Karpakavalli M et al.

/ Pharmacie Globale (IJCP) 2012, 5 (03)

Available online at www.pharmacie-globale.info

ISSN 0976-8157

Research Article

PHARMACIE GLOBALE INTERNATIONAL JOURNAL OF COMPREHENSIVE PHARMACY

MICROWAVE ASSISTED EXTRACTION AND ESTIMATION OF PIPERINE, ANDROGRAPHOLIDE USING HPLC TECHNIQUES
M Karpakavalli*1, K R Sini2 and I Arthi3
1Karpagam

College of Pharmacy, Othakalmandapam, Coimbatore, Tamilnadu, India. 2Grace College of Pharmacy, Palakkad, Kerala, India. 3Faculty of Pharmacy, Karpagam University, Coimbatore, Tamil nadu, India.

Received: 22 March 2012; Revised: 15 April 2012; Accepted: 27 April 2012; Available online: 5 May 2012

ABSTRACT

Plants belonging to the genus piper are reputed in the Indian ayurvedic system of medicine for their medicinal properties. Andrographis paniculata, commonly as "King of Bitters is a member of the plant family Acanthaceae. Research design: Indian herbal pharmacopoeia prescribes the extraction of piperine for 1 h and 3 h for andrographolide by reflux method followed by quantitative estimation using HPLC technique. In the present study, the extraction procedure of the above said phyto-constituents was planned to develop under microwave condition. Methods and Procedures: The method was focused for successful extraction with less volume of solvent, shorter duration and more yield than the reflux method. Several intensities and durations under microwave conditions were tried. Main outcomes and results: The higher yield of 2.12 % (at 500 W intensity & 16 min) of piperine and 0.20 % of andrographolide (at 500 W intensity & 21 min) were obtained in green technique as compared with the yield of 2.02 % of piperine and 0.18 % of andrographolide obtained by conventional technique. The novel green technique can be used in academics and industries for the extraction of phytoconstituents in comparatively very shorter duration with less consumption of solvents. Keywords: Microwave technique; Piper nigrum; piperine Andrographis paniculata; plant andrographolide; HPLC; green chemistry; economic; fast; safe. extract;

INTRODUCTION

Piperine (1-piperoylpiperidine), a nitrogenous pungent substance, is an alkaloid presents in the fruits of black pepper (Piper nigrum) and other piper species (family: Piperaceae). Since piperine has been recognized as a main alkaloid in these plants, numerous studies have focused on investigating the pharmacological activities of piperine. Recent pharmacological studies have shown that piperine possesses anti-inflammatory and analgesic effect1, anticonvulsant2, anti-ulcer3, anti-depressant effect4, cytoprotective effect and antioxidant activity5. Andrographis paniculata (Burm. F.) Nees, an important Indian medicinal herb, commonly known as King of Bitters, is a member of the plant family, Acanthaceae. It is used in traditional medicine against a variety of diseases including cold, fever, snakebite, diarrhea, and malaria.6-7 Indian Pharmacopoeia narrates that it is a predominant constituent of at least 26 ayurvedic formulations. Pharmacological research has demonstrated that Andrographis paniculata possesses anti-inflammatory, anti-allergic, immuno-stimulatory, antiviral, antioxidant, hepato-protective, cardiovascular activities, etc., and these studies have been well reviewed.8-9 It is one of the 32 Indian medicinal plants which have export values listed by
*Corresponding Author: M Karpakavalli Department of Pharmaceutical Chemistry, Karpagam Collage of Pharmacy, Othakalmandapam, Coimbatore -640032, Tamilnadu, India. Contact no: + 91-9842694947; Email: sreemenakag@gmail.com

National Medicinal Plants Board. Many of the older methods for the estimation of phytoconstituents include polarographic, iodometric, spectrophotometric, colorimetric methods and are found as the non-specific methods. [10-11] Nevertheless, UV spectrophotometry is still used by the manufacturers as it is rapid and cheap. However, there are circumstances in which a more specific determination of phytoconstituents is of value and thus the chromatographic method, principally reversed phase High Performance Liquid Chromatography (HPLC). Hence, the present study involves the HPLC estimation of phytoconstituents, those have been extracted by two different methods, viz., Conventional heating and microwave assisted extraction.12-13 Indian Herbal Pharmacopoeia14 prescribes a HPLC method for the quantitative estimation of some of the phytoconstituents involving a conventional extraction. The extraction and recovery of trace organic material from semi-solid and semi-solid matrices is often the slowest and most prone step of the analytical method. The conventional liquid extraction technique have two main disadvantages, viz., large volumes of organic solvents leading to sample contamination and losses due to volatilization during concentration steps. Recently, microwave energy is being used in chemical laboratories for many purposes. Microwave assisted Pharmacie Globale (IJCP), Vol. 03, Issue 05

