Identification and HPLC Purification of Aspirin (Acetylsalicylic Acid) from the Seed Coats, Leaves and Bark of Givotia Rottleriformis Griff.


 Aspirin (Acetylsalicylic acid; ASA) is an anti-inflammatory and anti-cancer molecule derived from salicylic acid (SA) and produced by different companies worldwide because of the wide range of therapeutic applications in human. Accumulated evidence suggest that ASA relieves pain, swelling, cold or flu, prevent colorectal cancer and cardiovascular diseases. ASA induces defense response against wide range of pathogens in plants. Recently, we reported benzoylsalicylic acid (BzSA) and other anti-cancer molecules such as gallic acid (GA) methyl gallate from the seed coats of G. rottleriformis 1-3. Here we are reporting the natural aspirin first time from the seed coats, leaves and bark of G. rottleriformis. HPLC chromatogram of hexane extract of seed coats, leaves and bark have shown a peak at RT 24.8min coeluted with the aspirin standard. The purified aspirin from the seed coats were subjected to 1H NMR and were confirmed as aspirin. These results suggest that aspirin biosynthesis is taking place in seed coats, leaves and bark of G. rottlerifirmis and supports the medicinal properties of G. rottleriformis. These results suggest that the seedcoats, leaves and bark of this plant contains the highly useful medicinal compounds to treat the rheumatism, cancer, cardiovascular, psoriasis, anti-inflammatory and other skin diseases.


Introduction
Givotia rottleriformis., is a commercially valuable tree belonging to the Euphorbiaceous family. It is a moderate sized tree grow in the forests of Andhra Pradesh, Karnataka, Tamil Nadu, and West Bengal 4 .
Because of the softness and light weight of the wood this plant has high value in toy industry. And the seeds and the bark have medical value and used for the treatment of rheumatism, dandruff, and psoriasis 5 . Aspirin also known as acetylsalicylic acid (ASA) and it is a nonsteroidal anti-in ammatory drug and it is a commonly used drug worldwide to reduce pain, fever and in ammation 6 . Aspirin is a potential drug used to prevent cardiovascular and cerebrovascular diseases (About 80 million pounds of aspirin are producing and 100 billion tablets consumed every year). Aspirin primarily works by blocking the action of cyclooxygenase1, which prevents the conversion of arachidonic acid (AA) into prostaglandins (PG), which further prevents the synthesis of thromboxane (Tx) [7][8][9] . Nonsteroidal antiin ammatory drugs (NSAIDs) block the COX enzymes and reduce prostaglandins throughout the body and thus reduce in ammation, pain, and fever 10,11 . PG are produced by the cells by the enzyme cyclooxygenase (COX) and carry out several important functions 11 . After more than a century of human use, researchers are still discovering how aspirin affects the body and the Scientists understood that SA was the component derived from plants that relieved pain fever and cancers [12][13][14][15][16] . However, long-term use of ASA in high doses causes stomach problems in some people 17 . In 1897 a chemist at a company called Bayer added a chemical modi cation called an acetyl group (CH 3 CO) to SA, turning it into ASA and Bayer called this new substance aspirin 18 . Willow bark has been used as a traditional medicine for more than 3500 years and determined salicylate contents in the food items 13,[18][19][20][21] . For centuries in Europe, people grew meadowsweet to treat pain and in ammation 18, 21-23 . Willow and meadowsweet contain high levels of aspirin-like compounds called salicin and methyl salicylate (MeSA) which would later form the basis for the discovery of aspirin 18,20 . The effect of aspirin still studying with the growing evidence of its chemo preventive effect against colorectal and other types of cancer 16,18,20,[24][25][26][27][28] .
Salicylic acid pathway is a well-studied defense responsive pathway against a broad range of bacterial, fungal and viral pathogens [29][30][31] . It is widely accepted that plants possess both an isochorismate synthase (ICS) and phenylalanine ammonia-lyase (PAL) pathway to synthesize SA both starting from chorismite 31,32 . SA levels increase in many plants upon infection with viruses, fungi, insects, and bacteria [33][34][35] and exogenous SA ptr-treatment boosts the defense system of the host plants [36][37][38] . Plants overexpressing NahG, a salicylate hydroxylase converts SA to catechol, are unable to accumulate SA upon pathogen infection and are impaired in their systemic acquired resistance (SAR), a broad-spectrum systemic defense response after a primary infection 39 .
plant have the ability to withstand environmental stress including seasonal changes 40,41 . Previous reports have suggested that SA and its analogues can confer stress tolerance in plants 42 . Literature suggested that the plant seeds imbibed in SA/ASA solution shown resistance to temperature, cold and drought stresses 43,44 and the seedlings also shown resistance to stress upon SA or ASA treatments. 45 SA and ASA plays defense signal role in plants 46,47 . SA and its derivatives are useful treat various tress in agriculture, horticulture and forestry 43,47 . Previous reports have shown that SA/ASA regulate the expression of stress responsive genes in plants 47 . Treatment of seeds of different plants SA/ASA overcome various stress 48,49 . In our previous study we have shown that pretreatment of SA, ASA, and BzSA (benzoylsalicylic acid) to tobacco induced SAR and offered better protection against tobacco mosaic virus 2, 3 .
In the present study we have identi ed and puri ed natural aspirin from the seed coats, leaves and bark of G. rottleriformis using HPLC using preparative column ( gure 1). The puri ed aspirin coeluted with the standard aspirin at RT24.8min and the puri ed aspirin was con rmed by 1 H NMR. Pre-treatment of tobacco plants with puri ed natural aspirin shown the similar effect in reducing TMV lesions as compared to standard ASA.

