INTRODUCTION:

Punarnava (Hogweed) Boerhavia diffusa (F– Nyctaginaceae) literally means ‘bring back to life’ or ‘renewer’. It grows wild in India and Brazil throughout year dries during the summer.¹ It has small fleshy leaves, bitter in taste, small reddish pink flowers and bears fruits in winter. It has cooling effect and it has very high medicinal value. Punarnava is beneficial in treating obesity.² Punarnava is effective in treating a disease called dropsy, a condition wherein excess of watery fluid gets accumulated in the tissues and body cavities.^3,4,6 A liquid extract of this plant stimulates urine secretion and discharge. It is also useful in treating Ascites, a condition caused by fluid accumulation in the abdominal lining and is particularly useful in treating specific types of Ascites caused by certain liver diseases.

The roots of the plant help in killing intestinal worms. It promotes mucous removal from bronchial tubes and hence beneficial in treating Asthma. When the paste made out of the roots of this plant, is applied externally on the skin, it forms a beneficial dressing for edematous swellings, ulcers and skin diseases.⁷

MATERIAL AND METHOD:

Chemicals

Working standards of punarnavin (98%) markers are obtained from Natural Remedy Pvt.Ltd, Bangalore India. The commercial crude powder of Punarnava roots was collected from Green Pharmacy, Pune and the formulation containing B. Diffusa was procured from the local market. All chemicals and reagents of analytical grade were purchased from Merk Chemicals, Mumbai, India.

Selection of analytical wavelength

Stock solutions of drugs were prepared in methanol separately. UV spectrum of 100 µg mL^-1 of each individual drug was taken.

Instrumentation and chromatographic conditions

The HPTLC plates were pre - washed with methanol and activated at 110⁰c for 5min prior to chromatography. The sample were spotted in the form of bands 6mm width with a CAMAG 100µL sample syringe (Hamilton, Bonaduz, Switzerland) on silica gel precoated HPTLC aluminium plate 60 F₂₅₄ [(20x10 cm) with 250µm thickness, E Merk, Darmstadt, Germany, supplied by ANCHROM technologies, Mumbai] using a CAMAG linomat V applicator (Switzerland). A constant application rate of 0.1µL/s was used and space between two bands of 6mm. linear ascending development was carried out in 20cm x 10cm twin through glass chamber (Camag, Muttenz, Switzerland) saturated with mobile phase. The mobile phase was consisted of toluene: ethyl acetate: formic acid in the ratio (8.0:1.5:0.7, v/v/v) and 20ml was used per chromatographic run. The optimized chamber saturation time with mobile phase was 30min using saturation pad at room temperature (25ᵒC ± 2). The length of chromatogram run was 80mm and run time was 45min. densitometric scanning was performed using a CAMAG TLC scanner III in the reflectance absorbance mode and operated by win CATs software (V 1.1.4, Camag). The slit dimension was kept at 5mm x 0.45mm and the scanning speed was 10mm/s. The source of radiation used was deuterium lamp emitting a continuous UV spectrum between 200 and 400nm. All determinations were performed at ambient temperature with a detection wavelength of 283nm. Concentration of the compound chromatographed were determined from the intensity of defused light. Evaluation was by peak area with linear regression.

Standard solutions and calibration graphs

Stock standard solution containing punarnavine (100µg/ml) was prepared by dissolving 1mg each of accurately weight markers in methanol up the volume to 10mL with methanol. Different volumes of stock standard solution applied on the HPTLC plate to obtained working standard in the concentration range of 300-1300ng/spot. Each concentration was applied six times on the HPTLC plate. The plate was then developed using the previously described mobile phase. The peak areas were ploted against the corrosponding concentration to obtained the calibration graph. Linear calibration curves were generated using least-squares linear-regression analysis.

Sample preparation

Estimation of punarnavine in commercial crude powder of B.diffusa roots

500mg of powder of plant material was extracted separately with (4×25)ml of water: methanol (70:30, v/v). The extract was centrifuge at 5000rpm for 10min and the supernatant was filtered through a 0.45µm nylon syringe filter before chromatographic analysis and analysed for the drug content. The analysis was repeated six times.

