Bicyclic Diterpenoid and Phytosterol Constituents from Andrographis Paniculata ( NEES )

A systematic phytochemical investigation of Andrographis paniculata led to the isolation of a bicyclic diterpenoid lactone andrographolide and a well known plant sterol b-sitosterol from the leaf extract. Andrographolide is one of the major diterpene lactone found in the pant. The structure was elucidated on the basis of spectroscopic analysis like 1H, 13C, 13C DEPT, 1H-1H COSY, FT-IR, UV-Vis and comparison with the known related compounds. It is very bitter in taste and has been reported to possess significant pharmacological activities like antioxidant, antitumor, anti HIV, cardioprotective and hepatoprotective activities. Recently several studies have been conducted to investigate the pharmacological activities andrographolide proved that it is the one of the main constituent attributed for the therapeutic potential of the plant A. paniculata.


INTRODUCTION
Andrographis paniculata has been used for centuries as a medicinal herb due to its unique ability to treat various ailments like jaundice, asthma, hypoglycemia, leprosy, gonorrhea, scabies, skin eruptions, hypertension, upper respiratory infections, fever, herpes and other chronic diseases 1,2 .In Ayurvedha more than 26 herbal formulations are mentioned from the plant revealed the widespread use for the treatment of human ailments.It is one of the commonly used plants as a popular remedy for the treatment of various liver disorders.The plant is known as the 'king of bitters' because it is extremely bitter in taste in every part of plant body 3 .On the basis of literature survey it has been observed that the aerial parts of the plants are usually used to extract the active phytoconstituents 4 .Previous phytochemical studies of this well studied herb showed that it is a rich source of bioactive flavonoids and diterpenoid lactones 5 .
A. paniculata was found to be used in traditional Indian system of medicines for lowering fever.The chief phytochemical constituent, andrographolide reported to possess antipyretic, antiulcerogenic and analgesics activity which is comparable to that of aspirin 6 .Different mechanisms have been proposed on anti-inflammatory activity of andrographolide involving modulating macrophage and neutrophil activity.Andrographolide is believed to be effective against various liver disorders like hepatitis; this may be responsible for the hepatoprotective avtivity of the plant 7 .
The purpose of this study is to identify and characterize the bioactive principles from the aerial part of A. paniculata.In this paper we report the isolation and characterization of known compounds from the plant namely andrographolide and b-sitosterol.

Plant materials
The fresh leaves of the plant were collected from different locations of Kerala, India in August 2014 and was taxonomically identified and authenticated by a recognized Medicinal Plants Research Institute, Thiruvananthapuram, Kerala.

Isolation of Andrographolide
Fresh leaves (500 g) of A. paniculata were cut into small pieces and powdered.The powdered material was first defatted with petroleum ether and then ethanol using soxhlet apparatus.Solvent evaporation under reduced pressure in a rotary evaporator yielded 2.8 and 5.6 g of petroleum ether and ethanol extracts respectively.Ethanol extract (5g) was fractionated using silica gel column chromatography (Mesh 60-120) using 100% petroleum ether, followed petroleum etherchloroform mixture by gradient elution technique 8 .Different fractions obtained were pooled on the basis of comparable R f values on TLC leading to a final three fractions (Fr.1 to Fr.3).Repeated column chromatography of Fr.1 and Fr.3 over silica gel (Mesh 100-200) yielded b-sitosterol (35 mg) and andrographolide (80 mg) respectively.

RESULTS AND DISCUSSION
b-sitosterol was obtained as colourless needle shaped crystals from the ethanol extract of A. paniculata (Fr.1).Its molecular formula was deduced as C 29 H 50 O from its EI-MS [M] + at m/z 414.It gave positive test for steroid in the Liebermann-Burchard reaction.UV spectrum showed l max at 202 nm indicating the presence of an olefinic group.On subjection to IR spectroscopic analysis, the observed absorption bands are appeared at 3421 (OH), 2958 (CH 3 ), 2850 (CH 2 ), 1460, 1053 (C-O) cm -1 .The compound was identified as stigmast-5-en-3b-ol (b-sitosterol) by comparison of its IR, melting point, mixed melting point and TLC data with authentic sample 9,10 .

CONCLUSIONS
The potential use of A. paniculata and its constituents in treating cancer, HIV infections and hepatitis were largely explored.Andrographolide one of its major constituent also reported to possess anticancer and immune stimulating potential 12,13 .Recent studies showed that andrographolide, a potent diter penoid lactone isolated from A. paniculata inhibited the in vitro proliferation of different tumor cell lines, representing various types of human cancers.The plant extract as well as the andrographolide were also reported to induce myeloid leukemia cell differentiation in mice.These investigations reveal the therapeutic use of andrographolide and other phytoconstituents reported from A. paniculata as a potential source against various human ailments.These facts emphasize to select A. paniculata as a suitable candidate for drug development with the greatest possibility of success.