Histology, Phytochemistry and bacterial activity of |Anise seed oil

Full text

Journal of Bacteriology & Mycology: Open Access
Histology, Phytochemistry and Bacterial Activity of Anise
(pimpinella anisum l.) Seed and Essential Oil
Submit Manuscript | http://medcraveonline.com
Introduction
Anise (Pimpinella anisum L.) (family: Umbelliferae) origin
is mediterranean region [1]. The major production area in
Sudan is Northern Sudan, while there is a very limit production
in Khartoum state [2]. Aniseeds contain 1.5-5% essential oil
        
gastrointestinal spasms. Consumption of aniseed in lactating
women increases milk and also reliefs their infants from
gastrointestinal problems [3]. In the food industry, anise is used as

and gums [4,5]. The composition of anise varies considerably
with origin and cultivation method. These are typical values for
the main constituents; Moisture: 9-13%, Protein: 18%, Fatty oil:
8-23%, Essential oil: 2-7%. Starch: 5%, N-free extract: 22-28%,
         
generally around 2-3% and anethole makes up 80-90% of the oil
   anethole, a phytoestrogen [7]. The essential
oil has reportedly been used as an insecticide against head lice
and mites. The oil is very famous oil throughout the world. It was
used in confectioneries, pharmaceutical, tooth paste, and other
industrial uses [8].
Anatomical features of anise are a vital process for clear
       
plant anatomy, phytochemistry and other recommendations.

quality control and quality assurance and other uses of essential
oil for medicinal and food purposes. Due to variation in climate,
topography, soil, and cultural practices; anise essential oil can be
differ, therefore more investigation are important to clarify this
changes. Morphological, structural and developmental features of
fruits and seeds are here studied, with the purpose to give a proper
    
future taxonomical and ecological studies.
Furthermore, some spices are reported to have bactericidal
or bacteriostatic activities. The inhibitory effects of spices are
mostly due to the volatile oils present in their composition [9].
The main factors that determine the antimicrobial activity are
the type and composition of the spice, amount used, type of
microorganism, composition of the food, pH value, temperature
of the environment, and proteins, lipids, salts, and phenolic
substances present in the food environment [10].

1Industrial Research and Consultancy Centre, Sudan
2University of Khartoum, Sudan
*Corresponding author: El Rasheed Ahmed Salim,
Industrial Research and Consultancy Centre, Ministry

Received:| Published: December 22,

Research Article

Abstract
This study was conducted to investigate the histology, phytochemistry and
antimicrobial activities of anise (Pimpinella anisum L.) seed and essential oil obtained
     
dehydration, clearing, wax embedding, sectioning, staining and mounting. Moisture
content performed according to FAO manuals. Oil extraction according to British
pharmacopeia. Test of the oil for antimicrobial activity conducted by cup-plate agar
     
activity of the prepared extracts. Standard microorganisms namely; Staphylococcus
aureus ATCC 25923 Gram (+ve) bacteria, Klebsiella pneumoniae   
(-ve) bacteria, Escherichia coli ATCC 25922 Gram (-ve) bacteria and Pseudomonas
aeruginosa ATCC 27853 Gram (-ve) bacteria were obtained from Medicinal and
Aromatic Plant Institute, National Centre of Research, Sudan. Two concentrations
used A (0.5 ml of anise essential oil in 4.5 ml solvent (methanol) to form (1:9) ratio
and Concentration B (2.5 ml of concentration diluted with 2.5 ml solvent (methanol)
to form (1:18) ratio). Histological features of anise under microscopical examining
showing hair (hr), epidermis (epi), mesocarp (mes), oil vittae (vit), endocarp (endc),
  
 
          
 
value (14.03). Microbiological activities of anise oil using concentration A and B
against Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa
showed moderate antibacterial effect. Concentration A showed low inhibition and
Concentration B showed no antimicrobial activities against Escherichia coli.
Keywords: Anise essential oil; Histology; Staphylococcus aureus; Klebsiella
pneumonia; Escherichia coli; Pseudomonas aeruginosa; Physico-chemicals