Analysis of commercial formulation B. diffusa :

To determine the content of punarnavine containing, the contents of twenty tablets were weighed, their mean weight determined and they were finely powdered.

Standard solutions and calibration graphs .

Standard stock solution containing punarnavine (100µg/ml) was prepared by dissolving 1mg each of accurately weight markers in methanol up the volume to 10mL with methanol. Different volumes of stock standard solution applied on the HPTLC plate to obtained working standard in the concentration range of 300-1300ng/spot. Each concentration was applied six times on the HPTLC plate. The plate was then developed using the previously described mobile phase. The peak areas were ploted against the corrosponding concentration to obtained the calibration graph. Linear calibration curves were generated using least-squares linear-regression analysis.

Sample preparation

Estimation of punarnavine in commercial crude powder of B.diffusa roots

500mg of powder of plant material was extracted separately with (4×25) ml of water: methanol (70:30, v/v). The extract was centrifuge at 5000rpm for 10min and the supernatant was filtered through a 0.45µm nylon syringe filter before chromatographic analysis and analysed for the drug content. The analysis was repeated six times.

Analysis of commercial formulation B. diffusa :

To determine the content of punarnavine containing, the contents of twenty tablets were weighed, their mean weight determined and they were finely powdered.

The weight of powder equivalent to tablet content was transferred into 50mL volumetric flask containing 20mL water: Methanol (70:30, v/v), sonicated for 30 min and diluted to 50 mL with water. Methanol [70:30, v/v]. The resulting solution was centrifuged at 3000 rpm for 15 min and supernatant was analysed for the said markers. The filtered solution was spotted on the HPTLC plate followed by development and scanning.

RESULTS AND DISCUSSION:

Optimization of procedure:

Initially, mobile phase was selected on the basis of previous reports of punarnavine . A common mobile phase consisting of ethyl acetate, methanol and formic acid was tried initially. Several modification were tried on trails, addition of toluene was found to be sutaible for the moment of punarnavine whereas Formic acid have effect on the peak shape of punarnavine . Hence the final mobile phase was optimized toluene: ethyl acetate: formic acid in the ratio (8.0:1.5:0.7, v/v/v) which was found to give desirable R_f value. The optimized mobile phase can able to give symmetrical, well resolved reproducible peaks with good shape and baseline separation. The R_f values obtained were 0.8 for punarnavine was selected.

Selection of analytical wavelength -

283nm was selected as scanning wave length. The identities of the bands from the sample extracts and commercial formulations were confirmed.

Fig 1 – HPTLC plates shows Rf values at 0.8 of punarnavine, in- house herbal formulation and marketed samples at 254 nm

Fig 2 – HPTLC plates shows Rf values at 0.8 of punarnavine, in- house herbal formulation and marketed samples at 366 nm

Table 1 – Shows content of punarnavine found in each marketed samples and in house formulations

S.No.	Samples (Marketed and Inhouse)	Content (% of Punarnavine found in each sample containing 1 gm of sample)
1.	M1	300.7 µg
2.	M2	289.7 µg
3.	M3	284.3 µg
6.	In – House preparation	287.4 µg

RESULTS AND DISCUSSION:

Analysis of the amount of punarnavine studied application of the developed method in commercial crude powder and commercial tablets, using the reference comparison method. The content of punarnavine for commercial crude powder of B. diffusa is found for M1 is 300.7 µg, For M2 is 289.7 µg, for M3 is 284.3 µg, for In – House preparation is 287.4 µg.

CONCLUSION:

HPTLC method for simultaneous estimation of punarnavine , the proposed method was found to be suitable for estimation of this markers in polyherbal formulation as it is proved to be reproducible,reliable and robust. Hence, this method can be used for as a rapid analytical tool in routine analysis to monitor loss or variation of the content of the markers in various herbal formulation

CONFLICT OF INTEREST:

None declared.

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Received on 18.01.2024 Modified on 22.04.2024

Research J. Pharm. and Tech 2024; 17(7):3427-3429.

DOI: 10.52711/0974-360X.2024.00536