PHARMACOGNOSTIC AND ANTIMICROBIAL EVALUATION OF HORDEUM VULGARE LEAVES

PHARMACOGNOSTIC AND ANTIMICROBIAL EVALUATION OF HORDEUM VULGARE LEAVES

Kiran, Vandana Garg * and Saloni Kakkar

Department of Pharmaceutical Sciences M. D. University, Rohtak, Haryana, India.

ABSTRACT: Hordeum vulgare (Barley) grass is the best functional food for providing nutrition and eliminating toxins from human cells; however, its functional ingredients have also played an important role in health benefits. Flavonoids, saponarin, lutonarin, K, Ca, Se, dietary fiber, polysaccharide, alkaloid, glycoside, tannin, metallothionein and polyphenols are all found in barley grass. Microscopical studies showed that stomata, epidermal cells and trichomes were conducted as part of a pharmacognostic investigation of the grass of H. vulgare. The qualitative assays were done to predict the presence of phenols, tannin and flavonoid content. The Minimum inhibitory concentration (MIC) method was used to screen H. vulgare leaf extract for antimicrobial potential against S. typhi, S. aureus, B. subtilis and E. coli and chloramphenicol was used as a standard. Amongst all the extracts, the chloroform extract of H. vulgare was found to be more effective as its MIC value is almost equal to the standard antibiotic chloramphenicol.

Keywords: Hordeum vulgare, Macroscopy, Microscopy, Taxonomy, Phytochemistry, Antimicrobial activity

INTRODUCTION: Hordeum vulgare Linn, also known as barley, is a member of the Poaceae family. Barley (Hordeum vulgare L.) is the world's fourth most important cereal crop, with the highest dietary fiber content. For nutritional peak, barley grass has young green leaves and stems of vegetative growth stage from a seedling at 10 days after sprouting (barley sprout) to elongation stage (barley green). Barley grass grows to be 0.5-1.2 m long and 0.5-0.1 cm thick. Plants contain a variety of constituents, including p-coumaroylagmatine, hordenin, pyrrolidine, luteolin glycoside, flavones glycosidesorientoside and orientin, 2- -Dglucopyranasyloxy, pangamic acid, protein, carbohydrates, calcium, phosphorus-glucan, phenolic acids, flavonoids, lignans, tocols,

phytosterols and folate. Beta-sitosterol, stigmasterol, campesterol and brssicastrol are major chemical constituents of barley ¹. Consuming whole grain barley and its hydroalcoholic extract regularly lowers the risk of chronic diseases. In India, barley has traditionally been used to treat atonic dyspepsia and to treat malaria ². It is used to treat respiratory and urinary tract infections in Argentina ³.

It is used to treat diabetes in China ⁴. In Pakistan, barley treats rheumatoid arthritis and joint pain ⁵. Barley is used to treating dysentery, diarrhoea and gastrointestinal disorders in the United States ⁶. Barley has antioxidant activity and has the potential to treat circulatory disorders, cancer, obesity, diabetes, arthritis, cholesterol and inflammation ⁷.

In the present work, we studied the various pharmacognostic parameter and antimicrobial activity of H. vulgare. Traditionally, H. vulgare is used in atonic dyspepsia, respiratory and urinary tract infections, diabetes and as anti-malarial.

MATERIAL AND METHODS: 

Plants Collection and Authentication: Hordeum vulgare (Barley) leaves are cultivated in Rohtak, Haryana, and the same was sent to NIScPR for authentication. Dr. Sunita Garg, Head, Raw Material Herbarium and Museum Division (RHMD) CSIR-National Institute of Science Communication and Policy Research (NIScPR), same was identified as Hordeum vulgare L. under reference number NIScPR/RHMD/Consult/2021/3951-52-2, 27/12/2021, Pusa Road, New Delhi.

Solvents and Reagents: Petroleum ether (60-80), Chloroform, ethanol, ascorbic acid, gallic acid, Folin Ciocalteu’sreagent, phosphomolybdenum, etc were procured from CDH. All chemicals used in the study were of all analytical grade.

