Al Ameen Arts Science and Commerce College, Bangalore -27

Minor Research Project

Project title:

“Phytochemical and pharmacological analysis of Ajwain and Kalonji

and antimicrobial analysis against nasal pathogens”

Ajwain Kalonji

Submitted to UGC

By

Ayesha Siddiqua Department of Microbiology

&

Dr.Sabiha Sultana Associate professor, Department of Botany

Declaration:

We, Ayesha Siddiqua and Dr.Sabiha Sultana have carried out the Minor

research project titled “Phytochemical and pharmacological analysis of

Ajwain and Kalonji and antimicrobial analysis against nasal

pathogens” and this has not been submitted elsewhere.

Date: Dec. 2016

Place: Bangalore

Ayesha Siddiqua

&

Dr.Sabiha Sultana

ACKNOWLEDGMENT

With a deep sense of gratitude we acknowledge the invaluable financial support provided by UGC to carry out this Minor research project and completing it on time. We wish to thank the Management of Al- Ameen Educational Society for their encouragement. we thanks Dr. B.M. Zakir, Principal, and Vice principle Dr. Rahmath Ataaz Al-Ameen College for their help and motivation.

Ayesha Siddiqua

& Dr.Sabiha Sultana

Al Ameen College, Bangalore.

DEDICATED

TO

OUR PARENTS

AND

AL-AMEEN COLLEGE, BANGALORE-560027

RESEARCH PAPERS PRESENTED IN NATIONAL AND INTERNATIONAL CONFERENCE IN THE YEAR.

Presented paper at UGC sponsored National Conference on

“Emerging Protein Technologies for Formulation Solutions” on

16th Oct 2014 at University college of Science, Tumkur University.

Analysis of Crude Protein in Trachyspermum ammi and their separation by TLC Method

Ayesha Siddiqua, Dr. Sabeeha Sultana, Omar Farooq*

*Faculty, Al-Ameen Arts, Science, Commerce College, Bangalore.

ABSTRACT

Plants provide us with nearly 30% of medicine and drugs that we use,

being nonnarcotic, having no side effects and easy availability at affordable

price makes these products sometime the only source of healthcare available to poor. In recent times there has been a market shift towards

herbal cures because of pronounced cumulative and irreversible ill effects of many modern drugs. Trachyspermum ammi, is commonly known as

Bishop’s weed, Carom seed (English names). According to Ayurveda, the

seeds are hot, pungent, stomachic, appetizer, aphrodisiac, carminative, laxative and diuretic, anti-inflammatory, anti-bacterial. Ajwain seed

analysis has revealed it to contain fibre (11.9%), carbohydrates (38.6%), tannins, glycosides, moisture (8.9%), protein (15.4%), fat (18.1%),

saponins, flavone and mineral matter (7.1%) containing calcium,

phosphorous, iron and nicotinic acid. The Ajwain fruits yields 2% to 4% brownish essential oil, with thymol as the major constituent (35% to 60%).

The present paper highlights on analysis and separation of crude protein

present in Trachyspermum ammi.

CONTENTS

Sl. No. Topic Page No.

01 Introduction 1

02 Aim and Objective of the Study 7

03 Review of Literature 8

04 Material and methodology 11

05 Result 15

06 Discussion 15

07 Reference 25

“Phytochemical and pharmacological analysis of Ajwain and Kalonji

and antimicrobial analysis against nasal pathogens”

Synopsis

Nigella sativa and Trachyspermum ammi were screened for phytochemical constituents. Tests for flavonoids, tannins, steroids were positive in both

spices except saponins. Hptlc and Tlc were performed where

chromatogram were obtained for both Chloroform, ethanolic extracts. Chloroform, ethanolic extracts of N. sativa and T. ammi were subjected to in

vitro antibacterial assay. Disc diffusion assay was adopted against Nasal bacteria. Among the four concentration 0.8gm/ml conc was found to have

antimicrobial effect on nasal microbes. These findings are still in infancy,

where in future these medicinal plants to be explored for their antimicrobial properties and used as alternative medicine.

Introduction Medicinal plants have been used as a possible source, for traditional treatments of many human diseases for thousands of years in many parts of the world. In rural areas of the developing countries, they continue to be used as the primary source of medicine. About 80% of the people in developing countries use herbal medicines for their health care. The natural products derived from medicinal plants have proven to be an abundant source of biologically active compounds, many of which have been the basis for the development of new lead chemicals for pharmaceuticals. With respect to diseases caused by microorganisms, the increasing resistance in many common pathogens to currently used therapeutic agents, such as antibiotics and antiviral agents, has led to renewed interest in the discovery of novel anti-infective compounds. As there are approximately 500 000 plant species occurring of which only 1% has been phytochemically investigated, there is great potential for discovering novel bioactive compounds from the plants. There have been numerous reports of the use of traditional plants and natural products for the treatment of oral diseases. Many plant-derived medicines used in traditional medicinal systems have been recorded in pharmacopeias as agents used to treat infections and a number of these have been recently investigated for their efficacy against oral microbial pathogens.

