Rosmarinus officinalis L. is an evergreen shrub that belongs to the Lamiaceae family.

Rosemary as it is commonly called, grows approximately one meter tall. Its young branches are

pubescent and turn woody on maturation. The leaves are simple, lineal, sessile, coriaceous,

perennial and their edges roll up. The upper and lower surfaces of the leaves are covered by

secretory hairs that give the plant a pleasant camphoraceous odor. The flowers of the Rosemary

plant are small, bilabiate, pale blue or lilac with violet spots, arranged in dense axillary or

terminal clusters [1]. The leaves of rosemary contain 1.0-2.5% essential oils and such

composition may vary according to the chemo type and the development stage at which the plant

has been harvested. The essential oil is almost colorless to pale yellow liquid with a

characteristic refreshing and pleasant odor [2].

The Rosemary plant is known to be grown widely in the Mediterranean basin and part of Europe,

but it is now cultivated through out the world. Rosemary which is a common house hold plant

have been found to have some many economical and medicinal medicinal importance for

centuries. Rosemary is commonly used as a spice, and flavoring agent in food processing [3];

used for its fragrance in soaps and cosmetics. World Health Organization (W.H.O) defined

medicinal plant as any plant which in one or more of its organs, contains substances that can be

used for therapeutic purposes or which are precursors for the synthesis of useful drugs. Among

the herbal extracts reported to have antioxidant activity, rosemary (Rosmarinus officinalis

L.) is one of the most widely commercialized plant extracts. Several studies have shown

that Rosemary also contains a large number of compounds which are responsible for its

significant antimicrobial [4], anti-inflammatory [5], antiviral [6], and anti-carcinogenic activities

[7]. The antioxidant and other biological activities were generally believed to be

attributable to the free radical scavenging properties of rosemary secondary metabolites

including polyphenolic acids, flavonoids, phenolic diterpenes, and essential oil. The

abietane diterpenes carnosic acid and carnosol, and the rosmarinic acid, are generally accepted as

the most antioxidant active compounds found in rosemary extract [8,9,10]. Carnosic acid is the

major phenolic diterpene present in rosemary leaves. Other antioxidant compounds such as

carnosol, rosmanol, and ros- mariquinone are formed as it undergoes an oxidative degradation

and rearrangement cascade within the plant [11]. It has also been reported that r osemary

extract shows inhibitory effects for human immunodeficiency virus (HIV) infection at

very low concentrations [6].

Metabolomics may be defined as the comprehensive quantitative and/or qualitative analysis of

all metabolites present in a bio-fluid, cell, tissue, or organism [12]. There are four conceptual

approaches in metabolomics: target analysis, metabolite profiling, metabolomics, and metabolic

fingerprinting [13]. Metabolite profiling is used in analyzing of a larger set of compounds, both

identified and unknown with respect to their chemical nature. This approach has been reported to

be used in the study of so many biological compounds. Metabolomics employs complementary

analytical methodologies such as mass spectrometry (MS) or nuclear magnetic resonance (NMR)

spectroscopy, in order to determine and quantify as many metabolites as possible, either

identified or unknown compounds [14]. In metabolic fingerprinting, a metabolic “signature” or

mass profile of the sample of interest is generated and then compared in a large sample

population to screen for differences between the samples; when signals that can significantly

discriminate between samples are detected, the metabolites are identified and the biological

relevance of that compound can be elucidated, greatly reducing the analysis time [14]. The term

‘metabolome’ has been used to describe the observable chemical profile or fingerprint of the

metabolites present in whole tissues [15]. Metabolome reflects the interaction between an
organism’s genome and its environment. Therefore an organism’s metabolome bridges the gap

between its genotype and phenotype.

Due to the complexity and diversity of plant metabolites it is unlikely that one single analytical

method could generate information about all metabolites present in a plant and it could probably

be necessary to perform a wide range of chemical analysis which should be both rapid and

reproducible. High-resolution 1H NMR spectroscopy is an important technique which is

currently being explored together with principal component analysis (PCA), the most common

method to analyze the variability in a group of samples. The signal intensities of NMR spectra

are based on molar concentrations and can therefore be compared directly. The combination of

NMR and PCA has been applied to the metabolic profiling of the biochemical relationship

between sugarcane callus tissues and their respective nutrient culture media [16].
Sample collection:
1. Pre-weight sample tube
2. Place the sample in preweighted tube and immediately immerse in liquid
nitrogen
3. Re weight the tube with sample (fresh weight)
4. Leave the sample 4 hrs in -20 o C and then add sample in the lyophilizer
until complete drying
5. Measure the dry weight of samples
6. Calculate water loss by subtracting dry weight from fresh weight
7. Calculate the water loss percentage as the following
Water loss= ( water loss / wet sample)X 100.

Metabolite extraction:
1. Homogenize the sample and ground well until convert to powder
2. weight 20 mg plant material ed for each sample/replicate
3. Calculate the solvent volumes using extraction calculator to achieve a
constant ratio of 2:2:1.8 of chloroform: methanol: water according to
Bligh and Dyer method (Bligh et al. 1959; Wu et al. 2008).
4. Rehydrate the dried plant material with the appropriate ice-cold
methanol: water solution.
5. Vortex-mixing for 20 s
6. Transfer the mixture into the ice-cold chloroform: water solution in glass
tubes. 7. Incubate the sample on ice for 10 min
7. Centrifuge for 10 min at 2000 x g at 4ºC.
8. Transfer The top layer, i.e. the hydrophilic extract, into a new
microcentrifuge tubes.
9. The hydrophilic extracts were dried using a Centrivap centrifuge for ~15
hr and stored at -20ºC until further preparation for NMR analysis.

NMR sample preparation and spectroscopy:

10.Re-suspend the dried hydrophilic extracts from each sample in 620 μl of
NMR buffer (100 mM sodium phosphate buffer (pH 7.3), 1 mM TMSP
(internal standard, 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid, CAS:
24493-21-8), and 0.1% sodium azide, in 99.9 atom % D2O)
11.Vortex-mixed and centrifuged to remove any remaining undissolved
material.
12.Add 600 μl into labelled NMR tube.
13.Run the sample on a BrukerAvanceTM III spectrometer operating at 700
MHz.
REFERENCES

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ed. Germany: Wiley VCH.

3. Saito, Y., Shiga, A., Yoshida, Y., Furuhashi, T., Fujita, Y. and Niki, E. (2004). Effects of
Novel Gaseous Antioxidative System Containing a Rosemary Extract on the Oxidation
Induced by Nitrogen Dioxide and Ultraviolet Radiation. Bioscience, Biotechnology, and
Biochemistry, 68, 781-786.

4. Bozin, B. Mimica-Dukic, N.; Samojlik, I.; Jovin, E (2007). Antimicrobial and antioxidant
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Lamiaceae) essential oils. J. Agric. Food Chem., 55, 7879–7885.

5. Englberger, W. Hadding, U. Etschenberg, E.; Graf, E. Leyck, S.; Winkelmann, J.

Parnham, M. J (1988). Rosmarinic acid: a new inhibitor of complement C3-convertase
with anti-inflammatory activity. Int. J. Immunopharmacol, 10, 729–737.

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Rosemary extract and its constituents, carnosic acid, carnosol and rosmarinic acid, in bulk oil and
oil- in-water emulsion. J. Agric. Food Chem. 44, 131-135.

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16. Mahmud I, Thapaliya M, Boroujerdi, A, Chowdhury K (2014). NMR-based

metabolomics study of the biochemical relationship between sugarcane callus tissues and
their respective nutrient culture media. Analytical and Bioanalytical Chemistry, 406(24),
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