Exploration and Phytochemical Estimation of Murraya koenigii Leaves for Pharmaceutical Applications

 

Radheshyam Sharma1*, Umesh Kumar2

1Azad Institute of Pharmacy and Research, Lucknow

2Hygia Institute of Pharmaceutical Education and Research, Lucknow

*Corresponding Author E-mail: radheshyamsharma1029@gmail.com

 

ABSTRACT:

The aim of current study was to evaluate so many pharmaceutical properties in the world of medicine. The purpose of the current study was to investigate the various phytochemicals present in Murraya koenigii leafs. The preliminary phytochemical studies with the help of Thin layer chromatography (TLC) method revealed that petroleum ether fraction contains volatile oil. The benzene fraction showed presence of fixed oil and alkaloid. Acetone fraction has confirmed presence of alkaloid. The fractions were further subjected to TLC in toluene: ethyl acetate (93:7) & ethyl acetate: methanol: water (76.5: 13.5: 10) solvent system with different detecting reagents for further confirmation of phytoconstituents. The alkaloid was prominently found in benzene, chloroform, acetone and methanol extract. The petroleum extract was found to contain volatile oil and fixed oil in benzene extract. The proximate analysis revealed that moisture content was 15%, total ash 73.33%, acid insoluble ash 26%, sulphated ash 80%, water soluble ash 89.5%, water insoluble ash 10.5%, acid soluble ash 74%. Several potent antioxidant compounds were extracted and their presences are confirmed by structure elucidation through various spectroscopic methods.

 

KEYWORDS: Exploration, Phytochemical Estimation.

 

 


INTRODUCTION:

Phytochemistry is the study of phytochemicals, which are chemicals derived from plants. Those studying phytochemistry strive to describe the structures of the large number of secondary metabolic compounds found in plants, the functions of these compounds in human and plant biology, and the biosynthesis of these compounds. Plants synthesize phytochemicals for many reasons, including protecting themselves against insect attacks and plant diseases.1 Phytochemicals in food plants are often active in human biology, and in many cases have health benefits. The compounds found in plants are of many kinds, but most are in four major biochemical classes, the alkaloids, glycosides, polyphenols, and terpenes. 2

 

Techniques:3, 4, 5, 6, 7

(a) Extraction:

Extraction in chemistry is a separation process consisting in the separation of a substance from a matrix. It includes Liquid-liquid extraction, and solid phase extraction. The distribution of a solid between two phases is an equilibrium condition described by partition theory. This is based on exactly how the analyte move from the water into an organic layer.

 

(b) Types of extraction:

There exist several types of extraction, including: liquid–liquid extraction, solid-phase extraction, and acid-base extraction. In liquid-liquid extraction compounds separate according to their relative solubility in two different immiscible liquid phases. This technique has been applied in several fields such as analytical chemistry and biology.

 

 

Figure 1:- Laboratory scale liquid-liquid extraction.

 

(c) Components of an extractive process:

Extractions often use two immiscible phases to separate a solute from one phase into the other. Typical lab extractions are of organic compounds out of an aqueous phase and into an organic phase. Common extractants are arranged from ethyl acetate to water (ethyl acetate < acetone < ethanol < methanol < acetone: water (7:3) < ethanol: water (8:2) < methanol: water (8:2) < water) in increasing order of polarity according to the Hildebrand solubility parameter. The extract can be put back to dried form using a centrifugal evaporator or a freeze- drier.

 

(d)  Modern variations and applications:

Techniques include supercritical carbon dioxide extraction, ultrasonic extraction, heat reflux extraction, mechanochemical-assisted extraction, microwave-assisted extraction, instant controlled pressure drop extraction (DIC), and perstraction.

 

Boiling tea leaves in water extracts the tannins, theobromine, and caffeine out of the leaves and into the water. Solid-liquid extractions at laboratory scales can use Soxhlet extractors (such as oil from olive cake see at right).