Numerous studies have shown that aromatic and medicinal plants are sources of diverse nutrient and non nutrient molecules, many of which display antioxidant and antimicrobial properties which can protect the human body against both cellular oxidation reactions and pathogens. Thus, it is important to characterize different types of medicinal plants for their antioxidant and antimicrobial potential. Aromatic and medicinal plants are known to produce certain bioactive molecules which react with other organisms in the environment, inhibiting bacterial or fungal growth (antimicrobial activity). The substances that can inhibit pathogens and have little toxicity to host cells are considered candidates for developing new antimicrobial drugs.

Spices and herbs have been used for thousands of centuries by many cultures to enhance the flavor and aroma of foods. Scientific experiments since the late 19th century have documented the antioxidant properties of some spices, herbs, and their components. Many studies reported the activities of spices and herbs on food borne pathogenic microorganisms. In

fact, plants produce a diverse range of bioactive molecules, making them a rich source of different types of medicines. Higher plants, as sources of medicinal compounds, have continued to play a dominant role in the maintenance of human health since ancient time Over 50% of all modern clinical drugs are of natural product origin and natural products play an

important role in drug development programs in the pharmaceutical industry.There has been a revival of interest in herbal medicines. This is due to increased awareness of the limited ability of synthetic pharmaceutical products to control major diseases and the need to discover new molecular structures as lead compounds from the plant.

Recently there has been a renewed interest in improving health and fitness through the use of more natural products. Herbs and spices are an important part of the human diet. They have been used for thousands of years to enhance the flavor, color and aroma of food. In addition to boosting flavor, herbs and spices are also known for their preservative and medicinal value which forms one of the oldest sciences.

A spice is a dried seeds; fruit, root or bark used in significant quantities as a food additive for flavor, color, or a preservative that kills the harmful microorganism or prevent their growth .From about 100 species of higher plants, there are 125 clinically useful drugs of known constitution have been isolated. About 5000 plant species have been studied in detail and it has been estimated that they can be used as a possible sources of new drugs. The bulk production of plant based drugs is one of the most important tasks for the pharmaceutical industry.

Nigella sativa L. – a Global Healer

Classification

Kingdom Plantae

Division Magnoliophyta

Class Magnoliopsida

Order Ranunculales

Family Ranunculaceae

Genus Nigella

Species Sativa

Binomial name Nigella sativa L

Nigella sativa L. is an annual herbaceous flowering plant and belongs to the family Ranunculaceae and native to southwest Asia. The seeds are small and black in color and possess aromatic odor and taste. It grows to 20–30 cm (7.9–12 in) tall, with finely divided, linear leave. The flowers are delicate, and usually colored pale blue and white, with 5–10 petals. The fruit is a large and inflated capsule composed of 3-7 united follicles, each containing numerous seeds. The seeds are tiny and hairy (1-2 mm long), black, 3 sided and look a bit like pieces of flint under a microscope. Nigella sativa L. is sometimes mistakenly confused with the fennel herb plant (Foeniculum vulgare). The flowers grow terminally on its branches while the leaves grow opposite each other in pairs, on either side of the stem. Its lower leaves are small and petiole and the upper leaves are long (6-10cm).

Origin

For thousands of years by various cultures and civilizations Nigella sativa L. (black cumin) has been used, around the world as a natural healing aid and as a good supplement in maintaining good health. Black cumin seeds were discovered in Tutankhamen's tomb, implying that it played an important role in ancient Egyptian practices.

