahlou M. The success of natural products in drug discovery. Pharmacology Pharmacol & Pharmacy.2013 2013; 4:17-31.

3.

Cragg GM, Newman DJ. Natural products: A continuing source of novel drug leads. Biochemical et biophysical acta. 2013; 1830:3670-3695.

4.

Fabricant DS, Farnsworth NR. The value of plants used in traditional medicine for drug discovery. Environmental Environ Health Perspectives. 2001; 109:69-75.

5.

Vimal K, Gogoi BJ, Meghvansi MK, Singh L, Srivastava RB, Deka DC. Determining the antioxidant activity of certain medicinal plants of Sonitpur, (Assam), India using DPPH assay. Journal J of Phytology. 2009;; 1:49-56.

1:49

6.

Swargiary

A,

Roy

B. In

vitro

anthelmintic

efficacy

of Alpinianigraand and

its

bioactive

compound,

astragalin

against Fasciolopsisbuski. International national Journal of Pharmacy and Pharmaceutical Sciences. 2015; 7:30-35.

www.tjprc.org

editor@tjprc.org

58

Dr. Jyoti Mehta & Iffet Aara 7.

Jones DP. Radical-free biology of oxidative stress. American Journal of Physiology-Cell Physiology. 2008; 295:C849-C868

8.

Sawa T, Nakao M, Akaike T, Ono K, Maeda H. Alkylperoxyl radical scavenging activity of various flavonoids and other phenolic compounds: Implications for the antitumor promoter effect of vegetables. Journal of Agricultural and Food Chemistry. 1999; 47:397-492.

9.

Branien AL. Toxicology and biochemistry of butylated hydroxyl anisole and butylated hydroxyl toluene. Journal of the American Oil Chemists Society. 1975; 52:59-63.

10. Ito N, Fukushima S, Haqlwara A, Shibata M, Ogiso T. Carcinogenicity of butylated hydroxyanisole in F344 rats. Journal of National Cancer Institute. 1983; 70:343-347.

11. Alho H, Leinonen J. Total antioxidant activity measured by chemiluminescence method. Methods Enzymology.1999; 299:3-15.

12. Abo KA, Ogunleye VO, Ashidi JS. Antimicrobial poteintial of Spondias mombin, Croton zambesicus and Zygotritonia crocea.

Journal of Pharmaceutical Research. 1991; 5(13):494-497.

13. Nweze EL, Okafor JL, Njoku O. Antimicrobial Activities of Methanolic extracts of Trume guineesis (Scchumn and Thorn) and Morinda lucinda used in Nigerian Herbal Medicinal practice. Journal ofBiological Research & Biotechnology. 2004; 2(1):3446.

14. Borhade SS. Chemical Composition and Characterization of Hemp Cannabis sativa Seed oil and essential fatty acids by HPLC Method. Archives of Applied Science Research. 2013; 5:5-8.

15. George ET, William CE. A textbook of Pharmacology. 1989; 743-748.

16. Dastur JF. Medicinal plants of India and Pakistan, First Indian edition, D.B. Taraporevala Sons and Company Private Ltd, Bombay.1962; 66-67.

17. Zhao J, Zhou L, Wang J, Shan T, Zhong L, Liu X, et al. Endophytic fungi for producing bioactive compounds originally from their host plants. Current research, technology and education topics in applied microbiology and microbial biotechnology.

2010; 1:567-576 18. Sikarwar MS, Patil MB. Antihyperlipidemic effect of ethanolic extract of Hibiscus rosa sinensis flowers in hyperlipidemic rats.

RGUHS Journal of Pharmaceutical Sciences. 2011; 1:117-122.

19. Olukoya DK, Idika N, Odugbemi T. Antibacterial activity of some medicinal plant from Nigeria. Journal of Ethnopharmacology. 1993; 39:69-72.

20. Sorachai K, Boonsom L, Saisunee L, Aphiwat T, Stephen G, Mary J. Antimalarial, anticancer, antimicrobial activities and chemical constituents of essential oil from the aerial parts of Cyperus kyllingiaEndl. Recordsof Natural Products.2011; 4:324-327.

21. Ghani A. Medicinal plants of Bangladesh with chemical constituents and uses. Asiatic. Society of Bangladesh, Dhaka.1998; 236.

22. Singh D, Goel RK. Anticonvulsant effect of Ficus religiosa: role of serotonergic pathways. Journal of Ethanopharmacology.

2009; 123:330-334.

23. Prasad PV, Subhaktha PK, Narayana A, Rao MM. Medico historical study of “asvattha” (sacred fig tree). Bull Indian Inst Hist Med, Hyderabad.2006; 36:1-20.