Citation:pimpinella anisum l.) Seed and Essential Oil. J
Bacteriol Mycol Open Access 3(4): 00070. DOI: 
Histology, Phytochemistry and Bacterial Activity of Anise (pimpinella anisum l.) Seed
and Essential Oil 2/6
Copyright:
©2016 Salim et al.
The objectives of this study are to investigate histology,
phytochemistry and antibacterial activities of anise oil on
Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli and
Pseudomonas aeruginosa using two concentrations (1:9 and 1:18).
Materials and Methods
Source of the plant
Anise seed was obtained from local Omdurman market, Sudan.
The production is area is the northern region of Sudan. The
original plant source was suggested to be brought from Egypt far
a year ago through River Nile valley movement.
Histology
Preparations: Soft tissue method adopted; seeds soaked into
running water for two days and then run in calm water not exceed

Fixation: Seeds were collected into formaline: acetic acid: alcohol
       
   

leaching of the oil from the plant.
Dehydration: Seeds were dehydrated through passing them into
a series of concentrations of ethyl alcohol from 50 to 95%.
Clearing: The seeds and stems after dehydration cleared by
passing them in a mixture of solutes; ethyl alcohol: cedar oil
         
(incubation) and the thirdly through pure xylene for overnight

Wax embedding: The above specimens were transferred from

Sectioning: The waxed specimens were sectioned using a
rotatory microtome (Letize 1512 - West Germany).
Staining: Dewaxing of sectioned specimens by xylene several
times to insure dewaxing. Then hydration of the dewaxed
specimens using decreasing dilutions of alcohol from 95 to 50%,
        
         
dehydration was conducted by passing the specimens through
        
       
transfers to xylene.
Mounting: The prepared sections were mounted on Canada
balsam, and then the sections were covered and placed
  
hours before microscopical examining.
Determination of moisture content
Moisture content was determined according to FAO manuals
[11].
Determination of oil Content (v/w)
Anise crushed to powders using a grinder. Essential oil
was obtained by hydrodistillation of the powdered dry fruits
according to the British Pharmacopeia Protocol. The oil phase
was separated, dried over anhydrous sodium sulfate, and kept in a

Physical and chemical properties
Physical and chemicals properties were determined according
to BS2073 [13] except odour and volatilization according to
British pharmacopeia [12].
Antimicrobial Activities
Preparation of extracts
Dilution of 0.5 ml of anise essential oil in 4.5 ml solvent
(methanol) (1:9) to make Concentration A., 2.5 ml of concentration
A was further diluted with 2.5 ml solvent (methanol) (1:18) to
make concentration B.
Source of microorganisms
Four standard organisms namely; Staphylococcus aureus
ATCC 25923 Gram (+ve) bacteria, Klebsiella pneumoniae ATCC
   , Escherichia coli ATCC 25922 Gram
(-ve) bacteria and Pseudomonas aeruginosa ATCC 27853 Gram
(-ve) bacteria were obtained from Medicinal and Aromatic Plant
Institute, National Centre of Research, Sudan.
Test of extracts for antimicrobial activity
To determine the effectiveness of anise oil against the above
four organisms’ cup-plate agar diffusion method was adopted,

of the prepared extracts [14]. Two ml of the standard bacterial
stock suspension (10-10) colony forming units per ml were
thoroughly mixed with 200 ml of sterile nutrient agar which was

       
with 0.1 sample of each of the concentrations using standard
Pasteur pipette and allowed to diffuse at room temperature
for two hours. The diameter of zone of inhibition (mean of two
replicates ± SD) as indicated by clear area which was devoid of
growth of microbes was measured to determine the antibacterial
activity. Two replicates were carried out for each extract against
each of the test organisms. After incubation the diameter of the
resultant growth inhibition zones were measured, averaged and
the mean value was tabulated. The experiment was replicated two

Results and Discussion
Histology
Anise histology with respect to oil structure, endodermis,

located embedded between the external epidermis of the seed
and mescarp. Other anatomical structures such as endodermis
and vascular bundle were occurred. Figure 2 should be anise seed;
the closer view showing the structure of oil bearing structure in
   
said that oil secretory structure of anise seeds was found in
secretory structure known as vitta. Also the above results were
coinciding with Parry [15] who illustrated the presence of oil
vittae in the mesodermis of anise seeds. The fruit “rind” consists
of the exocarp, represented by a periderm with lenticels, and