Composition

The oil of Nigella sativa L. can be divided into a solid and a volatile phase. The seeds contain both fixed and essential oils, proteins, alkaloids and saponin. The fixed, or fatty, oil is rich in unsaturated fatty acids, mainly linoleic acid (50 – 60 %), oleic acid (20 – 25 %), eicosadienoic acid (3 %) and dihomolinoleic acid (10 %) which is characteristic for the genus. Saturated fatty acids (palmitic, stearic acid) amount to about 30 % or less. Commercial Nigella oil may also contain parts of the essential oil, mostly thymoquinone, by which it acquires an aromatic flavour, but its content of the essential oil depends largely on the manufacturing, filtering, filling, and storing processes. The oil should be preferably stored in light-tight containers. Oxygen also impairs the quality of the stored oil. This influence is attributable to free oxygen radicals, which exhaust the radical scavenging potential of the oil in storage and thus diminish its biological efficacy in the essential oil (avr.0.5 %, max. 1.5 %), thymoquinone was identified as the main component (up to 50 %) besides p-cymene (40 %), apinene (up to 15 %), dithymoquinone and thymohydroquinone. Other terpene derivatives were found only in trace amounts: Carvacrol, carvone, limonene, 4-terpineol, citronellol. Furthermore, the essential oil contains significant (10%) amounts of fatty acid ethyl esters. On storage, thymoquinone yields dithymoquinonene and higher oligocondensation products (nigellone).

Trachyspermum ammi (Linn.) Sprague

Classification

Kingdom Plantae

(unranked) Angiosperms

(unranked) Asterids

Order Apiales

Family Apiaceae

Genus Trachyspermum

Species Ammi

Binomial name Trachyspermum ammi L.

Ajwain (Trachyspermum ammi L.) belonging to family Apiaceae, is widely used for curing various diseases in both humans and animals. Its other names commonly used in the literature are ajwan, ajowan, and Bishop’s weed, carom, or Ethiopean cumin. The most utilizable part of ajwain is the small caraway like fruit, which is particularly popular in Indian savory recipes, savory pastries, and snacks and as spice. Trachyspermum ammi L. is a small, erect, annual, herbaceous plant with branched leafy stems, feather like leaves (2.5 cm long), and 4-12 ray flower headsbearing 6 -16 flowers. The fruits are minute, greyish-brown coloured and egg shaped.

Flavour and Aroma

Raw ajwain smells almost exactly like thyme because it also contains thymol, but is more aromatic and less subtle in taste, as well as slightly bitter and pungent. It tastes like thyme or caraway, only stronger. Even a small amount of raw ajwain will completely dominate the flavor of a dish. In India, ajwain is never used raw, but either dry-roasted or fried in ghee or oil. This developed a much more subtle and complex aroma somewhat similar to caraway but little bit brighter smell.

Main Constituent

Ajwain seed analysis has revealed it to contain moisture (8.9%), protein (15.4%), fat (18.1%), fibre (11.9%), carbohydrates (38.6%), tannins, glycosides, saponins, flavone and mineral matter (7.1%) containing calcium, phosphorous, iron and nicotinic acid. T. ammi L. seeds contain 2.5-5% essential oil and the principal constituents of essential oil are phenolsthymol (35-60%), carvacrol (11%). The remainder of the oil is called thymine which contains p-cymene (50 - 55%), beta-pinene (4 - 5%), limonene with gamma-and betaterpinenes (30 - 35%). The essential oil (2.5 to 5% in the dried fruits) is dominated by thymol (2-isopropyl-5-methylphenol, 35 to 60%) α-pinene, p-cymene, limonene and γ-terpinene have been found. Essential oil distilled from aerial parts (flowers, leaves) of ajwain grown in Algeria, however, isothymol (50%) was found to be the dominant constituent before p-cymene, thymol, limonene and γ-terpinene. Isothymol, is not well defined and might refer to both 2-isopropyl-4-methylphenol and 3-isopropyl-6-methylphenol (carvacrol). From South Indian ajwain fruits, almost pure thymol has been isolated (98%), but the leaf oil was found to be composed of monoterpenoids and sesquiterpenoids: 43%cadinene, 11% longifolene, 5% thymol, 3% camphor and others.

Nasal Passageway The nose is one of the few openings that bacteria have direct access to get inside the body. The nasal passage is important for filtering the air that we breathe in and it also stops tiny foreign particles or microorganisms from entering the body. Many people do not know that the nose and nasal passages are, or can be, the perfect environment for some bacteria, good and bad. The nasal passageway is close to other niches like the lungs, mouth, and throat. The nasal passage is influenced by the bacteria colonies of Staphylococcus aureus. Other pathogens include such as Haemophilus influenzae, S. pneumonia, Nisseria meningitides, Moraxella catarrhalis and S. aureus. Successful colonization depends not only on the ability of S. aureus to survive host factors but also on coexistence with other bacteria. An airborne bacterium also causes allergies and irritation. Sinuses: The diseased maxillary and ethmoid sinuses are most commonly associated with affecting the nasal passages by causing inflammation and congestion. One of the diseases caused by the communities of bacteria is sinusitis. The ability of bacteria to infect the sinuses must first be set up by conditions that create a favorable environment in the sinus cavities. The inflammation in the sinuses is chronic do begin with. The causes for such chronic sinusitis cases are sometimes unclear. The Bacteria that causes the sinusitis are Streptococcus pneumonia, H. influenzae, and Moraxella