24. Govindarajan R, Pushpangadan P, Vijayakumar M. Antioxidant approach to disease management and the role of ′Rasayana′ herbs of Ayurveda. Journal of Ethnopharmacology.2005; 99:165.

Impact Factor (JCC): 5.9084

NAAS Rating: 3.80

Analysis of Phytochemicals, Antibacterial and Antioxidant Activities of certain Medicinal Plants of Garhwal against Human Pathogens

59

25. Arora D, Dubey SD, Ojha JK. Role of rasayana in preventing the complications of diabetes mellitus.Journal of Diabetes Association.1999; 39:47-50.

26. Singh DD, Singh B, Goel RK. Traditional uses, phytochemistry and pharmacology of Ficus religiosa: A review. Journal of Ethnopharmacology. 2011; 134(3):565-583 27. Ahmad M, Qureshi R, Arshad M, Khan MA, Zafar M. Traditional herbal remedies used for the treatment of diabetes from district Attock (Pakistan). Pakistan Journal of Botany. 2009; 41:2777-2782 28. Bag A, Bhattacharyya SK, Bharati P, Pal NK, Chattopadhyay RR. Evaluation of antibacterial properties of Chebulic myrobalan (fruit of Terminalia chebula Retz.) extracts against methicillin resistant Staphylococcus aureus and trimethoprimsulphamethoxazole resistant uropathogenic Escherichia coli. African Journal of Pediatric Surgery. 2009; 3: 2529.

29. Nadkarni AK. Terminalia chebula. In: Dr. K.M. Nadkarni’s. Indian Materia Madica, 3rd ed, Bombay: Popular Prakashan Pvt Ltd.1976; 1:1202‑1211.

30. Chattopadhyay RR, Bhattacharyya SK, Medda C, Chanda S, Datta S, Pal NK. Antibacterial activity of black myrobalan (Fruit of Terminalia chebula Retz.) against uropathogen Escherichia coli. Pharmacognosy Reviews. 2007;11:212-215.

31. Aneja KR, Joshi R, Sharma C. Potency of Barleria prionitis L. bark extracts against oral diseases causing strains of bacteria and fungi of clinical origin. New York Science Journal. 2010;3:512.

32. Habsah M, Amran M, Mackeen MM, Lajis NH, Kikuzaki H, Nakatani N, et al. Screening of Zingiberaceae extracts for antimicrobial and antioxidant activities. Journal of Enthnopharmacology. 2000;72:403-410.

33. Zhang S, Bi H, Liu C. Extraction of bio-active components from Rhodiola sachalinensis under ultrahigh hydrostatic pressure.

Separation and Purification Technology. 2007;57:275-280.

34. Chanda SV, Parekh J, Karathia N. Evaluation of antibacterial activity and phytochemical analysis of Bauhinia variegate L. bark. African Journal of Biomedical Research. 2006;9:53-56.

35. Parekh J, Chanda SV. In vitro antimicrobial activity and phytochemical analysis of some Indian medicinal plants. Turkish Journal of Biology. 2007;31:53-58.

36. Kumar GS, Jayaveera KN, Kumar CKA, Sanjay UP, Swamy BMV. Antimicrobial effects of Indian medicinal plants against acne-inducing bacteria. Tropical Journal of Pharmaceutical Research. 2007;6:717-723.

37. Onwukaeme DN, Ikuegbvweha TB, Asonye CC. Evaluation of phytochemical constituents, antibacterial activities, and effect of exudates of Pycanthus angolensis Weld Warb (Myristicaceae) on corneal ulcers in rabbits. Tropical Journal of Pharmaceutical Research. 2007;6:725-730.

38. Harborne JB. Phytochemical methods: a guide to modern techniques of plant analysis. Chapman and Hall Ltd., London: UK. 1973;49-188.

39. National Committee for Clinical Laboratory Standards (NCCLS). Methods for antimicrobial susceptibility testing of anaerobic bacteria. Approved Standard. 1993;M11-A3 40. Karamac M, Buci nski A, Pegg RB, Amarowicz R. Antioxidant and antiradical activity of Ferulates. Czech Journal of Food Sciences. 2005;23:64-68 41. Cleemput MV, Cattoor K, De Bosscher K, Haegeman G, De Keukeleire D, Heyerick A. Hop (Humulus lupulus)-derived bitter acids as multipotent bioactive compounds. Journal of Natural Products. 2009; 72(6):1220-1230.

www.tjprc.org

editor@tjprc.org

60

Dr. Jyoti Mehta & Iffet Aara 42. Kirby J, Romanini DW, Paradise EM, Keasling JD. Engineering triterpene production in Saccharomyces cerevisiae-betaamyrin synthase from Artemisia annua. FEBS Journal. 2008;275(8):1852-1829.

