International Standard Serial Number (ISSN): 2319-8141 .... RPA1300120.pdf · International...

International Standard Serial Number (ISSN): 2319-8141 International Journal of Universal Pharmacy and Bio Sciences 2(3): May-June 2013

INTERNATIONAL JOURNAL OF UNIVERSAL

PHARMACY AND BIO SCIENCES

Pharmaceutical Sciences Research Article……!!!

Received: 19-06-2013; Accepted: 25-06-2013

EXTRACTION AND SCREENING OF TRIMYRISTIN IN THE SEEDS OF

MYRISTICA FRAGNANS AND IN POLY HERBAL FORMULATIONS BY

SPECTROSCOPIC AND CHROMATOGRAPHIC TECHNIQUES

N. Hima Sai Bindu*, S. Rubesh Kumar, N. Duganath, N. Devanna

Department of Pharmaceutical Analysis, JNTUA - Oil Technological Research

Institute, Anantapur -515001, Andhra Pradesh, India.

KEYWORDS:

Trimyristin, API,

Solvent effect,

Poweromin.

For Correspondence:

N. Hima Sai Bindu*

Address: Department of

Pharmaceutical Analysis,

JNTUA - Oil

Technological Research

Institute, Anantapur,

Andhra Pradesh,

India-515001.

Email:

[email protected]

Mobile No: 8121900898

ABSTRACT

Nutmeg a well known spice in India is biologically termed as

Myristica fragnans Houtt. Apart from its use as a spice the plant has

several uses in the traditional alternative medicine mostly in

ayurvedic system. The components of the herbal plant such

trimyristin; myristicin and safrole are responsible for the various

pharmacological activities. The triglyceride Trimyristin which was

responsible for the anxiogenic activity was the present target of the

study. The drug Trimyristin was characterized by spectroscopic

methods like IR, UV and Thin layer chromatographic studies

including the solubility of the drug in various solvents. By the UV

studies the solvent effect of the drug was studied in various solvent.

A Standardized TLC procedure has been established for the

screening of the drug in plant extract and in polyherbal formulations

using the solvent system Dichloromethane: chloroform: ammonia

(6.5:3.5:0.1) with Rf value of 0.55, 0.564, 0.552 for API, extract and

polyherbal dosage form Poweromin. A Reverse phase UFLC method

developed using the mobile phase dichloromethane and acetonitrile

(99:5 v/v) showed retentions times of 0.904 and 0.906 for both the

API and extract.

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International Standard Serial Number (ISSN): 2319-8141

1. INTRODUCTION:

Myristica fragrans Houtt. commonly known as Jaiphal and Javitri in India, belongs to the

family Myristicaceae. It plant mainly has two spices, nutmeg seed and mace [1]. Nutmeg is the

seed kernel inside the fruit and mace is the fleshy red, net like skin covering (aril) on the kernel.

The most important part of the plant in terms of its pharmacological activity is the dried kernel

(seed), the nutmeg. The oil of nutmeg has been used for several medicinal as it contains the

pharmacologically active components. It is also used as a spice in various dishes, as

components of tea and soft drinks or mixed in milk and alcohol [2]. In traditional medicine

nutmeg is used as a stomachic, stimulant, carminative as well as for intestinal catarrh and colic,

to stimulate appetite, to control flatulence and it has a reputation.

The main constituents of M. fragrans have been found to be alkyl benzene derivatives

(myristicin, elemicin, safrole etc.), terpenes, alpha-pinene, beta-pinene, myristic acid and

triacylglycerols like trimyristin. Nutmeg contains about 10% essential oil, which is mostly

composed of terpene hydrocarbons (sabinene and pinenes; furthermore camphene, p-cymene,

phellandrene, terpinene, limonene, myrcene, together 60 to 80%), terpene derivatives (linalool,

geraniol, terpineol, together 5 to 15%) and phenylpropanoids (myristicin, elemicin, safrole,

eugenol and eugenol derivatives, together 15 to 20%) [3-5]. As earlier reported the essential oil

part of nutmeg was believed to be responsible for all the major pharmacological activity of the

drug but the fact is the major portion of the seed of the nutmeg yields fixed oil (60%) which

consists of a triglyceride named Trimyristin which was reported to have anti-depressant

activity, anti-inflammatory and anti-bacterial activity[6]. This is the principle need of using the

herbal drug in several poly herbal formulations. Trimyristin is an example of fundamental type

of fat known as a triglyceride. Hydrolysis of one mole of a triglyceride afford one mole of 1, 2,

3-propanetriol (glycerin) and 3 moles of fatty acids, which are carboxylic acids containing the

functional group at the end of a long alkyl chain. Chemically Trimyristin is called as 1,3Di

(tetradecanolyloxy) propan-2-yl tetradecanoate [7](Figure 1).

