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ISOLATION, CHARACTERIZATION AND ANTI TUBERCULAR SCREENING OF MAJOR PHYTOCHEMICAL MARKER FROM ORANGE PEEL


Manish Kashyap1, Sonal Dubey1*, Prabitha P1 and D.R. Harish Kumar2
1. Krupanidhi College of Pharmacy, Chikkabellandur, Carmelaram Post, Bangalore, India
2. Faculty of Pharmacy, M.S. Ramaiah University of Applied Sciences, Bangalore, India

Abstract

Current advancement in drug discovery technology and search for novel chemical diversity have intensified the efforts for exploring lead from “Ayurveda” the traditional system of medicine in India. Orange (Citrus sinensis) is an herbaceous member of the family Rutaceae. These are native to tropical and subtropical regions of India, China, Northern Australia and New Caledonia. Clinical trials and animal research support the use of orange peel for antityphoid, antidiabetic, anticarcinogenic, antiproliferative, antioxidant, antibiotic and antifungal activity. The major biochemical constituents of orange peel are identified as N-decylaldehyde-methyl ester of anthranilic acid, polymethoxy flavones, nobelitin, limonene, hesperidine etc. In our present investigation we have isolated  major phytochemical markers from methanolic extracts of peel of orange and characterized the isolated phytochemical markers by analytical methods. Antitubercular activity of the marker (OPE-1) obtained from the methanolic extract of Citrus sinensis peel was evaluated against Mycobacterium tuberculosis by ALAMAR blue assay method (MABA), using different solvent at varying concentration. Only water soluble fractions showed significant sensitivity.

Key words: Citrus sinensis peel, Column chromatography, Antitubercular activity.

Introduction

Citrus sinensis is a shrub that is subtropical rather than a tropical species. It has been used traditionally in Ayurveda, Unani and Siddha system of medicines since a long time. The various literature surveys related to orange plant show that the major compound which can be used for quality control, are not highlighted and there is a lack of analytical methods for them1. The literature surveys also indicate that there is also possibility of isolating new compounds from various parts of the fruits of citrus plant. Hence it was decided to carry out further research work in the field of isolation and standardization of chemical constituents from the peel of orange plant. Here in the present study attempts have been made to isolate and characterize the constituents which are present in Citrus sinensis peel 2, 3.  It is an important drug of Indian system of medicine and is being used since time immemorial. During the last two decades, the plant has been subjected to extensive phytochemical, pharmacological and clinical investigations

Fig1: Citrus sinensis plant

and many interesting findings have been reported in various fields. It has a long history of uses by indigenous and tribal people and in Ayurveda or natural herbal system of medicine 4, 5 

Distribution

The plant is indigenous to India, but it is also cultivated in south Spain, Caribbean Island, U.S.A., and New Zealand. In India it is grown in Maharashtra, Andhra Pradesh, Punjab, West Bengal and Kashmir.

Scientific classification

Kingdom – Plantae

Subkingdom – Tracheobionta

Division – Magnoliophyta

Class – Magnoliopsida

Subclass – Rosidae

Order – Sapindales

Family – Rutaceae

Genus – Citrus

Species – sinensis

Chemical composition

The peel of Citrus sinensis constitutes volatile oil in numerous schizo-lysigenous oil glands. They have glycosides mainly flavones glycosides; the most prevalent flavones present in Citrus sinensis are hesperidin, naringin, tangeretin, and nobelitin6. It also contains polymethoxyflavones (PMFs), hydroxylated polymethoxyflavones, hydroxylated polymethoxychalcones, along with monoterpines, sesquiterpines, gibberellic acid, phytol, amyrin, nomilin derivatives, carotinoids, alkaloids, brassinolides, castasterone, sitosterol, hydroquinone, vitamin C and pectin 7-10.

Materials & method

Collection of plant material

The dried and authenticated peels of Citrus sinensis were obtained from Green Chem Pvt. Ltd, Bangalore.