Pharmacognostic Evaluation: Pharmacognostic evaluation provides useful information about crude drugs' macroscopical, microscopical and physical properties.

Macroscopic Evaluation:  Macroscopic evaluation includes a complete evaluation of plants based on their external features. The plant material was evaluated in shape, size, color, odour, texture, and fracture characteristics ⁸.

Microscopic Evaluation: Microscopic evaluation includes the study of the transverse section and powder microscopy of H. vulgare leaves. After cutting the transverse section (T.S) of the plant was mounted with safranin to study the various characters. In powder microscopy, the plant powder was treated with glycerine, and chloral hydrate: HCL was used to mount various characteristics of plants like fibres, starch grain, calcium oxalate crystals, sclereid and many more ⁹.

Physicochemical Evaluation: Standardization parameter includes foaming test, ash values (total ash, water-soluble ash, acid-insoluble ash and sulphated ash) loss on drying,  extractive value, swelling index crude fibre content were determined by standard procedures ^{8, 10-11}.

Preparation of Extracts: Extraction of leaves of H. vulgare was done by two methods maceration and soxhlation. 500g of coarse powder of leaves of H. vulgare was weighed and treated with four solvents on the basis of polarity (low to high) to get petroleum ether, Chloroform, 80% ethanol, and water extract successively by triple maceration and soxhlation. Total 8 extracts were prepared and concentrated by rotary evaporator. These extracts were stored in an airtight container for further use.

Phytochemical Analysis: All extracts were chemically tested for the presence and absence of different phytoconstituents like alkaloids, flavonoids, tannins, glycosides, saponins, proteins and carbohydrates.

Quantitative Analysis: All extracts were tested for the different classes of phytochemicals present in them like total phenolic content, flavonoid content, and tannin content.

Total Phenolic Content: Total phenols present in all extracts viz., Petroleum ether, Chloroform, 80%, hydroalcoholic, and aqueous extract of leaves of H. vulgare were determined using Folin Ciocalteu’s reagent method without any modification ¹².

Total Tannin Content: Total tannin present in all extracts viz., Petroleum ether, Chloroform, 80%, hydroalcoholic and aqueous extract from leaves of H. vulgare was determined using Folin Ciocalteu’s reagent method without any modification ¹³.

Total Flavonoid Content: Total flavonoid present in all extracts viz., Petroleum ether, Chloroform, 80%, hydroalcoholic and aqueous extract from leaves of H. vulgare was determined using the Aluminium Chloride without any modification ¹⁴.

Antimicrobial Activity: Antimicrobial activity of all extracts viz., Petroleum ether, Chloroform, 80%, hydroalcoholic and aqueous extract from leaves of H. vulgare were determined using 96-well microtitration plate method, two-fold dilutions of the antimicrobial agent in a liquid growth medium are placed in smaller proportions in 96-well microtitration plate. After that, a microbial inoculum is added to each well. After thoroughly mixing, the inoculation in a 96-well microtitration plate is incubated (usually without agitation) under appropriate conditions depending on the test microorganism. After 24 to 27 hours of incubation, plates were stained with the Alamar blue dye (resazurin), which was a useful growth indicator ¹⁵.

RESULTS:

Pharmacognostic Evaluation: Pharmacognostic evaluation was screened to identify commercial varieties, substitutes, adulterants and the quality of drugs. It is a simple and dependable tool for obtaining information about raw drugs' biochemical and physical properties. Methods covered include macroscopic and microscopic analysis, organoleptic the character of plant powder and extracts. Phytoconstituents of plant powder were identified qualitatively and quantitatively with various chemical reagents.

Macroscopic Evaluation of H. vulgare: Macroscopic evaluation was done by studying the external features of the fruit of H. vulgare. H. vulgare in green colour, 0.7-1.8 m long and 0.1-0.6 cm thick and smooth texture.