 

Structure Elucidation:

In determining structures of chemical compounds, one generally aims to obtain, minimally, the pattern and multiplicity of bonding between all atoms in the molecule; when possible, one seeks the three dimensional spatial coordinates of the atoms in the molecule (or other solid). The methods by which one can elucidate the structure of a molecule include spectroscopies such as nuclear magnetic resonance (proton and carbon-13 NMR), various methods of mass spectrometry (to give overall molecular mass, as well as fragment masses), and x-ray crystallography when applicable. The last technique can produce three-dimensional models at atomic-scale resolution, as long as crystalsare available. When a molecule has an unpaired electron spin in a functional group of its structure, ENDOR and electron-spin resonance spectroscopes may also be performed. Techniques such as absorption spectroscopy and the vibrational spectroscopies, infrared and Raman, provide, respectively, important supporting information about the numbers and adjacencies of multiple bonds, and about the types of functional groups (whose internal bonding gives vibrational signatures), further inferential studies that give insight into the contributing electronic structure of molecules include cyclic voltammetry and X-ray photoelectron spectroscopy. 8 These latter techniques become all the more important when the molecules contain metal atoms, and when the crystals required by crystallography or the specific atom types that are required by NMR are unavailable to exploit in the structure determination. Finally, more specialized methods such as electron microscopy are also applicable in some cases.9

 

Chromatographic Techniques:

Chromatography is a laboratory technique for the separation of a mixture. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based is based on differential partitioning between the mobile and stationary phases. Subtle differences in a compound’s partition coefficients result in differential retention on the stationary phase and thus affect the separation.10

 

Phytochemicals:11, 12, 13

1.    Alkaloids:

Alkaloids are better-tasting chemicals, very widespread in nature, and often toxic. There are several classes with different modes of action as drugs, both recreational and pharmaceutical. Medicines of different classes include atropine, scopolamine, and hyoscyamine.

 

2.    Glycosides:

Anthraquinone glycosides are found in the laxatives senna, rhubarb and aloe. The cardiac glycosides are powerful drugs from plants including foxglove and lily of the valley. They include digoxin and digitoxin which support the beating of the heart, and act as diuretics.

 

3.    Polyphenols:

Polyphenols of several classes are widespread in plants. They include the colourful anthocyanins, hormone-mimicking phytoestrogens, and astringent tannins. In ayurveda, the astringent rind of the pomegranate is used as a medicine, while polyphenol extracts form plant materials such as grape seeds are sold for their potential health benefits. They have been continually studied in cell cultures for their different anti-cancer effects. Plant containing phytoestrogens have been used for centuries to treat gynaecological disorders such as fertility, menstrual and menopausal problems. Among these plants are pueraria mirifica, kudzu, angelica, fennel and anise.

 

4.    Terpenes:

Terpenes and terpenoids of many kinds are found in resinous plants such as the conifers. They are strongly aromatic and serve to repel herbivores. Their scent makes them useful in essential oils, or for aromatherapy. Some have had medicinal uses: thymal is an antiseptic and was once used as a vermifuge (anti-worm medicine).

 

Plant profile:14, 15

The curry tree (Murraya koenigii) is a tropical to sub-tropical tree in the family Rutaceae (the rue family, which includes rue, citrus, and satinwood), which is native to India and Sri Lanka.

 

 

Figure 2: Murraya koenigii tree

 

Scientific Classification:-

Kingdom:            Plantae

Subkingdom:      Tracheobionta

Division :              Magnoliphyta

Class:                   Magnoliopsida

Subclass:             Rosidae

Order:                 Sapindales

Family:                Rutaceae

Genus:                 Murraya

Species:                koenigii

 

Chemical constituents:

The chemical constituents present in Murraya koenigii leaves extract are  -  Koenimbine, koenine, koenigine, mahanimbine, murrayazolidine, murrayazoline, girinimbine, tocopherol, isomahanimbine, gurjunene, murrayanol, mahanine, bismurrayafoline E, euchrestine, bismahanine, bispyrafoline, isomahanine, O-methyl murrayamine A, O-methyl mahanine, lutein, carotene, caryophyllene, phellandrene.16

 

Stem bark of the Murraya koenigii contain girinimbine, murrayanine, marmesin 1’-O beta-D-galactopyranoside, mahanine, murrayacine, mukoeic acid, murrayazolinine, girinimbilol, mahanine, pyrafoline-D and murrafoline-I.

 

Seed of the Murraya koenigii contain koenoline, kurryam, koenine, koenimbine.