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process is a high speed technology that uses microwave irradiations as the energy source during solvent extraction leading to a faster processing time, improved yield and quality, direct extraction capacity, lower energy consumption, reduced solvent levels and lower capital investment when compared to conventional extraction methods. Thus, it is one of the simple, economic, safe, fast, clean, eco-friendly and efficient method for the synthesis of organic molecules and extraction of phyto-constituents. Microwave assisted extraction of Camptothecin from Nothapodytes foetida15, saponins from Chick pea16, taxanes from Taxus brevifolia17, limonoids from Azadirachta indica18, phenolic compounds from graph seeds19, are reported in the literatures. In microwave extraction, the speed of breaking up of plant cells and plant tissues in much higher and there is a less risk of decomposition or disintegration and oxidation of valuable plant constituents.20 Partial / multiple selective extractions can also be achieved by adjusting the MAP parameters.21 Thus, recalling the importance of extraction step in the analytical procedure. The aim of this study is to determine if MAE, compared to conventional, gives better yields with many other advantages and also to contribute the present effort available for routine analysis in the commercial as well as academic laboratories.

in a mechanical grinder and used. Analytical marker Analytical grade of Piperine and Andrographolide, synthesized and characterized by spectral studies, in JSS College of Pharmacy, Ooty, were used as markers. Extraction Conventional procedure: The powder of the dried fruits of Piper nigrum (0.5 g) and the powdered aerial parts of the dried plant Andrographis paniculata (1 g) were weighed and refluxed the dried fruit powder with ethanol (50 ml) for 30 min and the plant material with methanol (50 ml) for 60 minutes respectively. The extracts were filtered and the marc was re-extracted under similar conditions once for piperine and twice for andrographolide. The combined filtrates of piperine were evaporated to about 25 ml and then cooled. The resulting solution was then made up to 100 ml with the solvent. Transferred 20 ml of this solution to a 25 ml volumetric flask, added 2 ml of solution B of the standard preparation to the same flask and made up the volume. The solution was used for HPLC estimation.22 All the filtrates of andrographolide were combined and evaporated to dryness in a rotavapor at low temperature (40-50C) under reduced pressure. The residue was used for HPLC analysis of andrographolide as per Indian Herbal Pharmacopoeia. Microwave procedure: Microwave radiant technique was carried out in a sample vessel made of borosilicate glass. The powder of the dried fruits of Piper nigrum (0.5 g) and the powdered aerial parts of the dried plant Andrographis paniculata (1 g) were quantitatively transferred separately to two different sample vessels and added ethanol (25 ml) for Piper nigrum and methanol (25 ml) for Andrographis paniculata.

MATERIAL AND METHODS

HPLC grade solvents were procured from E. Merck (India) Ltd, Chennai. Water HPLC grade was obtained from a MilliQRP water purification system. All the other chemicals and solvents used were of analytical grade. A domestic microwave oven (Whirlpool, MT 243, 1000 W, 240 V/50Hz with an electronic timer) was used for all the microwave experiments. Shimadzu LC 2010A HT-HPLC system was used for HPLC analysis.

Collection and authentication For microwave irradiation, the subjective beaker was kept The dried fruits of black pepper (Piper nigrum) collected in a microwave oven and a glass tube was placed inside from an ayurvedic shop, Ootacamund and the aerial parts the beaker to avoid bumping. The beaker was covered of Andrographis paniculata collected from Dr S Rajan, with a Petri dish containing few pieces of ice. A 250 ml scientist, Medicinal plants collection unit, Government arts beaker containing water (200 ml) was placed in the oven college, Ootacamund and both were authenticated by to serve as a heating sink. Several reactions of microwave Botanical Survey of India, Coimbatore and a voucher irradiation were conducted for different MAP parameters specimen (Voucher no. BSI/SC/5/23/09-10/Tech-1354 such as heat intensities, durations and conditions like dated 12/01/10) was deposited in the herbarium of our continuous/ discontinuous heating by trial and error institute. The fruits were powdered and used for method which is tabulated in Tables 1 & 2. extraction. The whole plant was dried, powdered coarsely Table 1. The percentage yields and peak areas of piperine present in the conventional and microwave samples
Method Intensity (w) Duration (min) Conventional ---60 Microwave I 160 40 II 350 26 III 350 20 IV 500 16 Standard peak area 2890309 (1 g/ml); * - Average of three determinations Peak area 2914859 2926396 2947070 2903806 3059407 Yields (%) 2.02 2.03 2.04 2.01 2.12*

Table 2. The percentage yields and peak areas of andrographolide present in the conventional and microwave samples
Method Intensity (w) Duration (min) Conventional ---180 Microwave I 160 72 II 160 72 III 350 18 IV 350 33 V 500 18 VI 500 21 Standard peak area 2848248 (5 g/ml), *Average of three determinations Peak area 20567404 20216430 17907575 19501440 24484280 18330117 22138984 Yields (%) 0.18 0.18 0.16 0.17 0.18 0.17 0.20*