Figure 1: Chemical structures of Trimyristin


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The literature review reveals no analytical method for the identification of Trimyristin in API

and in seed extract. The previous reported works includes the quantification of the Trimyristin

by HPLC, in various vegetable oils [8]. Apart from trimyristin there were several works

reported on nutmeg which includes the isolation of other constituents like amino acids [9],

myristicin, safrole[10,11] etc along with the standardization of the whole herbal drug in unani

medicine. There were also reviews which explained the various pharmacological activities and

fatal activity of the drug [12-13]. So the present study was aimed to extract the active

pharmaceutical ingredient Trimyristin from the seed of nutmeg and to study solubility and

solvents effects in various solvents along with identification of Trimyristin by spectroscopic

and TLC method.

MATERIALS AND METHODS:

Plant material:

The seeds of Myristica fragrans were collected from the local drug store. The seeds were

crushed to fine powder and stored at 250C.

Chemicals:

Standard Trimyristin was obtained from Himedia (RM 1301-1G), India. Chloroform,

Dichloromethane, Ammonia were purchased from Merck Chemicals Ltd, SDFCL SD Fine

Chem. Limited and Mio Chem. Pvt. Ltd respectively. All other chemicals and reagents were of

HPLC grade. The polyherbal tablets Poweromin (Apex Laboratories Pvt Ltd) was obtained as

gift sample from the local ayurvedic store.

Instrumentation:

BRUKER FTIR –IFS 125 spectrometer was used for the scrutinization of the extract and API of

Trimyristin. ELICO-SL 244 UV / VIS Double beam spectrophotometer, (spectratreats) with

PMT detector, Shimadzu LC 20 AD UFLC system with 20 µL loop, LC 20 AD pump, SPD-

20A Photo- Diode array detector. Waters C8 (350µm, 4.6 x 150mm) column was used in the

analysis. LC solutions software was utilized for instrument control, data collection and data

processing using a Pentium (R) Dual –Core Processor was used for the estimation of trimyristin

in the API and extract. In addition, an electronic balance (Shimadzu AX200), an ultra sonicator

(Spincotech, model2200-MH SOLTECH), a hot air oven (Labhosp), UV chamber (Labhosp)

were used in this study.

Extraction of Trimyristin:

Crushed whole nutmeg seeds of 5 gms was weighed and transferred to a 50mL round-bottomed

flask. 50mL of hexane was added to the flask which was attached to a water-cooled condenser,

the mixture was heated under a gentle reflux for approximately 4 hrs at 60oC. The mixture was




cooled to room temperature. The hexane layer was separated out with pipette without disturbing

the settled solids. The hexane layer was evaporated by using rotavapour apparatus which on

evaporation yields a white crystalline powder. The crude extract was recrystallised by using

95% ethanol to yield pure trimyristin. Apart from the reflux method, the maceration method

was also tried for the extraction of the drug. An accurately weighed quantity of 10 Gms

powered material was taken in a 250 ml closed beaker and hexane was added until all the

powered material gets totally sinked in the solvent. The solution was macerated for a period 7

days with a frequent addition of 30 ml hexane in alternate days. After 7 days the supernatant

layer of the solution was carefully withdrawn with a pipette and transferred to a beaker and

evaporated until it yields white crystalline solid. The crude Trimyristin was recrystallized with

95% ethanol to obtain the pure form. This extracted trimyristin was characterized by IR, UV

and TLC.

SPECTRAL ANALYSIS:

IR spectral studies:

The extracted Trimyristin was confirmed by FTIR analysis using KBR. In this study, the IR

spectra of the isolated sample of Trimyristin were analyzed and compared with the IR spectrum

of standard Trimyristin.

UV- Visible Spectroscopic studies:

The spectroscopic studies were performed with API. A weighed quantity of 10 mg API was

dissolved in different solvents separately to a concentration of 10µg/ml and then scanned from

190-800 nm. The different solvents used in the study are acetone, acetonitrile, chloroform,

dichloromethane, Dimethyl formamide, hexane, isopropyl alcohol, tetrahydrofuran etc(Table 1).

These solvents were also used to study the complete solubility profile of the drug. Apart from

the single solvent system the co-solvency method was also tried using the solvents

dichloromethane: isopropyl alcohol.