 

Root of the Murraya koenigii contains mukoline.17

 

Pharmacological Activities:

In vivo studies 18, 19

Vasodilating activity- Crude aqueous leaf extract of M. koenigii was prepared which showed a dose dependent negative chronotropic effect on cardiovascular system of frog heart preparations which might be due to its direct actions on the heart and blood vessels.

 

Antidiabetic property- Mahanimbine a chemical constituent of M. koenigii was isolated from column chromatography of the petroleum ether extract of dried plant. The anti-diabetic activity was performed on the streptrozotocin induced wistar rats by using pure compound at a dose of 50 mg/kg and 100 mg/kg.

 

Hypocholesterolemic activity- Hypocholesterolemic activity was checked in aged mice, which was done by using crude ethanol extract of plant leaves of M. koenigii. The experiment was confirmed by observing a decrease in cholesterol level in dose dependent manner in aged mice.

 

Antiulcer Activity- Antiulcer activity of aqueous and ether extracts of M. koenigii was studied in reserpine induced gastric ulcer model in albino rats. Extracts were effective in gastric ulceration and suggested as protective as ranitidine.

 

Anti Diarrheal activity- The bioassay guided fractionation of the n-hexane extract of the seeds of M. koenigii resulted in the isolation of three pure compounds of bioactive carbazole alkaloids, kurryam, koenimbine and koenine.

 

Phagocytic activity- The methanol extract of M. koenigii leaves was evaluated on human oral and cell mediated immune response to ovalbumin, phagocytic activity by carbon clearance test, nitric oxide NO release from murine peritoneal macrophages and cyclophosphamide induced myclosuppression.

 

Analgesic and antinociceptive activity- The methanolic extract of leaves showed analgesic effect in hot plate model and formalin induced paw licking response in mice. The activity might be linked to the processes involved in the prevention of sensitization of nociceptors, down regulation of the sensitized nociceptors or blockade of the nociceptors at peripheral and central levels.

 

Anti-lipid peroxidative activity- The status of lipid peroxidation was investigated in rats fed with M. koenigii. The concentration of melondialdehyde showed a significant decrease, while hydroperoxides and conjugated dienes were significantly increased in liver and heart.

 

Radioprotective and chemoprotective activity- The study was aimed to investigate the radio protective and chemoprotective effect of M. koenigii methanolic extract after irradiation. It has demonstrated protection against radiation RT and cyclophosphamide CP induced chromosomal damage in vivo.

 

Antiamnesic activity- Transfer latency TL was measured using elevated plus model. Standard cholinergic agent, Piracetam 400mg/kg was comparable to the petroleum ether extract of plant leaves 300 and 500 mg/kg in improving the learning and memory of aged mice and was reversed from the effect of scopolamine.

 

Antihelminthic activity- The antihelminthic activity was performed on adult Indian earth worm ‘Pheretima posithuma’. The organism was chosen due to its resemblance with the intestine round worm parasites of human beings. Petroleum ether and alcoholic extracts were selected for the activity and Piperazine citrate as standard.

 

Memory enhancing- It was observed that ethanolic extract of leaves lowered serum cholesterol in mice, inhibited brain acetylcholinesterase enzyme and thereby elevated the acetylcholine concentration in brain homogenate and ultimately improved memory in aged mice.

 

Wound Healing effect- Male albino rates were used to check the wound healing activity by screening with ethanolic extract of leaves of M. koenigii. In the excision, wound healing model reveals that three groups which were taken for wound healing activity showed a decrease in wound area from day to day.

 

In vitro studies20,21

Cytotoxic Activity- The isolated carbazole alkaloid as Koenoline from root bark of M. koenigii exhibited the cytotoxic activity against KB cell culture system.  Carbazole alkaloids isolated from the stems of M. koenigii have effects on the growth of the human leukemia cell line HL-60.

 

Anti-Tumor assay- The three cell lines were used in this investigation comprised human breast MCF-7, human cervical HeLa and murine leukemia cell lines P388. The MCF-7, HeLa and P388 cells each were cultured as monolayers in RPMI-1640, supplemented with 10.0% v/v heat-inactivated FBS, 100U/mL penicillin and 100.0μg/mL streptomycin.