Extraction & selection of solvent

The shade dried peels obtained from Citrus sinensis were subjected to successive extraction using hexane, chloroform, ethyl acetate and methanol in an extractor. The process was first performed in small scale to find out the yield then taken for pilot scale.

Since methanolic extract of orange peel had shown good yield, it indicated the possibility of isolating new compounds as a marker; hence methanol was selected for lab scale and pilot scale extraction.

Requirements:

Fractionation

Stationary phase: Silica gel (60-200 mesh).

Mobile Phase: hexane, ethyl acetate, chloroform, methanol

HPLC Analysis

Column: Phenomenex, C-18, 250 x 21.2 mm.

Mobile Phase:  Pump A- Water + H3PO4 100 μl

Pump B- Acetonitrile

Elution: Gradient.

Flow rate: 1.5 ml/min.

Injection vol: 20 μl

Detector: SPD-M10 Photoiodide detector at 280 nm

Fractionation and isolation of methanolic extract of Citrus sinensis  peel

Procedure: The column was charged with methanolic extract of Citrus sinensis  peel and first eluted with 100% pet ether. The polarity of mobile phase was gradually increased to chloroform; ethyl acetate and then methanol. The fractionations done from the column is given in the flowchart (Fig 2).

Fig 2 : Fractionation of methanolic extract of Citrus sinensis

Similar spots were obtained in good yield in the fractions 6,7, 8 using the solvent pet ether and chloroform in varying concentration. These three fractions were combined with a yield of 27.2 g orange peel exracts (OPE-1).

Quantitative Analysis of Isolated Compound in Methanolic Extract by HPLC

Sample preparation – 50.7 mg of OPE-1 of Citrus sinensis was weighed and dissolved separately in 25 ml of HPLC grade methanol with the help of sonicator.

Standard preparation – The standard for the estimation of these extract was weighed 6.7 mg dissolved in 25 ml HPLC grade methanol.

The peaks were observed in methanolic extracts in the similar retention time corresponding to the values as observed in analytical HPLC of OPE-1. It confirms the presence of the compound in the methanolic extract of Citrus sinensis thus, the process can be said as standardized. The assay can be estimated using formula:

In the HPLC chromatogram of the methanolic extract the area of OPE-1 was found to be 1329163. By calculating according to the formula given OPE-1 present in methanolic extract was found to be 2.34%.

Evaluation of Anti-tubercular activity of methanolic extract of peel of Citrus sinensis

The anti-mycobacterium activity of the methanolic extract was assessed against Mycobacterium tuberculosis using Alamar Blue Assay (MABA) method. This methodology is non-toxic, uses a thermally stable reagent and shows correlation with proportional and BACTEC radiometric method. 

Procedure

Briefly, 200 μl of sterile deionised water was added to all outer perimeter wells of sterile 96 well plates to minimize evaporation of medium in the test wells during incubation. The 96 well plates received 100 μl of the Middlebrook 7 H9 broth and serial dilution of compound were made directly on plate. The final drug concentrations tested were 100 μg /ml to 0.8 μg /ml. Plates were covered and sealed with paraffin and incubated at 37oC for five days. After this, 25 μl of freshly prepared 1:1 mixture of Alamar Blue reagent and 10% tween 80 was added to the plate and incubated for 24 hrs. Here MIC is defined as the lowest drug concentration which prevents the colour change from blue to pink.

RESULTS & DISCUSSION

The phytoconstituents from methanolic extract of Citrus sinensis peel gave positive results for the following tests:

  1. Carbohydrates ( Molisch’s test, Fehling’s test, Benedict’s test, Barfoed’s test)
  2. Alkaloids(Dragendroff’s test, Wagner’s test, Mayer’s test, Hager’s test)
  3. Sterols & steroids (Libermann Burchard test, Salkowski test)
  4. Glycosides ( Legal’s test, Baljet test, Borntrager test, killer killani test)
  5. Saponins ( Foam test, Shinoda test, lead acetate test)

 TLC of the isolated compound OPE-1 is given in  Fig 3. The TLC was carried out using the solvent system toluene: ethyl acetate: formic acid in the ratio 7: 3: 0.5

A-OPE-1

B– Orange peel extract

Fig 3: Visualization UV 366 nm

The HPLC chromatograms for OPE-1 are shown in Fig 4 & Fig 5 and the results are summarized under the Table 1. 