catarrhalis. The symptoms of this disease are severe headache, pain or pressure in the areas of the face, cough, and fever. It could also be damaging to the mucous membrane if left untreated. Bipolaris specifera is a fungus that also causes sinus. This fungus is very fast growing and they grow at 25 degrees Celcius. In their growing process they change their shape and color too. Microbes present in the Nasal Passageway Even though we cannot see them with our naked eye, microscopic organisms are living inside our nasal passageways. Although most of these bacteria are non-pathogenic, some may induce illness if they successfully break through the body’s defense systems. Some examples of these non-pathogenic bacteria are Streptococcus, Neisseria, Haemophilus, and Micrococcus. Some of the pathogenic examples include Staphylococcus aureus, Corynebacterium diphtheriae, Streptococcus pneumoniae, and Haemophilus influenzae among others.

Streptococcus Haemophilus

Corynebacterium diphtheriae

Aim and Objective

Aim and Objective:

• To study two (Ajwain and Kalonji) important Indian spices for the evaluation of their medicinal potential.

• To extract the compounds in different solvents.

• To perform HPTLC and TLC and identify compounds of medicinal value from these spices

• Screening of these spices for the antimicrobial property.

Review of Literature

In the last two decades, antibiotic resistance is an emerging problem

worldwide (Walsh, 2000; Cohen, 2002). This has lead to the search for new, safe and effective antimicrobial agents from alternative natural resources

like plant products.

The typical Indian spices and herbs like cumin, black cumin, mustard, fenugreek, ajowain, curry-leaf, nutmeg and henna are usually used in curries, pickles, sauces etc. These spices are also known to have some ethno-medicinal or anti-microbial properties (Singh et al., 2002). Plants traditionally used for medicinal purpose in different parts of the world have been screened for possible antimicrobial action by several workers (Bonjar, 2004). Antibacterial activities of extracts of different plants against various microorganisms have been reported by many scientists (Chaudhury and Tariq, 2008; Gutierrez et al., 2008). Some spices were specifically tested for anti-microbial activities (Sagdic et al., 2003).

Nigella sativa Medicinal uses

Nigella sativa L. in Islam, it is regarded as one of the greatest forms of healing medicine available, in the Unani Tib-e-Nabvi system of medicine, Black cumin is regarded as a valuable remedy for the cure of diseases (Source: http://www.crescenlife.com/dietnutrition/Kalonji.htm). In the Middle East and Southeast Asian countries, the seeds have been traditionally used to treat ailments including asthma, bronchitis, rheumatism and related inflammatory diseases, to increase milk production in nursing mothers, to promote digestion and to fight parasitic infections. Its oil has been used to treat skin disease eczema and when the oil is little bit warm it is used to treat cold symptoms. Many researchers have recently also studied it's effectiveness towards cancer, and it's said to have many anti-cancer compounds. Its many uses have earned Black cumin seed the Arabic approbation 'Habbatul barakah', meaning the seed of blessing. It’s therapeutic used was initiated after the advent of Islam, since; Prophet Muhammad (pbuh) mentioned its therapeutic efficacy and potential of cure. Over 200 studies have been carried out since 1959, at international universities and articles published in various journals have shown remarkable results supporting its traditional uses. Recent research on the Nigella sativa L. as an anti-biotic, anti-tumor, anti-inflammatory,

anti-histaminic, anti-bacterial, anti-bronchial and immune boosting agent has shown great promise. The popularity of the plant was highly enhanced by ideological belief in the herbs as a cure for multiple diseases. Due to the sayings of the Holy Prophet Mohammed (Peace be upon him) that the plant is full of medicinal value it gained immense popularity. Consequently, Kalonji has been extensively studied particularly in the Islamic world which justifies its broad traditional therapeutic value.

Effects against Microorganisms: The plant extract and its constituent have been extensively studied for their antimicrobial effect against a wide range of bacterial, fungal and parasitic organisms. The methanolic extract of N. sativa L. seeds was found to exhibit antiplaque action by potently inhibiting Streptococcus mutans, thus also preventing dental caries (Namba et al., 1995). The alcoholic and ether extract showed in vitro antibacterial activity against Gram positive and Gram negative bacteria (Sokmen, 1999). In another study, it was found to exhibit antibacterial activity especially against Bacillus pumilus, B.subtilis, Streptococcus mutans, taphylococcus lutea, Staphylococcus aures and Pseudomonas aeruginosa (El-Kamali et al., 1998). The ethanolic extract was found to posses’ anticestodal effect in children and the essential oil showed in vitro antifungal against Aspergillus species and Carvularia lumanta (Agarwal et al., 1979) as well as against pathogenic yeast Candida albicans (Hanafy and Hatem, 1991). The aqueous extract of the seeds possess potent in-vivo antifungal activity against Candidiasis in mice (Khan et al., 2003).