FIG. 1: REPRESENTATIVE PHOTOGRAPH OF H. VULGARE

Microscopic Evaluation of H. vulgare: Different photographs showed the presence of stomata’s, epidermal cells and trichomes (T). Fig. 2 represents the T. S. of H. vulgare leave A) Epidermal Cell (EC) (S), B) Open Guard cell (OGC), C) Stomata, D) Upper epidermal cell (UEC). Fig. 3 represents the powder microscopic characteristics of  H. vulgare leave A) Small barb-like trichome (SBT), B) Bundle of fiber (BF), C) Sieve tube (ST), D) Trichome (T), E) Epidermal Cell (EC), F) Sieve cell (SC).

FIG. 2: REPRESENTATIVE PHOTOMICROGRAPH (X400) OF T.S OF  H. VULGARE LEAVE A) EPIDERMAL CELL (EC) (S), B) OPEN GUARD CELL (OGC), C) STOMATA, D)  UPPER EPIDERMAL CELL (UEC)

FIG. 3: REPRESENTATIVE PHOTOMICROGRAPH (X400) OF THE POWDER MICROSCOPY OF  H. VULGARE LEAVE A)  SMALL BARB LIKE TRICHOME (SBT), B) BUNDLE OF FIBER (BF), C) SIEVE TUBE (ST), D) TRICHOME (T), E) EPIDERMAL CELL (EC), F) SIEVE CELL (SC)

Standardization of Plant Material: Plants standardization entails confirming their identity and determining their quality, purity, and chemical compounds to establish a minimum standard of quality.

The total ash value denotes the purity of the material. In contrast, acid-insoluble, water-soluble, and sulphated ash values denote the presence of siliceous, earthy and inorganic material in the plant.

The crude fiber content of a material indicates the amount of indigestible cellulose, pentosans, and lignin present. Extractive values indicate the class of phytoconstituents present in plants. The swelling index measures the amount of mucilage in plant material.

The results showed that barley does not contain mucilage. Foaming index is used to identify the presence of saponin in the plant. The results show the presence of saponinin the barley. Table 1 summarized the results of various standardization parameters of the plant.

TABLE 1: STANDARDIZATION PARAMETER OF H. VULGARE

Phytochemical Analysis: Petroleum ether, Chloroform, 80% ethanolic and water extracts of leaves of H. vulgare were dissolved in respective solvents and tested for the presence of various chemical groups of compounds. Table 2 summarises the results of the phytochemical screening.

TABLE 2:  PHYTOCHEMICAL SCREENING OF PETROLEUM ETHER, CHLOROFORM, 80% ETHANOLIC AND, WATER EXTRACTS OF LEAVES OF H. VULGARE

Total Phenolic Content: Phenols can reduce free radicals; the more phenols present in the plant, more its antioxidant potential will be.

All the extracts (8) obtained from H. vulgare were prepared by different extraction techniques (maceration and soxhlation) using different solvents like petroleum ether, Chloroform, 80% ethanolic, and water extracts of leaves of  H. vulgare were screened for total phenolic content.

Amongst all extracts, hydroalcoholic extract (Soxhlation) of leaves of H. vulgare contains maximum phenolic content is shown in Table 3.

TABLE 3: TOTAL PHENOLIC CONTENT OF ALL 8 EXTRACTS OF H. VULGARE

Total Tannin Content: All the extracts (8) were prepared by different extraction techniques (maceration and soxhlation) by using different solvents like petroleum ether, Chloroform, 80% ethanolic, and water extracts of leaves of  H. vulgare were screened for total tannin content. Among all extracts, the aqueous extract (Soxhlation) of leaves of H. vulgare contains maximum tannin content, are shown in Table 4.

TABLE 4: TOTAL TANNIN CONTENT OF ALL 8 EXTRACTS OF H. VULGARE

Total Flavonoid Content: All the extracts (8) were prepared by different extraction techniques (maceration and soxhlation) by using different solvents like petroleum ether, Chloroform, 80% ethanolic and water extracts of leaves of  H. vulgare were screened for total flavonoid content. Amongall extracts, the aqueous extract (Soxhlation) of leaves of H. vulgare contains maximum flavonoid content, is shown in Table 5.