Material And Methods
General details 1 HNMR (400 MHz) spectra were recorded on Bruker-AC-200 and Bruker-Avance-400 spectrometer with chloroform-d as solvent and TMS as reference (d = 0 ppm). The chemical shifts were expressed in d down eld from the signal of internal TMS. Thin layer chromatography (TLC) was carried out using silica gel plates (Merck 60F254) and the compounds were visualized by irradiation with UV light and/or by iodine vapor. Column chromatography was carried out using (acme's) silica gel (100-200 mesh).

Plant Materials
Mature, dry seeds, bark and leaves of G. rottleriformis (A voucher specimen No. PARC/2011/2140) were collected from trees available at Regional Forest Research Centre (RFRC), Rajahmundry, Andhra Pradesh, India. Tobacco seeds (VT-1158, NN gene type, resistant to TMV) were obtained from the Central Tobacco Research Institute, Rajahmundry, Andhra Pradesh, India.

Statement of plant material Identi cation
The plant material used in this study were collected from the trees available at the Regional Forest Research Centre, Rajahmundry, Andhra Pradesh, India. I thank to Dr. Vara Prasad for helping the identi cation of plant material and allowing to use the plant material the study.
Compound extraction from seed coats, leaves and bark Mature seeds leave and bark of G. rottleriformis were dried at room temperature for 3 days, the seed coat consisting of epicarp and mesocarp were removed manually using cutter. Dried seed coats, leaves and bark was ground into a ne powder separately using a grinder. The powder was soaked in methanol repeatedly for 3 times for 2days and the total compounds were extracted. The extract was vacuum dried with the help of rotary evaporator with vacuum under the heating condition.