Ajwain Medicinal uses

Traditionally, ajwain known as a digestive aid which is used to relief abdominal discomfort occur due to indigestion. In southern parts of India, dry ajwain seeds are powdered and soaked in milk, which is then filtered and fed to babies. Many assume it relieves colic in babies, and for children it also improves digestion and appetite

A study conducted using the essential oil suggests that it has some use in the treatment of intestinal dysbiosis. (Hawrelak et al., 2009) reported its benefit to inhibit the growth of undesired pathogens while not adversely affecting the beneficial flora. T. ammi L. has been shown to possess anti-aggregatory effects; anthelmintic (Lateef et al., 2006); antihyperlipidaemic (Javed et al., 2006); antifilarial (Mathew et al., 2008); insecticidal (Chaubey, 2008); kidney stone inhibitory (Kaur et al., 2009); molluscicidal (; Singh and Singh, 2000); mosquito repellent (Pandey et al., 2009); and nematicidal activities (Park et al., 2007).

Antimicrobial actions in vitro: The antimicrobial action of T. ammi L., in the protection of foodstuffs against microbial spoilage, conducting

laboratory assays of antimicrobial efficacy in vitro was studied. The active principles thought to be responsible for the antimicrobial activity of ajwain were reported to be carvacol and thymol. Antifungal action of volatile constituents of T. ammi seeds on ten fungi (Acrophialophora fusispora, Curvularia lunata, Fusarium chlamydosporum, F. poae, Myrothecium roridum, Papulaspora sp., Alternaria grisea, A. tenuissima, Drechslera tetramera and Rhizoctonia solani) were found to inhibit the growth of all test fungi by 72-90% (Singh et al., 1979). (Caccioni et al., 2000) reported the antibacterial property of phenolic compounds, such as thymol and carvacol, depending on the concentration used.

MATERIALS AND METHODOLOGY

MATERIALS AND METHODS Collection and of Medicinal Plant Seeds

The seeds of N. sativa (Kalonji) and T. ammi (Ajwain) were purchased. The dried plant seeds were grinded using pestle and mortar and packed in polythene bags and placed in a dried place for further extractions. Extract preparation

Soxhlet extraction assembly was used for this purpose. Each of 100 g dried and powdered seeds were mixed with 250 ml chloroform, ethanol and continuous extraction was done for 5 to 6 h for three consecutive days. After that extracts were filtered, aqueous extracts were dried by, the organic extracts were dried in a rotary evaporator at reduced pressure. Finally powdered extracts was weighed and transferred in clean and dried vial, then stored in refrigerator at 4°C until use. Phytochemical screening

Chemical tests were carried out to evaluate the presence of the phytochemicals Alkaloids, Flavonoids, Saponins, Tannins and Sterols in the selected spices; using standard procedures described by Sofowora (1993); Trease and Evans (1989).

Phytochemical examinations were carried out for all the extracts as

per the standard methods. Detection of alkaloids (Evans, 1997)

Solvent free extract (50 mg) was stirred with few milliliters of dilute hydrochloric acid and filtered. The filtrate was tested carefully with various alkaloidal reagents as follows: Mayer’s test (Evans, 1997)

To a few mL of filtrate, a drop or two of Mayer’s reagent was added by the sides of the test tube, a white creamy precipitate indicated the test as positive.

Mayer’s Reagent

Mercuric chloride (1.358 g) was dissolved in 60 mL of water and potassium chloride (5.0 g) was dissolved in 10 mL of water. The two solutions were mixed and made up to 100 mL with water. Wagner’s test (Wagner, 1993)

To a few ml of filtrate, few drop of Wagner’s reagent were added by the side of the test tube. A reddish-brown precipitate confirmed the test as positive. Wagner’s reagent

Iodine (1.27 g) and potassium iodide (2 g) were dissolved in 5mL of water and made up to 100mL with distilled water. Hager’s test (Wagner et al., 1996)

To a few mL of the filtrate, 1 or 2 mL of Hager’s reagent (saturated aqueous solution of picric acid) was added. A prominent yellow precipitate indicated the test as positive. Detection of saponins by foam test (Kokate, 1999)

The extract (50 mg) was diluted with distilled water and made up to 20 mL. The suspension was shaken in a graduated cylinder for 15 min. A two cm layer of foam indicated the presence of saponins. Detection of phytosterols (Finar, 1986) Libermann-Burchard’s test

The extract (50 mg) was dissolved in 2 mL of acetic anhydride. To this, one or two drops of concentrated H2SO4 were added slowly along the sides of test tube. An array of colour changes showed the presence of phytosterols.

