Solicitation of HPLC and HPTLC Techniques for Determination of Rutin from Polyalthia longifolia Thwaites.

BACKGROUND: Polyalthia longifolia Thwaites is an important traditional plant in India. Rutin, an active constituent has been reported to possess good amount of pharmacological as well as therapeutic potential.
OBJECTIVE: The aim of the present study was to find out by analytical techniques how much percentage of rutin is present in the plant leaves' ethanolic extract by analytical techniques.
MATERIALS AND METHODS: Shade dried leaves of Polyalthia longifolia were subjected to cold ethanolic extraction followed by monitoring the isolated rutin high-pressure liquid chromatography (HPLC) and high performance thin layer chromatography (HPTLC) after carrying out preliminary phytochemical screening.
RESULTS: Extraction yield was found to be 13.94% w/w. Phytochemical screening of the extract showed the presence of flavonoids, steroids, diterpenoids, alkaloids, saponins, tannins and phenolic compounds and mucilage. From the Rf value, the ethanolic extract was found to be having constituent identical to rutin. By HPTLC and HPLC the amount of rutin was found to be 11.60% w/w and 4.03% w/v, respectively.
CONCLUSION: The active constituent isolated was found to be equal to rutin.

INTRODUCTION

In an agricultural country like India, where medicinal plants are abundantly and easily available at relatively low cost, there is every virtue in exploiting such local and traditional medicine of vegetable origin for human and animal use. India has the knowledge and skill to develop its research and development capabilities in herbal research.[123] In recent times, focus on plant research has increased all over the world and evidence has been collected to show immense potential of medicinal and local plant utility traditional systems of herbal medicines.[45]

Polyalthia longifolia Sonn. Thwaites (Order: Magnoliales; Family: Annonaceae) is an evergreen, tall, handsome plant commonly used as an ornamental street tree due to its effectiveness in combating noise pollution. Polyalthia is the Greek word for poly, meaning much or many and althia from althea, meaning to cure. It is large shrub of genes including 120 species. India has 14 species of Polyalthia. Among the several species of Polyalthia longifolia cv. Pendula grown in India is mostly used in indigenous medicine, whereas, Polyalthia longifolia var. Pendula is used mostly in traditional medicine. Leaves are aromatic and used as essential oils.[67]

Various chemical constituents identified from the plant leaves are namely, azafluorene alkaloid namely polylongine and three new Aporphine N-oxide alkaloids. In addition, leaf oil has been reported to contain a vast number of sesquiterpenes such as allo-aromadendrene, caryophyllene oxide, β-caryophyllene, β-selinene, α-humulene, α-pinene and camphene. Activity guided fractionation of ethanolic extract has revealed the presence of flavonoid components in the extract such as quercetin, quercetin-3-o-β-glucopyranoside, kaemperfol-3-o-α-rhamnopyranosyl-β-glucopyranoside, kaempferol-3-o-α-rhamnopyranosyl-(1-6)-β-glucopyranoside, rutin and allantoin from butanol fraction. Ethanolic extract has found to contain bulbocapnin, steroids such as β-sitosterol, stigmasterol and campesterol constituents.[89101112]

MATERIALS AND METHODS

Collection, authentication and extraction

Fresh leaves of Polyalthia longifolia (Asapalav) were collected from Mumbai local market in month of April-May and shade-dried. The leaves were authenticated by Agarkhar Research Institute, Pune. A voucher specimen (No. 3/187/2013/Adm. 1692/080) was deposited in the botany department of Agharkar Research Institute, Pune. Further, they were subjected to cold extraction procedure[13] as follows:

Step 1: To 600 gm of leaves powder, 5 L 70% ethanol was added

Step 2: The sample was soaked for 12 hrs in an orbital shaker at 50 revolutions/min

Step 3: The extracts were filtered using Whatman Filter No. 1

Step 4: The concentrate was evaporated to dryness under reduced pressure using rotary evaporator at 40°C

Step 5: The extract was collected and stored in an air-tight amber colored glass container.

This ethanolic extract of Polyalthia longifolia leaves was subjected to analytical studies after carrying out preliminary phytochemical screening as described[1415] by comparing it with standard biomarkers.[16] Standard biomarker and analytical grade solvents used for identification purpose were obtained from Sigma-Aldrich Private Limited, India. The general selection criterion for high-Pressure liquid chromatography (HPLC) and high performance thin layer chromatography (HPTLC) methods is to optimize the separation and identification of the bioactive compounds from the extract and to check the comparison of accuracy of the results obtained by these two widely used techniques. The basic advantage in selecting these powerful visualization techniques was their accuracy, preciseness, specificity, sensitivity and reproducibility.[17]

Analytical studies

Analytical studies are comprised of:

Thin Layer Chromatography (TLC) for identification of the constituents

High Performance Thin Layer Chromatography (HPTLC) for quantization and to identify the active constituents

High Pressure Liquid Chromatography (HPLC) for determining the percentage of the active constituents.

Thin layer chromatography

Mobile Phase: Methanol: Glacial Acetic Acid: Formic Acid: Water (3: 0.9: 0.9: 0.5)

Standards: Rutin dissolved in ethanol.

Extract: 10 mg in 1 ml of ethanol. The sample was filtered before spotting on to the plate.

Chamber Saturation Time: 30 min

Visualization: The TLC plate was exposed to ammonia and visualized under UV lamp.