43. Yadav VR, Prasad S, Sung B, Kannappan R, Aggarwal BB. Targeting inflammatory pathways by triterpenoids for prevention and treatment of cancer. Toxins (Basel). 2010;2(10):2428-2466.

44. Sawai S, Saito K. Triterpenoid biosynthesis and engineering in plants. Frontiers in Plant Science. 2011;2:25.

45. Monika, Kour N, Kaur M.Antimicrobial analysis of leaves of cannabis sativa. Journal of Sciences. 2014; 4(2):123-127 46. Ohkawa H, Onishi N, Yagi K. Assay of lipid peroxidation in animal tissue by thiobarbituric acid reaction. Analytical Biochemistry. 1979; 95:351-358.

47. Patra B, Schluttenhofer C, Wu Y, Pattanaik S, Yuan L. Transcriptional regulation of secondary metabolite biosynthesis in plants. Biochimica et Biophysica Acta. 2013;1829:1236-1247.

48. Prakash V, Gandotra S, Kumar P, Singh N. Phytochemical Screening and Antimicrobial Activity of Ficus religiosa. Journal of Pharmaceutical Sciences& Research. 2017;9(2):100-101 49. Ogunlowo O, Arimah B, Adebayo M. Phytochemical analysis and comparison of in-vitro antimicrobial activities of the leaf, stem bark and root bark of Ficus benghalensis. IOSR Journal of Pharmacy. 2013; 3:33-38.

50. Uma B, Prabhakar K, Rajendran S. In vitro antimicrobial activity and phytochemical analysis of Ficus religiosa and Ficus benghalensis against diarrhoeal enterotoxigenic E. coli. Ethnobotanical leaflets. 2009; 4-7 51. Kumari OS, Rao DNB, Reddy DVB. Phyto-chemical analysis and anti-microbial activity of hibiscus rosa-sinensis. World journal of pharmacy and pharmaceutical sciences. 2015; 4(5):766-771.

52. Bhaskar A, Nithya V, Vidhya VG. Phytochemical screening and in vitro antioxidant activities of the ethanolic extract of Hibiscus rosasinensis L. Annals of Biological Research. 2011; 2(5):653-661.

53. Malik, M. A., Bhat, S. A., Fatima, B. I. L. Q. U. E. E. S., Ahmad, S. B., Sidiqui, S., & Shrivastava, P. U. R. N. I. M. A. (2016).

Rheum emodi as a valuable medicinal plant. International Journal of General Medicine and Pharmacy, 5(4), 35-44.

54. Subba B, Sharma A, Budhathoki A. Assessment of phytochemical content, antioxidant and antibacterial activities of three medicinal plants of Nepal. Journal of Medicinal Plants Research. 2016; 10(45):829-837 55. Patel R, Patel A, Desai A, Nagee A. Study of secondary metabolites and antioxidant properties of leaves, stem and root among Hibiscus rosa-sinensis cultivars. Asian Journal of Experimental Biological Sciences. 2012; 3:719-725.

56. Gauthaman KK, Saleem MTS, Thanislas PT, Prabhul VV, Krishnamoorthy KK, Devaraj NS, et al. Cardioprotective effect of the Hibiscus rosa-sinensis flowers in an oxidative stress model of myocardial ischemic reperfusion injury in rat. BMC Complementary and Alternative Medicine. 2006; 6:32.

57. Tiwari U, Yadav P, Nigam D. Study on phytochemical screening and antibacterial potential of methanolic flower and leaf extracts of Hibiscus rosa sinensis. International Journal of Innovative and Applied Research. 2015; 3(6):9-14.

58. Udo IJ, Ben MG, Etuk CU, Tiomthy AI. Phytochemical, proximate and antibacterial properties of Hibiscus rosa-sinensis L. leaf. Journal of Medicinal Plants Studies. 2016; 4(5):193-195.

59. Baliah NT, Astalakshmi A. Phytochemical analysis and antibacterial activity of extracts from Terminalia chebula Retz.

International Journal of Current Microbiology and Applied Sciences.2014; 3(3):992-999 60. Tandon C, Mathur P, Sen M, Khatoon S. Cannabis sativa L. (Bhang): A possible source of new antibacterial medicament.

International journal of pharmaceutical sciences review and research.2017; 44:221-227

Impact Factor (JCC): 5.9084

NAAS Rating: 3.80

Analysis of Phytochemicals, Antibacterial and Antioxidant Activities of certain Medicinal Plants of Garhwal against Human Pathogens

61

61. Appendino G, Gibbons S, Giana A, Pagani A, Grassi G, Stavri M, et al.Antibacterial cannabinoids from Cannabis sativa: a structure-activity study. Journal of Natural Products. 2008; 71(8):1427-1430.