Detection of flavanoids Alkaline reagent test

An aqueous solution of the extract was treated with 10% ammonium hydroxide solution. Yellow fluorescence indicated the presence of flavanoids. HPTLC and TLC Study

Chromatography is one of the fast emerging tools by which the quality control and fingerprint of herbs can be maintained. Using this technique, the identification of various chemical markers of the herbal drugs can be easily done and it also helps to identify the same herbs in combination. Application of TLC/HPTLC methodology in testing of phytoconstituents from individual herbal drugs and fingerprint characteristic of Ajwain and kalonji have been done. Popularity of TLC/HPTLC analytical method for analysis of herbal drugs due to economic, rapid, simultaneously screening of large number of herbal samples and less time consuming methods. Many aqueous extract or alcoholic extracts, hydro alcoholic extracts are used in manufacturing Ayurvedic and herbal formulations. If the phytochemical profile of the plant or its part is known an appropriate kind of extract can always be used by selection for a particular purpose. A TLC or HPTLC profile of the phytochemical can be employed for the similarity or dissimilarity or to find out the presence or absence of the certain phytochemicals TLC/HPTLC has excellent resolution and, therefore, permits simultaneous identification of a wide range of substances in a single run. Antimicrobial Assay Disc diffusion bioassay

Kirby-Bauer method was followed for disc diffusion assay. In vitro antimicrobial activity was screened by using Mueller Hinton Agar (MHA) obtained from Himedia (Mumbai). The MHA plates were prepared by pouring 15 ml of molten media into sterile petriplates. The plates were allowed to solidify for 5 min and nasal secretions from volunteers was

swabbed uniformly and the inoculum was allowed to dry for 5 min. sterile 6-mm Whatman no.1 filter paper discs were aseptically placed on plates. Spice decoctions or extracts of standard concentrations (0.8gm/ml and 0.4 g/ml) were aseptically poured on the discs along with sterile double distilled water. The different concentrations of extracts were loaded on discs. The loaded discs were placed on the surface of medium and the compound was allowed to diffuse for 5 min and the plates were kept for incubation at 37°C for 24 h. Negative control was prepared using respective solvent. At the end of incubation, inhibition zones formed around the disc were measured with transparent ruler in millimeter. The inhibition zone diameters were measured three times and means were represented to nearest mm. Figure 3 Discs with the different concentration of extract

Figure 4 Discs with the different concentration of extract and the nasal swabs

RESULTS AND DISCUSSION

1. Phytochemical Analysis

Table 1 Results of Phytochemical Analysis of Ajwain, Kalonji.

Variable tested Ajwain

Kalonji

Alkaloids + +

Tannins ++++ +++

Steroids +++ +++

Flavonoids ++++ ++

Saponins - -

Phytochemical constituents such as alkaloids, flavonoids, tannins,

phenols, saponins, and several other aromatic compounds are secondary metabolites of plants that serve a defense mechanism against prediction by many microorganisms, insects and other herbivores. The present study

carried out on the plant samples revealed the presence of medicinally active constituents. The phytochemical constituents of the selected plants investigated are summarized in Table-1. Analysis of plant extracts revealed the presence of, Alkaloids, flavonoids, phenols, saponins, steroids and tannins in the selected plants which could be responsible for the observed antimicrobial property. These bioactive compounds are known to act by different mechanism and exert antimicrobial action. Tannins bind to proline rich proteins and interfere with the protein synthesis. Flavonoids are hydroxylated phenolic substance known to be synthesized by plants in response to microbial infection and it should not be surprising that they have been found in vitro to be effective antimicrobial substances against microorganisms. Their activity is probably due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell walls. Antimicrobial property of saponin is due to its ability to cause leakage of proteins and certain enzymes from the cell. Steroids have been reported to have antibacterial properties, the correlation between membrane lipids and sensitivity for steroidal compound indicates the mechanism in which steroids specifically associate with membrane lipid and exerts its action by causing leakages from liposomes.