Results And Discussion
We have initiated out study to re-establish G.rottleri rmis plant populations, and developed an e cient micropropagation method 4 . While cutting the seeds we observed a bulk amount of compound in the seed coats. Based on the medicinal properties of this plant, we have directed to characterize the medicinally important compounds form the seed coats, leaves and the bark of this plant. And we isolated important bioactive molecules 1-3 . In our previous studies, we repot benzoyl salicylic acid (BzSA) for the rst time from the seed coats of G. rotteriformis and proved as a potential defense inducer against Tobacco mosaic virus (TMV) as a compared to salicylic acid a well-known plant hormone induces disease response against a wide range of pathogens 2 . BzSA, salicylic acid (SA) and its precursors such as cinnamic acid (CA), benzaldehyde (BD) and benzoic acid (BA) are puri ed from the seed coats of G. rottleriformis using preparative HPLC 2 . The biosynthesis of SA was reported in different plants are takes place via cinnamic acid 32,50 . In our previous study we report that SA is further converted to BzSA using benzoyl-CoA 2 . In addition we also reported the puri ed gallic acid (GA) and methyl gallate (MG) from the seed coats of G.rottleriformis 1 . The presence of GA, MG, SA, SA-analogues and BzSA in the seed coats are suggest that the existence of phenylpropanoid pathway in the seed coats of this plant 1-3 . A peak eluted at RT 24.8 min from the 10% fraction of seed coats co-eluted with standard aspirin (Figure 2). In order to determine the presence of aspirin in the leaves and bark of this plant, A 10% fraction of leaf/bark from the open silica column were resolved on preparative HPLC using the same HPLC program and the HPLC chromatogram showed the elution of peak at RT 24.8min corelates with the standard aspirin ( Figure 3&4). The peak eluted at RT 24.8 min was puri ed and subjected to 1 HNMR and con rmed as aspirin ( Figure 5). Tobacco plants that were pre-treated with puri ed natural ASA showed similar effect in decrease in lesion size as compared with standard ASA ( Figure S1). The amount of aspirin detection was signi cantly high in the leaves as compared with the seed coats and the bark (Figure 2-4). SA and related compounds are produced by plants as part of their defense systems against pathogen attack and environmental stress 20 . So far, no reports on the biosynthesis of aspirin in the plants are published. First time we are reporting the aspirin in the seed coats, leaves and the bark of G. rottleriformis. HPLC analysis of fruits and vegetables provide unknown amounts of aspirin and no aspirin detected in foods by HPLC 21 . It was reported that ASA concentrations were too low in volunteers eating a verity of diets 15,21,51,52 .
Our results strongly suggest that the biosynthesis of aspirin is taking place in the seed coats, leaves, and the bark of this plant. The biosynthesis of aspirin in plants require SA as a precursor was detected in the seed coats 2 . BzSA puri ed from the seed coats of this plant also required SA as a key precursor 2, 3 . The chemical synthesis of BzSA was achieved using SA and benzoyl chloride 3 . Previous reports suggest that pre-treatment of aspirin induces systemic acquired resistance (SAR) in plants against a broad spectrum of pathogens 2, 3, 53-55 . Aspirin has been used for >100 years for pain relief and to treat in ammatory conditions and fevers 56,57 . Aspirin is effective in the prevention of cardiovascular disease and several cancers [58][59][60][61][62][63][64][65][66] . Cyclooxygenase-1 (COX-1) produce prostaglandins (PGs) and thromboxane (TxA2) and regulate the gastrointestinal, renal, vascular and other physiological functions whereas Cyclooxygenase-2 (COX-2) produce PGs and involved in in ammation, pain and fever 57, 67-70 . The development of selective COX-2 inhibitors are therapeutic advantage whereas COX-1 inhibitors causes adverse side 71 . It was reported that a small daily dose of aspirin (100mg) helps to reduce the risk of myocardial infarction and stroke 72 . COX-1supports the bene cial homeostatic functions, whereas COX-2 induced by in ammatory mediators and involved in the in ammatory diseases such as rheumatoid and osteoarthritis 7,73,74 . The puri ed BzSA from the seed coats of G. rottleriformis inhibits higher COX-2 than COX-1(data not shown) and the existence of aspirin in this plants support the anti-rheumatism medicinal properties of G. rottleriformis 7,75 . The seeds and bark of this plant used for the treatment of psoriasis [76][77][78] . A Randomized trial of low-dose Aspirin to reduce vascular endothelial In ammation in Psoriasis [76][77][78] . The plant G. rottleriformis grown particularly in hill forests and exposed to high temperatures may be because of high temperature stress and other abiotic and biotic stresses this plant synthesizes a lot of stress and other compounds to overcome biotic/abiotic stresses. Several studies reported that SA and ASA protect plants from biotic/abiotic stresses both in plants and animals 41,[79][80][81][82] . Aspirin is metabolized to SA and salicyluric acid (SU) in humans 83 . Aspirin rapidly hydrolyzed to SA with an half-life of 5 to16 min 84 and the hydrolysis of aspirin takes place in liver and stomach 84 . Both aspirin and SA are bound to serum albumin 84 and the serum half-life of aspirin is more or less 20 min 84 . Acetylation of COX-1 and COX-2 IKK-complex inhibit the enzyme activity and are important to treat pain, fever, in ammation, cardiovascular and various cancers and the excess of aspirin deacetylate into SA and excreted 7,85 . The fall in aspirin concentration is associated with a rapid rise in SA concentration 83 . SA is renally excreted in part unchanged and the rate of elimination is in uenced by urinary pH, the presence of organic acids, and the urinary ow rate 15,84 . Our results summarizing that identi cation of natural aspirin in G. rottleriformis suggest that both plants and animals utilizing aspirin and the use of salicylates in rheumatic diseases supports the medicinal properties of G. rottleriformis. And identi cation of SA, ASA, BzSA, BA, BD,GA and MG in this plants indicating that phenylpropanoid biosynthesis pathway is highly active in this plant 1-3, 86, 87 and based on our results we suggesting that seed coats, leaves and the bark of G. rottleriformis contains aspirin and are useful to treat cancer, cardiovascular anti-in ammatory, psoriasis and rheumatic diseases.