Fig 4: HPLC Chromatogram for OPE-1

Fig 5: HPLC Chromatogram for methanolic extract of Citrus sinensis peel

Fig 6: Structure of OPE-1 

SPECTROSCOPIC ANALYSIS OF THE ISOLATED COMPOUNDS

All the spectroscopic data obtained for the compound OPE-1 suggested that the structure of the compound may be the following:

IR Spectra: 33475 cm-1(O-H stretch), C-H (aliphatic stretch) 2919 cm-1, 1646 cm-1(C=O stretch), 1444 cm– 1(C=C stretch), 1204 cm-1(C-O stretch).

Mass peaks: The mass spectra of compound displayed a molecular ion peak at m/z 303.4.

1H-NMR: δ 12 (2H, s, OH), 9.05(1H, s, Ar), 6.94-6.88(2H, m, Ar), 6.14-6.11(1H, s, OH), 3.35(s,1H,OH),1.08(3H, d, J=6 Hz).

13C-NMR: δ 17.78(s), 55.66(s), 66.01(s), 68.27(s), 69.56(s), 70.67(s), 72.03(s), 72.95(s), 75.49(s), 76.24(s), 78.33(s), 95.50(s), 96.34(s), 99.42(s), 100.57(s), 103.28(s), 112.04(s), 114.11(s), 117.89(s), 130.88(s), 146.43(s), 147.93(s), 162.46(s), 163.00(s), 165.10(s), 196.97(s).

Table 1: HPLC peak results for methanolic extract of Citrus sinensis peel

Evaluation of antitubercular activity of methanolic extract of Citrus sinensis

The anti mycobacterium activity of the methanolic extract of Citrus sinensis peel was assessed against Mycobacterium tuberculosis by Alamar Blue Assay (MABA) method. The results obtained are given in Table 2.

Table 2: Antitubercular activity results of methanolic extract of  Citrus sinensis  peel  

  Final drug concentration(µg/ml)
S.No Solvents 100 50 25 12.5 6.25 3.125 1.6 0.8
1 Water S S R R R R R R
2 Methanol S R R R R R R R
3 Ethanol S R R R R R R R
4 Chloroform S R R R R R R R
5 Diethyl ether S R R R R R R R

R- Resistance, S- Sensitive 

Higher concentration of 100 μg/ml all the solvent showed sensitivity against Mycobacterium tuberculosis while lowering the concentration shows resistance by the bacteria. Water soluble portion showed significant activity at 50 μg/ml.

CONCLUSION

The peels of Citrus sinensis fruit belonging to the family Rutaceae were selected for isolation of phytoconstituents. The targeted marker OPE-1 was isolated from methanolic extract using column chromatography. HPLC method was developed for the assessment of the purity and this was found simple and accurate. The isolated purified compound was characterized by determining its physical properties like solubility, melting point and by subjecting it to NMR (1H and 13C), mass spectroscopy and IR spectroscopy. From the spectral data, the isolated compound OPE-1 was identified as (2R, 3R)-2-(3, 4-dihydroxyphenyl)-3,7-dihydroxy-4-oxo-2,3-dihydrochromen-5-olate.  In this work we concluded that the column chromatography method employed for isolation of OPE-1 was found to be suitable for commercial scale. The isolated compound was present in sufficient quantity in the methanolic extract of Citrus sinensis peel. It is suitable to call it as marker.

The methanolic extract of peels of Citrus sinensis was evaluated for antitubercular activity against Mycobacterium tuberculosis by ALAMAR blue (MABA) method in different solvents with varying concentration. Highest concentration of all the dried extract (100 μg/ml) in all the solvent showed sensitivity against Mycobacterium tuberculosis while lowering the concentration shows resistance by the bacteria. Only water as the solvent showed the sensitivity of upto 50 μg/ml.

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