TLC STUDIES:

Slide preparation:

TLC plates were prepared by mixing the adsorbent Silica gel G, with a small amount of inert

binder calcium sulphate (gypsum) and water. This mixture was spread as thick slurry on a glass

slide of dimensions 10 x 5 cm. The thickness of the adsorbent layer is typically around 0.1-

0.25mm. The plate was dried and activated by heating in an oven for thirty minutes at 110 .

Preparation of mobile phase:

Mobile phases were selected basing on polarity of solvents. Different mixtures of mobile phases




of different composition were prepared and used for elution of TLC slides. Accurate volumes of

the solvent were measured using a measuring cylinder and kept for saturation for at least 30

minutes by covering the TLC chamber with a lid.

Preparation of Standard Solutions:

Stock solution of standard was prepared in dichloromethane to a concentration of 1000µg/ml.

This solution was further diluted to a concentration of 100µg/ml with the same solvent.

Preparation of extract and Tablet Solutions:

An accurately weighed quantity of the recrystallised Trimyristin was dissolved in

dichloromethane to a concentration of 1000µg/ml and further diluted to a concentration of 100

µg/ml. A weighed quantity of 5mg tablet was powered and dissolved in dichloromethane by

sonicating for 30 minutes at 600C. This solution was filtered through Whatmann filter paper and

used for the spotting.

Application of sample:

Sample solutions of the drug and reference standard are spotted on TLC slide at a distance 1cm

from the bottom of slide using a capillary tube..

Development of slides:

The TLC plates were developed in TLC chamber. The composition of mobile phase was

optimized by using different solvents of varying polarity. The TLC runs were made under

laboratory conditions of 25 ±50 and 50% relative humidity. After development, the plate was

removed and dried and kept in Iodine chamber. The brown spots indicates the presence of

Trimyristin, the Rf value was calculated.

UFLC analysis:

An isocratic Chromatographic separation was developed by using UFLC Shimadzu LC 20 AD

, column used was Pre-packed Waters Symmetry C8, 250 x 4.6mm, 5µm. The mobile phase

system consists of Dichloromethane: Acetonitrile (95:5 v/v), with a flow rate of 1.0 ml/min and

injection volume was 20 l with a vacuum degasser and PDA detector. LC solutions software

was utilized for instrument control, data collection and data processing.

Preparation of standard and extract stock solution:

An accurately weighed quantity of 10mg API and extract was transferred into two separate

10ml volumetric flask and dissolved in dichloromethane to 10ml to give a concentration of

1000µg/ml. This solution was further diluted to a concentration of 10 µg/ml.

RESULTS AND DISCUSSION:

When the two extracting methods were compared the maceration of the drug for seven yielded

more components than the reflux method. But when coming to the extraction of Trimyristin




yield was more in the case of reflux method with hexane. The percentage yields of reflux and

maceration method with hexane was 22% and 14.3% respectively.

IR Spectroscopic studies:

The extract and the API of Trimyristin were characterized using FTIR Spectrometer. The

isolated fractions gave IR spectra similar to that of the standard Trimyristin. Thus the results

indicated a strong similarity between both the standard and extracted compound. The API and

extracted compound showed the similar bands at 2900 -2700 cm-1 (

C-H stretching), 1729 cm-1

(C=O stretching vibration of ester), 1388 cm-1

( symmetrical deformations of CH3 group),

1226cm-1

, 1172 cm-1

, 1111 cm-1

( asymmetrical stretching of ester C-O-C, common to all long

chain triglycerides of long chain fatty acids), 1035 cm-1

(symmetrical stretching of ester C-O-

C), 714 cm-1

(wagging of CH2 group)(Figure 2).

Figure 2: IR Spectrum of Standard showing the characteristic peaks

Figure 3: IR Spectrum of Sample showing the characteristic peaks

UV -Visible Spectroscopic studies:

Before performing the UV-Visible spectroscopic studies the solubility of the drug in various

solvents has been tested (Table 1).The λmax of the API and extracted Trimyristin was

determined in different solvents using double beam UV -Visible Spectroscopy. The change of




maximum wavelength was observed with the change of solvent. This was due to the solvent

effect that is the interaction of the drug with the solvent system. These interactions were due to

the hydrogen bonding of the oxygen in the drug with the solvent molecules. In the present study

there was a shift of λmax towards the left (blue shift) and right (red shift) of the spectrum. This

is because in the solvent with higher polarity or dielectric constant if the ground state molecule

is better stabilized than the excited state this results in the blue shift and it is vice versa, if the

excited state molecule is better stabilized it results in red shift. This shift is mainly based on the

hydrogen bonding of the molecule; it may be either intermolecular hydrogen bonding or intra-

molecular hydrogen bonding. When it is intermolecular hydrogen bonding the energy shift is

more which makes the excited state molecule stable indicating the bathochromic shift where as

the intermolecular hydrogen bonding with less polar solvent or intra-molecular hydrogen

bonding makes the ground state molecule stable resulting in hypsochromic shift. Due to the

shifts the drug showed different λmax in different solvents. The solvents in which the drug

showed prominent peaks are mentioned in (Table 2) (Figure 4-12).