 

Antimicrobial Activity- Benzoisofuranone derivatives along with six known carbazole alkaloids and three known steroids were isolated from stem bark of M. koenigii. These compounds are found to be effective in range 3.13 - 100μg/ml concentration.

 

Antioxidative property- Isolated carbazole alkaloids from dichloromethane extract of leaves of M. koenigii were evaluated on the basis of oil stability index together with their radical scavenging ability against DPPH radical on the basis of lag time to reach a steady state.

 

Skin pigmenting- The formulation of cream of essential oil of leaf of M. koenigii was found to have sun protection factor.

 

MATERIALS AND METHODS:

Materials requirements:

Fresh curry leaf was collected from available sources, India. Petroleum ether (Merck, Germany), hexane (Merck, Germany), chloroforms (Merck, Germany), absolute alcohol (Jiagsu Huaxi, China), sodium sulphate (Na2So4, (Qualigens, India), TLC plate (Merck, Germany). Acetone (Merck, Germany) Tris. Hcl (Loba, India) were used.

 

Extraction with soxhlet apparatus:

A soxhlet extractor is laboratory apparatus invented in 1879 by Franz von Soxhlet. It was originally designed for the extraction of a lipid from a solid material, thought it is not limited to the extraction of lipids. Typically, a Soxhlet extraction is required only when the desired compound has high solubility in a solvent, and the impurity is insoluble in the solvent. If the desired compound has a high solubility in a solvent then a simple filtration can be used to separate the compound from the insoluble substance. 22 Normally a solid material containing some of the desired compound is placed inside a thimble made from thick filter paper, which is loaded into the main chamber of the soxhlet extractor. The Soxhlet extractor is positioned into a flask containing the extraction solvent. The Soxhlet is then fitted with a condenser. 23

 

The solvent is heated to reflux. The solvent vapour travels up a distillation tube and floods into the chamber housing the thimble holding the solid. The condenser ensures that solvent vapour condenses and drips back down into the chamber housing the solid material. The chamber containing the solid material slowly fills with warm solvent. Some of the desired compound then dissolves in the warm solvent. When the soxhlet chamber is almost full, the chamber is automatically emptied by a siphon side arm, with the solvent running back down to the distillation flask. This cycle may be allowed to repeat many times, over hours or days.

 

During each cycle, a portion of the non-volatile compound dissolves in the solvent. After many cycles the desired compound is concentrated in the distillation flask. The advantage of this system is that instead of many portions of warm solvent being passed through the sample just one batch of solvent is recycled. After extraction the solvent is removed typically by means of a rotator evaporator yielding the extracted compounds. The non-soluble portion of the extracted solid remains in the thimble, and is usually discarded.24

 

Fresh curry leaves obtained from available sources, India were washed and cleaned thoroughly under running water. The excess water was drained and the leaves were dried in vaccum for one week. Dried curry leaves were powdered in a multi-mill fitted with sieve to obtain a coarse powder

 

 

Figure 3: Rotatory evaporator setup

 

Two solvents namely chloroform and ethanol was used for the extraction. The extraction from 35 g curry leaf powder with 250 ml of the solvent was carried out at 600C for a period of 10 h. The solvent was evaporated using a rotator evaporator. The evaporator was maintained below 500C. 7 gm of chloroform extract of crude and 5 gm of ethanol extract of crude were obtained. 25, 26

 

Isolation of compounds from the extract 27

 

Figure 4: Schematic diagram of extraction and isolation of biologically active compound from plant.

RESULT AND DISCUSSION:

Curry leaf (Murraya koenigii) belongs to Rutaceae family and is a very important medicinal plant. The dried leaves of this plant are also used as an flvouring agent in cooking in India. It is also used in many of the Indian ayurvedic and unani medicine.

 

A variety of chemical compounds from very part of the plant have already been isolated. The major compounds responsible for its characteric features are alkaloids, glycosides, steroids, flavonoids, carbohydrates and starch. The plant is rich in carbazole alkaloid. The plant poses tonic and stomachic properties. Murrya koenigii is credited with numerous chemical compounds that interact in a complex manner to stimulate its pharmacodynamic response.