2. HPTLC & TLC

HPTLC study of Kalonji extracts

Development of chromatographic HPTLC fingerprint profile of ethanol and chloroform extract of Nigella sativa (kalonji). Both the extracts of Nigella sativa was subjected to HPTLC fingerprint profile to check the presence of different phytopharmaceuticals. Numbers of peaks, peak areas, Rf values were recorded. Different combination of solvent systems were tried to get good separation and sharp peaks. The adequate resolution was obtained in the solvent system toluene: ethyl acetate : formic acid (5:4:1) and it was found to be the best for separation of compounds present in both the extracts of Nigella sativa. HPTLC Fingerprinting was carried out by using CAMAG HPTLC system (Switzerland) with a Linomat 5 sample applicator. The whole investigation process was carried out in air-conditioned room maintained at 22ºC and 55% humidity. TLC was also done on pre-coated silica gel HPTLC aluminium plates 60F254 (20 cm×20 cm, 0.2mm thickness, 5-6 μm particle size, E-Merck, Germany). 5 μL of extracts were spotted as bands of 6 mm width by using the auto sampler fitted with a 100 μL Hamilton syringe. The plates were established using toluene: ethyl acetate : formic acid (5:4:1) as a solvent system. The solvent system was

transferred in CAMAG twin-trough plate development chamber which was lined with filter paper and pre-saturated with 30 ml mobile phase. The resulted plates were dried in air and scanned. A spectrodensitometer (Scanner 3, CAMAG) equipped with ‘win CATS’ planar chromatography manager software was used for the densitometry measurements, spectra recording and data processing. Absorption/remission was the measurement mode at a scan speed of 20 mm/s. Chromatograms were recorded at 254 and 366 nm. The Rf value of each compound which were separated on plate and data of peak area of each band were recorded.

Result:

In HPTLC analysis composition of mobile phase was optimized by testing different solvent system of varying polarity and the best results were obtained by using by using toluene: ethyl acetate : Formic acid (5:4:1). The chromatograms of ethanol extract at 254nm revealed the presence of total 11 compounds with 3 components of Rf= 0.47 and 0.78 and 0.81 at a very high concentration along with 8 component in small concentration, Whereas chloroform extracts exhibited presence of 10 compounds with 2 components at 254 nm Rf= 0.47, 0.72 at a very high concentration along with 7 component in small concentration.

Table no 1 .1

Sl no

Name of the extract

No of peak Rf value Peak area 254nm 366

nm 254nm 366 nm 254nm 366 nm

1 Ethanol 11 8 0.04,0.09, 0.13,0.21, 0.35,0.41,0.47,0.65, 0.68.0.78, 0.81

0.08,0.09, 0.36,0.42, 0.59,0.67, 0.72, 0.81

120.95,306.12, 354.57, 676.37, 698.12,1400.07, 5134.44, 5902.22, 6872.84,9018.87, 9609.56

210.54, 278.93, 290.37, 1970.62, 6600.00, 7609.4, 8170.44, 9814.71

2 Chloroform 10 7 0.05,0.08, 0.12,0.35,0.41,0.47, 0.53, 0.58, 0.68.0.75

0.08, 0.42, 0.59,0.67, 0.72, 0.78, 0.81

129.95, 385.57, 976.37, 1060.12,1423.07, 2545.44,4069.22, 5266.84,6108.87, 7606.56

210.54, 562.37, 627.62, 1709.00, 2606.4, 5617.44, 8114.71

Chromatographic profile of ethanol and chloroform extracts at 254nm.

Chromatographic profile of ethanol and chloroform extracts at 366 nm

3D Chromatogram of Ethanol extract at 254 nm

Chromatogram of chloroform extract at 366 nm

HPTLC study of Ajwain extracts

Development of chromatographic HPTLC fingerprint profile of chloroform and ethanolic extract of Ajwain. Both the extracts of Ajwain was subjected to HPTLC fingerprint profile to check the presence of different phytopharmaceuticals. Numbers of peaks, peak areas, Rf values were recorded. Different combination of solvent systems were tried to get good separation and sharp peaks. The adequate resolution was obtained in the solvent system toluene: ethyl acetate (93:7) and it was found to be the best for separation of compounds present in both the extracts of Ajwain. HPTLC Fingerprinting was carried out by using CAMAG HPTLC system (Switzerland) with a Linomat 5 sample applicator. The whole investigation process was carried out in air-conditioned room maintained at 22ºC and 55% humidity. TLC was also done on pre-coated silica gel HPTLC aluminium plates 60F254 (20 cm×20 cm, 0.2mm thickness, 5-6 μm particle size, E-Merck, Germany). 20 μL of extract was spotted as bands of 6 mm width by using the auto sampler fitted with a 100 μL Hamilton syringe. The plates were established using toluene: ethyl acetate (93:7) as a solvent system. The solvent system was transferred in CAMAG twin-trough plate development chamber which was lined with filter paper and pre-saturated with 20 ml mobile phase. The resulted plates were dried in air and scanned. A spectrodensitometer (Scanner 3, CAMAG) equipped with ‘win CATS’ planar chromatography manager software was used for the densitometry measurements, spectra recording and data processing. Absorption/remission was the measurement mode at a scan speed of 20 mm/s. Chromatograms were recorded at 254 and 366 nm. The Rf value of each compound which were separated on plate and data of peak area of each band were recorded.