TABLE 5: TOTAL FLAVONOID CONTENT OF ALL 8 EXTRACTS OF H. VULGARE

Antimicrobial Activity: All 8 extracts were screened for antimicrobial activity against S. typhi, S. aureus B. subtilis and E. coli using the MIC method, and chloramphenicol was taken as standard. Among all extracts, H. vulgare chloroform extract shows maximum antimicrobial potential. Results of antimicrobial activity are reported in Table 6 and Fig. 4.

TABLE 6: ANTIMICROBIAL POTENTIAL OF ALL 8 EXTRACTS OF H. VULGARE

FIG. 4: REPRESENTATIVE PHOTOMICROGRAPH OF ANTIMICROBIAL ACTIVITY OF DIFFERENT EXTRACTS OF H. VULGARE AGAINST SELECTED STRAINS (S. TYPHI, S. AUREUS, B. SUBTILIS, E. COLI). Barley Maceration Petroleum ether extract (B1), Barley Soxhlation Petroleum ether extract (B2), Barley Maceration Chloroform extract (B3), Barley Soxhlation Chloroform extract (B4), Barley Maceration Hydroalcoholic extract (B5), Barley Soxhlation Hydroalcoholic extract (B6), Barley Maceration Aqueous extract (B7), Barley Soxhlation Aqueous extract (B8).

DISCUSSION: Histological evaluation showed that diacytic stomata is present in theH. vulgare. Standization of leaves of H. vulgarereveled that plant material is free from erarthy material, silicons, carbonates and inorganic material which assuer the quality and purity of the leaves. Leaves cantain 18% of dietry fiber which is good for consumption as its normalized the bowl movement. Literature reveled the presence of  phenolic compounds such as benzoic acid, cinnamic acid, ferulic acid vannlic acid, flavonoids (cyanidin, delphindin, pelargonidin, catchin and myricitin, phytosterols like beta – sitosterol, stigmasterol, canpesterol, brssicastrol are present in leaves of H. vulgare. Phytochemicals studies showed the presence of amino acid, carbohydrates, steroides, flavaonids, phenols, alkaolids and tannin in the leaves of H. vulgare. Quantity of the phenols, flavonids and tannin content in the leaves of H. vulgare were also estminated; Barley Soxhlation Hydroalcoholic (BSHA) extract contain the maximum phenols i.e., 87.2 GAE/g, Barley Soxhlation Hydroalcoholic (BSHA) extract contain the maximum flavanoids i.e., 82.08 GAE/g, Barley Soxhlation Aqueous(BSA) extract contain the maximum tannin i.e., 58.75tannin acid/g. Results of quantitative analysis suggested that H. vulgare leaves may have good antioxidant potential as its contain a good amount of phenols and flavonoids. Therefore, H. vulgare leaves were evaluated for antimicrobial activity. Chloroform extract of H. vulgare showed maximum antimicrobial activity amongst all 8 extracts.

CONCLUSION: The data gathered from the current study on taxonomy, macroscopy and microscopy, preliminary phytochemical screening, and phytochemical evaluation of the plant will aid in the correct identification of H. vulgare. It is also concluded that H. vulgare leaves have antimicrobial potential against S. typhi, S. aureus, B. subtilis and E. coli. With MIC values of 62.5, 31.25, and 62.5, chloroform extract of Barley leaves showed the highest antimicrobial activity against S. aureus, B. subtili and E. coli. The current study suggested that H. vulgare chloroform extract could be a good candidate for a natural antimicrobial agent because it has activity against gram-positive and gram-negative microbes.

ACKNOWLEDGEMENTS: The authors are thankful to the M.D. University Rohtak, for providing necessary Lab facilities.

CONFLICTS OF INTEREST: All the authors have no conflicts of interest to declare.

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    How to cite this article:

    Kiran, Garg V and Kakkar S: Pharmacognostic and antimicrobial evaluation of Hordeum vulgare leaves. Int J Pharm Sci & Res 2023; 14(7): 3414-20. doi: 10.13040/IJPSR.0975-8232.14(7).3414-20.

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