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In each case, the mixture was filtered and the marc was used for re-extraction once for Piper nigrum and twice for Andrographis paniculata under similar conditions. The combined filtrates of Piper nigrum extract were evaporated to about 25 ml and then cooled. All the three filtrates of Andrographis paniculata were combined and evaporated to dryness in Rota vapor at low temperature (40 50C) under reduced pressure. The residue was used for HPLC analysis of andrographolide as above. Preparation of solutions Piperine Standard solution: A solution (25 ml) containing 2 ml of solution A (1mg / ml of piperine in ethanol) and 2 ml of Solution B (0.5 mg / ml of p-dimethylamino benzaldehyde in ethanol) and sufficient ethanol was used to make up the volume. Piperine Sample solution: The solution (25 ml) obtained from both the techniques was then made up to 100 ml with ethanol. Transferred 20 ml of this solution to a 25 ml volumetric flask, added 2 ml of solution B of the standard preparation to the same flask and made up the volume. Andrographolide Standard solution: A solution of standard andrographolide (100 g / ml) in methanol was used. Andrographolide Sample solution: The solutions of 25 ml of extracts obtained from both the techniques were prepared using methanol as solvent. HPLC analysis Chromatographic separation was performed on a Shimadzu liquid chromatographic system (LC-2010A, HT) was equipped with multi-wavelength UV array detector, a column oven (35C), and a degasser, an auto-injector and low pressure gradient quaternary pumps. A reverse phase C-18 column (250 x 4 mm, 5 l (waters phenomenex) was used for the separation. Class VP series version 6.01 (Shimadzu) data station was applied for the data collecting and processing. Mobile phase of a mixture of methanol and water (65:35) was delivered at a flow rate of 1.5 ml min-1 with detection at 343 nm for piperine and 1 ml min-1 with detection at 223 nm for andrographolide. The column temperature was maintained at 25C. 50 l of standard preparation was injected using a rheodyne syringe and the chromatogram was recorded. The process was repeated for sample preparation. Peaks corresponding to the retention time of phytoconstituents were identified and their percentage calculated by the formula.

developed process can replace the Indian herbal pharmacopoeia method for the extraction of piperine before its HPLC estimation and can be used routinely in the laboratories. Figure 1. HPLC chromatogram of piperine standard sample

Figure 2. HPLC chromatogram conventional sample

of

piperine

Figure 3. HPLC chromatogram of piperine microwave sample

RESULTS AND DISCUSSION

A typical HPLC chromatogram for phytoconstituents standard and phytoconstituents obtained by conventional and microwave method is shown in figures 1-6 respectively. The amount of piperine determined by HPLC when isolated conventionally by refluxing for 2 X 30 min was found to be 2.02%. Under microwave conditions, at 160W intensity and heating for duration of 20 min almost same yield was obtained. At 350W intensity and heating for 2 X 13 and 2 X 10 min, similar yields were obtained. However, a higher yield of 2.12 % was obtained at 500W intensity and for 2 X 8 min duration. The experiment was repeated thrice to confirm the results. Saving of 44 min is achieved. The HPLC chromatograms of both conventional and microwave extracts (Figure 2 and 3) were found to be the same that of the standard (Figure 1) indicating the suitability of the procedure. Hence, the

The amount of andrographolide found by HPLC when isolated conventionally by refluxing for 180 min was found to be 0.18 %. Under microwave irradiation at 160 W for 72 min, the yield of andrographolide was found to be 0.18%. At 350W intensity for 27 min and 33 min durations, 0.17 and 0.18 % of andrographolide was obtained respectively. Similarly, at 500W intensity for 18 min duration, 0.17 % of andrographolide was obtained. However, 500W intensity and irradiation for 21 min produced 0.20 % andrographolide. The yield was confirmed by repeating the experiment thrice. Longer heating durations involved in the extraction costs the curriculum and the industry, where time is important as money. In the present study, better yields were produced at 500W intensity and heating for 21 min when compared with the conventional method of extraction of andrographolide from Andrographis paniculata. The HPLC chromatograms of both conventional and microwave extracts were found to be almost the same that of the standard, which are shown in Figures 5, 6 and 4 respectively, indicating the suitability of the procedure. Pharmacie Globale (IJCP), Vol. 03, Issue 05

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Figure 4. HPLC chromatogram of andrographolide standard sample

Figure 6. HPLC chromatogram of andrographolide microwave sample

Figure 5. HPLC chromatogram of andrographolide conventional sample

CONCLUSION

Longer heating durations involved in the extraction costs the curriculum and the industry, where time is important as money. Hence, following this method, a saving of 159 min can be achieved in each standardization process.

ACKNOWLEDGEMENT

The authors wish to place on record their heartfelt thanks to Jagadguru Sri Sri Shivarathreeshwar Deshikendra Mahaswamigalavaru of Suttur Mutt, India, for providing the facilities for part of the work and Karpagam College of Pharmacy, Coimbatore.

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