Result :

In HPTLC analysis composition of mobile phase was optimized by testing different solvent system of varying polarity and the best results were obtained by using by using (toluene: ethylacetate; 93:7). The HPTLC fingerprinting of ethanol and pet ether extracts of Ajwain was developed in the solvent system (toluene: ethylacetate; 93:7) were scanned at two wavelengths UV 254 nm and UV 366 nm. At UV 366 nm, 12 peaks in chloroform and 7 peaks in ethanol were observed in chromatogram. Major peaks with high intensity of compounds were observed at Rf values 0.78, 0.70, 0.63, 0.65, 0.54, 0.48 and 0.42 in Chloroform; and 0.78, 0.71, 0.60, 0.56, 0.52 in ethanol extract.

Table no 1.2

Sl Name of No of peak Rf value Peak area

no the extract

254nm

366 nm

254nm 366 nm 254nm 366 nm

1 Chloroform

8 12 0.28,0.36, 0.38,0.41, 0.43,0.48, 0.55,0.59

0.29, 0.30 0.38, 0.40 0.42, 0.48, 0.54, 0.58, 0.63, 0.65, 0.70. 0.78,

201.54, 289.93, 652.37, 1027.62, 4007.00, 5606.4, 7917.44, 8414.71

102.95,305.1, 307.57, 406.37, 556.12,614.07, 785.44, 879.22, 3002.84,6118.8,7996.56, 8904.67

2 Ethanol 5 7 0.41, 0.47, 0.58, 0.65, 0.68.

0.48, 0.52, 0.54, 0.56, 0.60, 0.71,0.78,

512.95, 782.12, 793.57, 1006.37, 7006.12

201.54,65 2.93, 1002.37, 2097.62, 4527.00, 6003.4, 6917.44.

Chromatographic profile of ethanol and chloroform extracts at 254nm

Chroatographic profile of ethanol and chloroform extracts at 366 nm

Chromatogram of ethanol extract at 366 nm

Chromatogram of chloroform extract at 366nm

3. Antimicrobial Analysis:

Zone of inhibition (Avg diameter in mm) of nasal swabs with different

concentration of extracts

Table 2

Volunteers

Kalonji -ethanol(0.8g

/ml)

Ajwain –Ethanol(0.8g

/ml)

Kalonji- Chloroform(0.4

g/ml)

Ajwain –- Chloroform(0.4

g/ml)

1 7mm 15mm NA NA 2 5mm NA 7mm NA 3 14mm NA 7mm 10mm 4 7mm NA 8mm NA 5 12mm NA NA 5mm 6 10mm 10mm 8mm 7mm 7 NA 14mm NA 7mm 8 13mm 11mm NA NA 9 NA 11mm NA NA

10 1mm 16mm 7mm NA

Figure 1 Plates with Zone of inhibition –Volunteers 1-5

Figure 2 Plates with Zone of inhibition –Volunteers 6-10

The results of the antimicrobial activity of plant extracts tested against microorganisms by disk diffusion method are shown in Table 2. Among the plants screened, the ethanol extract(0.8gm/ml) of Kalonji and

ajwain, showed significant inhibition against microbes present in nasal swab, with less activity shown by the chloroform extracts as seen in table 2, the plant extracts showed antibacterial activity.

Conclusion: Ajwain and Kalonji have shown antimicrobial activity against

nasal bacteria (Not identified).The significance of the present work is to evaluate the importance of spices as plant remedies for various diseases.

There is a clear need for exploration of new antimicrobial agents with novel mode of action from plant sources and to study the potentiality for

applications in food systems. Considering the impact of the antibiotics on

pathogens and normal flora, search for potential antimicrobial agents from plant sources is a good alternative aspect. Pharmacological evaluations and

understanding the mechanism of these biologically active compounds in the inhibition of the pathogens is a promising area of research. Future

research is also necessary to use the phytochemicals as food additives or as

functional ingredients in food.

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