 

The human body experiences several cellular and metabolic injuries daily either due to external factors such as environmental pollutions or pathogen invasion or due to the internal metabolism. This stress is associated with the generation of reactive free radicals within the body that causes oxidation of biomolecules and thus renders weakened immune system. To combat the ill effects of free radicals in the human body, supplementation of antioxidants in a diet could be a long-term strategy to prevent life style-induced non communicable diseases such as hypertension, cardiovascular disease and cancers. Addition of fresh curry leaves in the diet of albino rat showed an alteration in peroxidation level to a remarkable extent. Thus, antioxidant activity of the plant with potent bioactives has been reported by a number of workers.

 

Preliminary phytochemical screening of Curyy leaves in different solvent extract showed the presence of alkaloids, glycosides, steroids, flavonoids, carbohydrates and starch. Phytochemical analysis of the four different extracts of Murraya koenigii revealed the presence of alkaloids, glycosides, steroids, flavonoids, carbohydrate and starch.

 

Quantification of phytochemicals:

Due to the vast differences in the nature of the phytochemical constituents found in a plant, there is no particular solvent that is known to extract all the compounds. Therefore, in this study solvents like ethanol, water, acetone and hexane are used for extraction to accommodate the range of polarities of the compounds present in Murraya koenigii leaves. The results showed that the choice of these various solvents play a crucial role in the quantitative analysis of different phytochemicals extracted from the leaves of Murraya koenigii.

 

Quantification of compounds obtained from the extract varies greatly among the four solvents, which is an indication that solvents have different extracting capacity for phytochemicals.

Preliminary phytochemical screening:

Standard screening test of the leaf extract was carried out for various plant constituents. The powder was screened for the presence or absence of secondary metabolites such as alkaloids, flavonoids, tannins, glycosides, saponins etc. using standard procedure.

 

Table 1: Standard screening tests

S. No

Chemical tests

Observation

Inference

1

Wagnor’test

+

Alkaloids present

2

Salkowski test

+

Steroids present

3

Liebermann test

+

Steroids present

4

Shinoda test

+

Flavonoids present

5

Borntragger’s test

+

Glycosides present

 

Table no. 2: Successive solvent extraction of leaves of Murraya koenigii

Solvent used

Colour and consistency

Average extractive values in % w/w in dry weight basis

Petroleum ether

Yellow green oily mass

2.20%

Benzene

Green sticky and oily mass

4.50%

Chloroform

Dark green residue

3.20%

 Acetone

Brown sticky mass

2.20%

Methanol

Dark green residue

3%

Ethanol

Dry brown mass

7.88%

 

Successive solvent extraction values in various organic solvent were observed as petroleum ether 2.21%, benzene 4.50%, chloroform 3.20%, acetone 2.20%, and methanol 3% as shown in table.

 

All extracts subjected to chemical evaluation and results were shown in table. The petroleum ether volatile oil test was positive. In benzene extract phytosterols and fixed oil were prominently seen. The chloroform extract showed presence of alkaloid only. Whereas methanol exract was found to contain alkaloid and saponin. The ethanol extract was observed with saponin and flavonoid respectively. The preliminary phytochemical studies with the help of Thin layer chromatography (TLC) method revealed that petroleum ether fraction contains volatile oil. The benzene fraction showed presence of fixed oil and alkaloid. Acetone fraction has confirmed presence of alkaloid. The fractions were further subjected to TLC in toluene: ethyl acetate (93:7) and ethyl acetate: methanol: water (76.5: 13.5: 10) solvent system with different detecting reagents for further confirmation of phytoconstituents. The alkaloid was prominently found in benzene, chloroform, acetone and methanol extract. The petroleum extract was found to contain volatile oil and fixed oil in benzene extract. The proximate analysis revealed that moisture content was 15%, total ash 73.33%, acid insoluble ash 26%, sulphated ash 80%, water soluble ash 89.5%, water insoluble ash 10.5%, acid soluble ash 74%.