Table 1: Showing the solubility of the drug in various solvents.

SOLVENT SOLUBILITY SOLVENT SOLUBILITY

Acetonitrile Soluble On Heating Isopropanol Soluble

Acetone Soluble Methanol Insoluble

1- Butanol Soluble Petroleum Ether Soluble

Chloroform Soluble Tetrahydrofuran Soluble

Dichloromethane Soluble DMSO Soluble

Diethyl Ether Soluble Cyclo Hexane Soluble

Dimethyl Formamide Soluble On Heating 0.1 N H2SO4 Insoluble

Ethanol Soluble On Heating 0.1N NaOH Insoluble

Hexane Soluble Water Insoluble




Table 2: Showing the λmax of the drug in various solvents

S.No Solvent λmax(nm)

1 Acetone 262

2 Acetonitrile 206, 277

3 Chloroform 232, 278

4 Dichloromethane 229.5

5 Dimethyl formamide 266.5

6 Hexane 279.5

7 Isopropyl alcohol 211.5

8 Tetrahydrofuran 214.5

9 Dichloromethane: isopropyl alcohol (2:8) 222

Figure 4: Spectrum of Trimyristin API in Acetonitrile

Figure 5: Spectrum of Trimyristin API in Acetone




Figure 6: Spectrum of Trimyristin API in Chloroform

Figure 7: Spectrum of Trimyristin API in Dichloromethane

Figure 8: Spectrum of Trimyristin API in Dimethyl formamide




Figure 9: Spectrum of Trimyristin API in Isopropyl alcohol

Figure 10: Spectrum of Trimyristin API in Hexane

Figure 11: Spectrum of Trimyristin API in Tetrahydrofuran

Figure 12: Spectrum of Trimyristin API in Dichloromethane: Isopropyl alcohol




TLC Studies:

The TLC studies for the poly herbal formulation, API and extract were performed in different

solvent system. Among them the suitable solvent system that can be used for the identification

of the drug Trimyristin consists of chloroform: dichloromethane: ammonia in the ratio of

6.5:3.5:0.1. The spots were visualized using Iodine chamber. The Rf value of the drug was

found to be 0.55. A few different solvent systems used were diethyl ether: chloroform: acetic

acid (4:4:2), acetone: acetic acid: dichloromethane (2:2:6), chloroform: ethyl acetate:

dichloromethane (6:2:2), dichloromethane: chloroform (5:5) (Figure 13).

A B C D E

Figure 13: TLC slides showing the spots of Trimyristin

S:Standard API, T: Tablet, E: Extract

A: Dichloromethane: chloroform: ammonia (6.5:3.5:0.1), B: Dichloromethane: chloroform

(5:5), C: Diethyl ether: chloroform: acetic acid (4:4:2), D: Acetone: acetic acid:

dichloromethane (2:2:6), E: Chloroform: ethyl acetate: dichloromethane (6:2:2).

UFLC Analysis:

An isocratic Chromatographic separation was developed by using UFLC Shimadzu LC 20 AD,

column used was Pre-packed Waters Symmetry C8, 250 x 4.6mm, 5µm. The mobile phase

system consists of Dichloromethane: Acetonitrile (95:5 v/v), with a flow rate of 1.0 ml/min and

injection volume was 20 l. The system was injected with standard and extracted Trimyristin.

The retention time of Trimyristin for standard and extract was found to be 0.904minutes, 0.906

minutes respectively (Figure 14a, 14b).




Figure 14a, 14b: Showing the chromatogram of Trimyristin API and extract respectively.

CONCLUSION:

The developed analytical methods are simple and can be used for the screening of Trimyristin

from the extract and in polyherbal formulations. These spectroscopic and chromatographic

methods are precise and cost effective. The solubility profile, UV spectroscopic and UFLC

characterization has an impetus in the further studies of analytical method development for the

drug by various analytical techniques.

ACKNOWLEDGEMENTS:

The authors are thankful to Mr. Dr. N.M. Dakshina Murthy, Rural Medical Officer, Anantapur,

for providing the physician samples of poly herbal formulation Poweromin.

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