Citation:pimpinella anisum l.) Seed and Essential Oil. J
Bacteriol Mycol Open Access 3(4): 00070. DOI: 
Histology, Phytochemistry and Bacterial Activity of Anise (pimpinella anisum l.) Seed
and Essential Oil 3/6
Copyright:
©2016 Salim et al.
by the parenchymatic mesocarp, with branched secretory ducts
          
endocarp, destituted of secretory ducts, and derived from the
activity of a ventral meristem, which emerges early in the fruit
        
   

more details about the oil vitae. Oil vittae was located embedded
between the external epidermis of the seed and mesocarp other
anatomical structures such as vascular bundle was occurred. Oil
vittae embedded between the external epidermis of the seed and
mesocarp. Other anatomical structures were such as endocarp,
sclerenchyma, cuticle, endosperm, sclerenchyma, crystals and

of anise seeds agreed with Parry [15] who illustrated the presence
of oil glands on stem exodermis of mint leave (Appendix 1).
Figure 1: Transverse section (10X) through the anise seed showing

Figure 2: Transverse section (40X) through the anise seed showing
hair (hr), epidermis (epi), mesocarp (mes), oil vittae (vit), endocarp
(endc), cuticle (cu), endosperm (ends), sclerenchyma (sch), crystals

Figure 3: Transverse section (40 X) through the anise seed showing

Plate 1: Antimicrobial activity of anise against Staphylococcus
aureus.
Plate 2: Antimicrobial activity of anise against Klebsiella
pneumoniae.

Citation:pimpinella anisum l.) Seed and Essential Oil. J
Bacteriol Mycol Open Access 3(4): 00070. DOI: 
Histology, Phytochemistry and Bacterial Activity of Anise (pimpinella anisum l.) Seed
and Essential Oil 4/6
Copyright:
©2016 Salim et al.
Phytochemistry
 
gravity, odor and taste, acid and ester values of fresh herb of anise
were shown in Table 1. Moisture content was less than what

a minimum of the range determined by Guenther [8] who reported
     
due to climatic, cultural or post harvest conditions. Refractive


oil was 0.9825 and fell in the range mentioned by Guethner [8]
which ranged between 0.980 - 0.990. The oil odour was similar to
anithole; which constitute the major party of anise oil; indicating

oil was present in anise oil (Figure 4).
Table 1: Purity, moisture content, oil content, physical and chemical
properities of anise seed and oil.
Properties Values
Purity 
Moisture content 
Oil content 1.5% (v/w)
Refractive index 
 0.9825
Odor and taste Anithole odor
Evaporation No trace left
Acid value 1.29
Ester value 14.03
Plate 3: Antimicrobial activity of anise against Escherichia coli.
Plate 4: Antimicrobial activity of anise against Pseudomonas
aeruginosa.
Figure 4: Cross section through anise seed showing oil vittae and
other histological structure [15].

Citation:pimpinella anisum l.) Seed and Essential Oil. J
Bacteriol Mycol Open Access 3(4): 00070. DOI: 
Histology, Phytochemistry and Bacterial Activity of Anise (pimpinella anisum l.) Seed
and Essential Oil 5/6
Copyright:
©2016 Salim et al.
Acid value and ester value of anise oil were 1.29 and 14.03
respectively and they are lower than what mentioned by Guenther
[8] who mentioned that anise acid value and ester value were
         
cultural practices and post harvest factors. It is well documented

yield of volatile oils produced by medicinal plants [17-19].
Microbiology
Antibacterial effects of anise oil both concentrations (1:9)
and (1:18) was illustrated in Table 2. The results showed that
concentration A (1: 9) of anise seed oil showed moderate
inhibitory effect against Staphylococcus aureus ATCC 25923 Gram
(+ve) bacteria. And concentrations B of anise seed oil (1:18) had
low effect against Staphylococcus aureus ATCC 25923 Gram (+ve)
bacteria.
Table 2: Antibacterial activity of anise volatile oil using two diluted
concentrations.
Microorganisms
Inhibition zones (mm) mean
Concentration
A (1:9)
Concentration
B (1:18)
Staphylococcus aureus 13.5 (M) 15 (L)
Klebsiella pneumoniae 17 (M) 17.5 (M)
Echerichia coli 14 (L) -
Pseudomonas aeruginosa 15.5 (M) 15.5 (M)
Where:
Test volume of extract = 0.1 ml/cup.
18 mm > high (H). 15-17mm moderate (M), 12 - 14mm low (L) and 11<
none (N).
Concentration A and B showed moderate inhibitory effect against
Klebsiella pneumoniae  and Pseudomonas
aeruginosa ATCC 27853 Gram (-ve) bacteria. While Concentration A (1:9)
of anise seed oil showed low inhibitory effect against Escherichia coli ATCC
25922 Gram (-ve) bacteria.These results partially agree with Gangrad [20]
who mentioned that anise essential oil has antibacterial activity against
Staphylococcus aureus and Escherichia coli.
Conclusion
We can conclude that the anatomy, phytochemistry and
microbiology of anise seed and oil postulates the followings;
Anatomy of aniseed exhibits the nominal histological structures
of anise seed likes the oil vittae, hair, epidermis, mesocarp,
      