 

 

Table no. 3: Chemical examination of various extracts of leaves of Murraya koenigii

Constituents

Extracts

P           B          C          A            M             E              

Alkaloids

-             -           +          +             +               -

Carbohydrates

-             -           -           -              -               -

Phytosterols

-             +           -           -              -               -

Proteins and amino acids

-             -           -           -              -               -

Saponin

-             -           -           -              +              +

Fixed oil/fat

-             +           -           -              -               -

Gums/mucilages

-             -           -           -              -               -

Flavonoids

-             -           -           +              -               +

Volatile oil

+             -           -           -              -               -

P= Petroleum ether, B= benzene, C= chloroform, A= actone, M= methnol, E= ethanol extracts


 

Table 4: TLC screening of various crude extract of leaves of Murraya koenigii

Solvent

System Used

Detection

Reagent

Observation

Inference

P

B

C

A

M

E

Ethyl

Acetate

Methanol:

Water

(75.5:13.5:10)

KOH

Red (Vis)

Yellow

Anthraquinone

Anthrone

-

-

-

-

-

-

Vanillin sulphuric acid

Red/yellow/brown/blue-green

Bitter principle

+

+

+

-

+

-

Dragendorffs reagent

Orange Red (vis)

Alkaloid

+

+

+

+

+

-

NP/PEG and UV

Yellow/green/orange

Flavonoid

-

-

-

+

-

+

 

VS reagent

Blue (vis)

Saponin

-

-

-

-

-

-

Toluene:

ethyl acetate

(93: 7)

VS reagent

Red/yellow/brown/bluegreen

Essential oil

+

+

-

-

-

-

Hcl/acetic acid

Blue brown

Valepotriate

-

-

-

-

-

-

NH3 / KOH

Light Blue brown

Coumarin

-

-

-

-

-

-

P=petroleum ether, B=benzene, C=chloroform, A=acetone, M=methanol, E=ethanol extracts

 


 

Table 5: Evaluation of leaves Murraya koenigii

Name of Analysis

Values obtained on dry weight basis (w/w)

Moisture content

15%

Total ash

73.33%

Acid insoluble ash

26%

Sulphated ash

80%

Water soluble ash

89.5%

Water insoluble ash

10.5%

Acid soluble ash

74%

 


The isolation and study of antioxidant activity of the two compounds - caryophyllene, and phellandrene isolated from Murraya koenigii are given in the following text. These two bioactive compounds are simple in structure and were characterized using FT-IR, 1H-NMR, GC, and GC-MS. These simple bioactive compounds can be used as food supplements and as natural antioxidants.

 

Structural elucidation of compounds:

Chloroform extract from Murraya koenigii:

The chloroform extract was separated using column chromatography with silica gel as packing material, and hexane as a solvent. The polarity of the solvent was increased step up step using hexane and acetone. The first two fractions of the extract were obtained using difference in polarity of the eluent. The compound was isolated using a mixture of 99.5% hexane and 0.5% acetone. Compound were analysed by FTIR, GC, GC MS, as described below.

 

Compound from chloroform extract:

IR spectrum (ѵ, cm-1) of compound revealed bands at 719, 813, 927.79 cm-1, 1020, 1055, 1107 cm-1 (-C=C-mono subs), 1278, 1303, 1363, 1380 cm-1 (-C-C- str), 1460, 1514 cm-1 (H-C-H bend), 2869, 2925, 2960, 3016, 3049 cm-1 (H-C-H Asy str). The chloroform extract of compound was tested using a GC and the compound yielded a single peak confirming the presence of only one compound. The retention time was 22.5 min., with 98.74% purity. The spectral data of compound was found to be in good agreement with the reported value for caryophyllene. In the 1H-NMR spectrum of compound, the peaks appearing at 1.29 and 1.96 indicate the presence of the CH2 group in the molecule. The peak appearing at 2.73 shows the presence of a cyclobutane linkage between the five-membered rings and the six-membered rings. The peaks appearing in the range of 4.75 and 5.25 are due to presence of ethylene linkage.

 

GC-MS studies revealed a sharp peak with a retention time of 8.4 minutes, with m/z value of 221 which corresponds to the molecular weight of caryophyllene. From the GC-MS and 1H-NMR data we conclude that the isolated compound is caryophyllene, whose structure is given below in Figure.

 

 

Figure 5: Structure of caryophyllene

 


 

 

A

 

B

 

 

C

Figure 6: Characterization of caryophyllene isolated from Murraya koenigii leaf using chloroform as solvent

A=FTIR; B=GC; C=GC MS

 


Ethanol extract from Murrya koenigii:

UV spectrum of the ethanol extract of the Murraya koenigii leaf revealed the presence of eight peaks at 671, 610, 510, 490, 417, 298, 263.4 and 223.8 nm, corresponding to eight different compounds. Of these, we were interested in studying only the one major compound whose absorption peaks were at 263.4 nm.