62. Naveed M, Khan TA, Ali I, Hassan A, Ali H, Din ZU, et al. In vitro antibacterial activity of Cannabis sativa leaf extracts to some selective pathogenic bacterial strains. International Journal of Biosciences. 2014; 4(4):65-70 63. Stavroula, M. A. M. O. U. C. H. A., & Rahul, J. I. T. I. N. (2017). Mediterranean climate affects the biosynthesis of secondary metabolites in common medicinal plants. Int. J. Bot, 6, 17-28.

64. Hemaiswarya S, Poonkothai M, Raja R, Anbazhagan C, Hemaiswarya S, Poonkothai M, et al. Comparative study on the antimicrobial activities of three Indian medicinal plants.Egyptian Journal of Biology. 2009; 11:52-57 65. Jiwala SA, Bagul MS, Parabia M, Rajani M. Evaluation of free radical scavenging activity of an ayurvedic formulation.Indian Journal of Pharmaceutical Sciences.2008;70:31-35.

66. Valsaraj R, Pushpangadan P, Smitt UW, Adersen A, Nyman U. Antimicrobial screening of selected medicinal plants from India.Journal ofEthnopharmacology. 1997; 58:75-83.

67. Mousa O, Vuorela P, Kiviranta J, Wahab SA, Hiltohen R, Vuorela H. Bioactivity of certain Egyptian Ficus species. Journal ofEthnopharmacology. 1994; 41:71-76.

68. Begum, S. S., Raju, K., & Gangadhar, B. Medicinal and phytochemical properties of selected native plants of andhra pradesh: a review.

69. Farrukh A, Iqbal A. Broadspectrum antibacterial and antifungal properties of certain traditionally used Indian medicinal plant. WorldJournal of Microbiology and Biotechnology. 2003; 19:653-657.

70. Uma B, Prabhakar K and Rajendran S. In vitro antimicrobial activity and phytochemical analysis of Ficus religiosa L. and Ficus bengalensis L. against diarrheal enterotoxigenic E. coli. Ethno botanical Leaflets.2009; 13:472-474.

71. Aswar M, Aswar U, Wagh A, Watkar A, Vyas M, Gujar KN. Antimicrobial activity of ficus benghalensis.Pharmacology online2008;2:715-725 72. Murti K, Kumar U.Antimicrobial activity of Ficus benghalensis and Ficus racemosa roots L. Pharmacology online.2011; 3:218-223 73. Afify AMMR, Hassan HMM. Free radical scavenging activity of three different flowers-Hibiscus rosasinensis, Quisqualis indica and Senna surattensis. Asian Pacific Journal of Tropical Biomedicine. 2016; 6(9):771-777.

74. Naik GH, Priyadarsani KI, Naik DB, Gangabhagirathi R, Mohan H. Studies on the aqueous extract of Terminalia chebula as a potent antioxidant and a probable radioprotector. Phytomedicine. 2004; 11:530-538 75. Rajani, N., Sireesha, G., & Hephsibah, S. Efficacy of garlic and cinnamon on type 2 diabetes.

76. Kibiti CM, Afolayan AJ. Preliminary phytochemical screening and biological activities of Bulbineabyssinicaseed in the folk medicine in the Eastern Cape Province, South Africa. Journal of Evidence-Based Complementary and Alternative Medicine.

2015; 2:1-12.

77. Ravi, N., & Lean, H. T. B. Phytochemicals screening, antioxidant activity and frying quality as affected by aqueous extract of malaysianseraikayu (eugenia polyantha).

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Dr. Jyoti Mehta & Iffet Aara

AUTHOR'S PROFILE

Name: Dr. Jyoti Mehta Educational Qualification: Ph.D. in Microbiology Publications: Dr Jyoti Mehta, Mr. Mohd Akhlak. Isolation and identification of bacterial contamination from commonly used household surfaces. Journal of Emerging Technologies and Innovative Research. 2018; 5(12): 697-705.

ISSN: 2349-5162 Dr Jyoti Mehta. Synergistic effect of some spices in combination with antibiotics against drug resistant pathogenic microorganisms. International Journal of Pharmacy and Biological Sciences. 2019; 9(1): 824-830. ISSN: 2230-7605 Dr Jyoti Mehta and Shah Shahista. Studies on the screening of phytochemical, antioxidant and antibacterial activities of certain medicinal plants of Kashmir. International Journal of Biological Research and Development (IJBRD) journal. 2019; 9(2): 1-14. ISSN: 2250-0022

Iffet Aara, Department of Microbiology, Uttaranchal College of Science and Technology, Uttarakhand, India Educational Qualification: M.Sc in Microbiology from Uttaranchal college of science and Technology

Impact Factor (JCC): 5.9084

NAAS Rating: 3.80