       
Sudanese aniseseed. The physicochemical properties of the
         
their pharmacognosy properties. Antibacterial activity of anise
essential oil was reported against Staphylococcus aureus ATCC
25923 Gram (+ve) bacteria, Klebsiella pneumoniae 
Gram (-ve) bacteria, Escherichia coli ATCC 25922 Gram (-ve)
bacteria and Pseudomonas aeruginosa ATCC 27853 Gram (-ve)
bacteria.
Acknowledgement
I wish to express my thanks to the staff of Medicinal and
Aromatic Plants Institute (NCR, Sudan) for their support during
tests antimicrobial. My thanks extend to Department of Botany,
Faculty of Science, and University of Khartoum for their assistance
in anatomical work to complete this study with special thanks to
Mr. Kamel. Also my thanks due to Department of Food Research
Industries, Industrial Research and Consultancy, Sudan for their
generous help in conducting extraction and chemical tests.
References
1. Abu zeid EN (1992) Aromatic plants and their agricultural and
pharmaceutical products. (1st edn), Al Dar Al Arabia for Printing
and Distribution, Cairo, Egypt, pp. 473.
2. El Hussein SA (1983) Essential oil crops of Sudan (Technica
communication of ISH). International Society of Horticultural
Crops. Acta Horticulture 143: 213.
3. 
4. Salehi Surmaghi MH (2010) Medicinal plants and phytotherapy.
In: Mobarakeh HI (Eds.), Effect of Zeravschania membranacea
alcoholic extract on some components of complement system and

5. Ö       
antifungal effect of anise (Pimpinella anisum L.) fruit oil at ripening

6. 
Delhi, India, pp. 19-21.
7. Albert Puleo M (1980) Fennel and anise as estrogenic agents. J
Ethnopharmacol 2(4): 337-344.
8. Guenther E (1975) The essential oils. D Van Nostard Company Inc,

9. Arora DS, Kaur J (1999) Antimicrobial activity of spices. Int J

10. Sagdic O (2003) Sensitivity of four pathogenic bacteria to Turkish
thyme and wild marjoram hydrosols. Lebensmittel-Wissenschaft

11. 
Organization of United Nations, Rome, Italy, pp. 205, 227, 228, 232,
258.
12. British Pharmacopoeia (1988) Anonynous. Appendix XI F, HMSO,
UK, pp. A138.
13.           
standard Institute, UK.
14. Kavangh F (1972) Analytical Microbiology. In: Favanagh F (Ed.),
Academic Press, London, UK, pp. 11.
15.        


16. Mourão KS, Beltrati CM (2000) Morphology and anatomy of
developing fruits and seeds of Mammea americana L. (Clusiaceae).


Citation:pimpinella anisum l.) Seed and Essential Oil. J
Bacteriol Mycol Open Access 3(4): 00070. DOI: 
Histology, Phytochemistry and Bacterial Activity of Anise (pimpinella anisum l.) Seed
and Essential Oil 6/6
Copyright:
©2016 Salim et al.
17.           
(Mentha ssp.) in Northern Ostrobothnia. In: British Pharmacopoeia
       
728.
18.          
methods on the essential oil content and composition of Roman
.
19. Ramezani S, Ramezani F, Rasouli F, Ghasemi M, Fookian MH (2009)
Diurnal variation of essential oil of four medicinal plants species
in central region of Iran. Research Journal of Biological Science 4

20. Gangrad SK, Shrivastava RD, Sharma OP, Moghe MN, Trivedi
KC (1990) Evaluation of some essential oils for antimicrobial
properties. India perfumer 34(3): 204-208.