 

The extract was separated using a silica gel column, employing hexane as the eluent initially. The polarity of the solvent was increased gradually using a mixture of hexane and acetone. The first two fractions of the extract were obtained using difference in polarity of the eluent. Column chromatography was continued using hexane: acetone (95:5 v/v) mixture for the isolation of the compound. On UV spectrophotometery, the compound exhibited an absorption maximum of 263.4 nm.

 

Compound from ethanol extract:

The ethanol extract of compound gave a single peak on analysis with GC, confirming the presence of a single compound with a retention time of 5.916 minutes and 97.44% purity. IR spectrum (ѵ, cm-1) of compound revealed bands at: 927.79 cm-1, 1058 cm-1 (-C=C-mono subs), and 1126 cm-1 (-C-C- str).

 

In 1H-NMR spectrum of compound, the peaks at 1.50 clearly showed the presence of the CH group in the molecule. The peak around 2.15 indicated the presence of the CH group of an aliphatic molecule. The peak around 3.93 in the spectrum indicated the presence of a C-C linkage between the five-membered ring and a six-membered ring.

 

The peak at 7.32 was due to the presence of an aromatic ring in the compound.

 

GC-MS studies revealed a sharp peak with an m/z value of which corresponds to the molecular weight of phellandrene. The NMR and GC-MS data taken together confirms that the compound is phellendrene. The GC, FTIR, NMR and GC MS spectra are shown in figure.

 

 

Figure 7: Chemical structure of phellandrene


 

 

A

 

 

B

 

C

 

D

Figure 8: Characterization of phellendrene isolated from Murraya koenigii leaf using ethanol as solvent

A= FTIR; B= GC; C=1H NMR; D=GC MS

 


CONCLUSION:

IR spectrum (ѵ, cm-1) of chloroform extract revealed bands at 719, 813, 927.79 cm-1, 1020, 1055, 1107 cm-1 (-C=C-mono subs), 1278, 1303, 1363, 1380 cm-1 (-C-C- str), 1460, 1514 cm-1 (H-C-H bend), 2869, 2925, 2960, 3016, 3049 cm-1 (H-C-H Asy str). The chloroform extract was tested using a GC and the compound yielded a single peak confirming the presence of only one compound. The retention time was 22.5 min., with 98.74% purity. The spectral data of chloroform extract was found to be in good agreement with the reported value for caryophyllene. In the 1H-NMR spectrum of chloroform extract, the peaks appearing at 1.29 and 1.96 indicate the presence of the CH2 group in the molecule. The peak appearing at 2.73 shows the presence of a cyclobutane linkage between the five-membered rings and the six-membered rings. The peaks appearing in the range of 4.75 and 5.25 are due to presence of ethylene linkage.28

 

GC-MS studies revealed a sharp peak with a retention time of 8.4 minutes, with m/z value of 221 which corresponds to the molecular weight of caryophyllene. From the GC-MS and 1H-NMR data we conclude that the isolated compound is caryophyllene.

 

The ethanol extract of compound gave a single peak on analysis with GC, confirming the presence of a single compound with a retention time of 5.916 minutes and 97.44% purity.

 

IR spectrum (ѵ, cm-1) of compound revealed bands at: 927.79 cm-1, 1058 cm-1 (-C=C-mono subs), and 1126  cm-1 (-C-C- str). 29

 

In 1H-NMR spectrum of compound, the peaks at 1.50 clearly showed the presence of the CH group in the molecule. The peak around 2.15 indicated the presence of the CH group of an aliphatic molecule. The peak around 3.93 in the spectrum indicated the presence of a C-C linkage between the five-membered ring and a six-membered ring.

 

The peak at 7.32 was due to the presence of an aromatic ring in the compound.

 

GC-MS studies revealed a sharp peak with an m/z value of which corresponds to the molecular weight of phellandrene. The NMR and GC-MS data taken together confirms that the compound is phellendrene. 30

 

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Received on 13.05.2019        Accepted on 11.06.2019

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 2019; 9(3):159-168.

DOI: 10.5958/2231-5691.2019.00025.X