High performance thin layer chromatography

The HPTLC was performed at the Radiant Research Laboratories Private Limited, Bangalore. The analysis was carried out by application of the sample and standard dissolved in methanol on HPTLC plate’s silica gel 60 F254 (20 × 10 cm). Spots of extract (6 and 9 μg/l) and standard (3, 6 and 9 and 12 μg/l) were applied on the plates. The PL leaves extract (47.5 mg) and standard (5.16 mg) were dissolved in (ratio of 1:10 in terms of weight in mg) 10ml of the solvent. Scanning of the developed plates was carried out at 333 nm (before) and 550 nm (after) with following details:

Instrument: CAMAG Linomat 5

Linomat 5 application parameters

Spray gas: Inert gas

Sample solvent type: Methanol

Dosage speed: 150 nl/s

Predosage volume: 0.2 μl

Sequence

Syringe size: 100 μl

Number of tracks: 10

Application position Y: 12.0 mm

Band length: 8.0 mm

Calibration parameters

Calibration mode: Single level

Statistics mode: CV

Evaluation mode: Area and peak height

Formula:

High pressure liquid chromatography analysis

Chemicals: Rutin Standard, buffers, methanol, plant extract

Instrument and other details:

HPLC instrument make: Shimadzu LC-10 ATVP

Software: Chromtech N 2000 data

Detector: UV Wavelength 280 nm

Flowrate: 1.5 ml/min

Injection volume: 20 μl

Column dimensions: RP C-18, 250 × 4.6mm, 5 μ

Procedure:

Mobile Phase: Methanol: Phosphate Buffer (pH 3) in ratio 60:40.

2 mg of standard and 5 mg of sample were dissolved in 2 ml and 5 ml of solvent respectively.

Formula:

RESULTS

Extraction yield

The extraction yield of the dried powder of Polyalthia longifolia (PL) leaves for ethanolic extract was found to be 13.94% w/w [Table 1].

Table 1

Percent yield of the extract

Preliminary phytochemical analysis of the plant extract

The plant ethanolic extract was found to be present positive for flavonoids, steroids, diterpenoids, alkaloids, saponins, tannins and phenolic compounds and mucilage [Table 2].

Table 2

Preliminary phytochemical analysis of the plant extract

Chromatographic analysis of extract

TLC reports

Rf value (standard Rutin) = 0.78

Rf value (ethanolic extract) = 0.69

By comparing the Rf value [Figure 1], the sample was found to be identical to Rutin as active constituent.

Figure 1

TLC of Polyalthia longifolia (PL) ethanolic extract

HPTLC reports

The ethanolic extract of PL leaves has shown well resolved spots on the HPTLC plate at Tracks 5 and Track 6 in comparison to standard Rutin at Tracks 1-4 and 7-10. The images were obtained under UV visible wavelength at 333 nm but well resolved spots were obtained only after derivatization at 550 nm. Quantization by HPTLC and by comparing the Rf value at 0.36 (start),0.41(maximum) and 0.44 (end) confirms that the spots resolved were of rutin present in extract identified by comparison with standard biomarker as dark green paste spots [Table 3 and Figures 2-13].

Table 3

HPTLC analysis of Polyalthia longifolia and rutin

Figure 2

Track 1 - HPTLC peak of standard Rutin

Figure 13

HPTLC image after (550 nm) derivatization

Track 2 - HPTLC peak of standard Rutin

Track 3 - HPTLC peak of standard Rutin

Track 4 - HPTLC peak of standard Rutin

Track 7 - HPTLC peak of standard Rutin

Track 8 - HPTLC peak of standard Rutin

Track 9 - HPTLC peak of standard Rutin

Track 10 - HPTLC peak of standard Rutin

Track 5 - HPTLC peak of Polyalthia longifolia

Track 6 - HPTLC peak of Polyalthia longifolia

HPTLC image before (333 nm) derivatization

By HPTLC studies, the amount of rutin present in the extract was found to be 11.60% w/w (5.51 mg of rutin present in 47.5 mg of PL leaves extract).

HPLC reports

The ethanolic extract of PL leaves has shown well resolved peak at 280 nm (retention time is 3.8 min) in comparison to rutin at a flow rate of 1.5 ml/min using methanol and phosphate buffer (pH 3) in the ratio of 60:40 when HPLC studies were undertaken [Table 4 and Figures 14 and 15].

Table 4

HPLC analysis of Polyalthia longifolia and Rutin

Figure 14

HPLC peak of standard Rutin

Figure 15

HPLC peak of Polyalthia longifolia

By HPLC estimation, the amount of rutin in ethanolic extract of PL leaves was found to be 4.03% w/v.

DISCUSSION AND CONCLUSION

Thus, rutin is an important active constituent isolated from many plant extracts. Further to our consideration, it is widely known for its immense pharmacological potential like anti-inflammatory, anticancer, antiarthritic etc. Our research article emphasis on the studies related to isolation of rutin in the cold ethanolic extract which was monitored by sophisticated chromatographic methods such as HPTLC and HPLC. Research on the extract gave us well resolved spots in HPTLC and good peak in HPLC. Hence, we urge the young readers to step up in taking the task for exploring the extract of the local plant for isolating the other constituents and undertake studies on various pharmacological interventions by using different animal models.