Ehsst.org

Pleione 7(1): 234 - 240. 2013.
ISSN: 0973-9467
East Himalayan Society for Spermatophyte Taxonomy
Effect of solvent, temperature and time on extraction process
of anti ulcer compound present in Amaranthus spinosus
Linnaeus
Debiprasad Ghosh1, Prasenjit Mitra1,Tanaya Ghosh1, Arabinda Ray2,
Krishna Chandra Sarker2, Abhaya Prasad Das3 and Prasanta Kumar Mitra1
1Department of Biochemistry, 2Department of Physiology, North Bengal Medical College, Sushrutanagar – 734012 Dist. Darjeeling, West Bengal, India 3Taxonomy & Environmental Biology Laboratory, Department of Botany, North Bengal University, Siliguri 734013, West Bengal, India 4 Corresponding author; Present address: Department of Medical Biotechnology, Sikkim Manipal Institute of Medical Sciences, Gangtok,Sikkim; E-mails: [email protected] [Received revised 12.06.2013; Accepted 20.06.2013] Abstract
Effect of solvent, temperature and time on extraction process of anti ulcer compound present inthe leaves of Amaranthus spinosus Linnaeus was studied. Results showed that extraction ofleaves of Amaranthus spinosus with ethanol (50 %, v/v) at 40° C for one hour had maximum antiulcer effect against indomethacin induced gastric ulceration in albino rats.
Key words: Extraction process, Anti ulcer effect, Amaranthus spinosus,Indomethacin
INTRODUCTION
Amaranthus spinosus Linnaeus, a medicinal plant of the family of Amaranthaceae, isdistributed in hot tropical to middle hills (upto 1500 m amsl) of entire country includingEastern Himalaya. The plant grows in cultivated areas as well as in waste places. Leaves ofAmaranthus spinosus Linnaeus are stacked and alternate. The plant is known as “pricklyamaranthus” in English and “ban lure”or “dhuti ghans” in Nepali and “Kanta Note” inBengali. Medicinal uses of Amaranthus spinosus as mentioned in Ayurveda (Chopra &Chopra1958; Das et al 2010) are: (i) leaf infusion is diuretic and used in anemia; (ii) rootpaste is used in gonorrhea, eczema, menorrhea etc. Ethnic uses of Amaranthus spinosus ismainly with the villagers of Sikkim who use leaf infusion of the plant in stomach disorderspecially in case of indigestion and peptic ulcer (Gurung 2002).
Recently we observed anti-ulcer activity of the leaves of Amaranthus spinosus against indomethacin induced gastric ulcer in albino rats. Tempted with this observation we undertookstudies for isolation of the active compound present in Amaranthus spinosus responsiblefor anti gastric ulcer activity. In this communication we report effects of solvent, temperatureand time on extraction process of anti ulcer compound from the leaves of Amaranthusspinosus Linnaeus.
METHODOLOGY
Collection of plant material
Leaves of Amaranthus spinosus Linnaeus were collected from the medicinal plant garden
of the University of North Bengal and its identity has been confirmed in the Taxonomy and
Environmental Biology Laboratory of the Department of Botany, North Bengal University.
A voucher specimen of the plant has been preserved in the Biochemistry department of the
North Bengal Medical College for future references.
Preparation of leaves for anti-ulcer screening
Leaves of Amaranthus spinosus were sundried and powdered. 10 grams of this powder
was separately extracted with 100 ml of different solvents at different temperatures and
time on a rotary shaker. The extract was filtered and the solvent was evaporated to dryness
in vacuo with rotary evaporator at 40 – 50 degree Celsius. A brownish mass was obtained.
This mass was kept for anti-ulcer screening.
Effect of solvents in extraction process
Distilled water as well as 50 % (v/v) of acetone, ethanol, methanol and petroleum ether
were used separately in extraction process.
Effect of time in extraction process
Extraction processes were done separately for 30, 60, 90 and 120 minutes after adding the
solvent.
Effect of temperature in extraction process
In separate experiments extraction processes were done at 30, 40 and 50 degree Celsius.
Reagents
All reagents required for the experiment were procured from Merck, USA.
Experimental animals
Wistar strain albino rats of both sexes were used for the study. The animals were housed in
colony cages (5 rats/ cage) and were kept for at least one week in the experimental wing of
the animal house (room temperature 25 – 28º C and humidity 60 – 65 % with 12 hours light
and dark cycle) before experimentation. Animals were fed on laboratory diet with water ad
libitum. For each set of experiment10 animals were used. The animal experiment was
approved by the ethics committee of the Institute.
Indomethacin
Indomethacin was collected from Torrent Research Centre, Gandhinagar, India.
Test drug
Brownish mass obtained from extraction process was used as the test drug.
Production of Indomethacin induced gastric ulcer (Parmar & Desai 1993)
Indomethacin (10 mg/kg) was given orally through feeding tube to rats in two doses at an
interval of 15 hours. One hour after the last dose of indomethacin feeding, animals were
sacrificed by cervical dislocation and the stomach was taken out and incised along the greater
curvature. Stomach was then examined for the presence of ulcers.
236 Extraction of anti ulcer compound present from Amaranthus spinosus
Evaluation of ulcer index (Szelenyi & Thiemer 1978)
Gastric lesions were counted and the mean ulcerative index was calculated as follows:
I - Presence of edema, hyperemia and single sub mucosal punctiform hemorrhageII – Presence of sub mucosal hemorrhagic lesions with small erosionsIII – Presence of deep ulcer with erosions and invasive lesions Ulcer index = (number of lesion I) x1 + (number of lesion II) x2 + (number of lesion
III) x 3.
Anti gastric ulcer study
Test drug in the dose of 1g/ kg body weight was given orally through feeding tube 30 minutes
prior to administration of each dose of indomethacin.
Statistical analysis
The values were expressed as mean ± SEM and was analyzed using one-way analysis of
variance (ANOVA) using Statistical Package for Social Sciences (SPSS) 20th versions.
Differences between means were tested employing Duncan’s multiple comparison tests and
significance was set at p < 0.05.
Table – 1 shows the effect of solvents on extraction process for isolation of anti gastric ulcercompound from the leaves of Amaranthus spinosus Linnaeus. It was found that ethanol (50%, v/v) extract of the leaves of Amaranthus spinosus had maximum anti ulcer activityagainst indomethacin induced gastric ulcers in albino rats. Ulcer index was calculated to only10.8 ± 0.7 when indomethacin induced gastric ulcer index in rats was 26.6 ± 1.3. The resultwas statistically significant up to the level of p<0.001. Acetone as well as water extracts hadalso good anti ulcer activity. Anti ulcer activities came 15.5 ± 0.9 for acetone extract and18.3 ± 1.0 for water extract. Methanol and petroleum ether extracts had, however, nosignificant anti gastric ulcer activity against indomethacin induced gastric ulcers in albinorats.
Table 1. Anti ulcer activity of extract of leaves of Amaranthus spinosus Linnaeus against
indomethacin induced gastric ulcers in albino rats : Effect of solvents on extraction process.
Ulcer index
Data shown in mean ± SEM (n = 10). Indomethacin inducedulcer index was 26.6 ± 1.3 *p<0.001 Effect of time on extraction process for isolation of anti gastric ulcer compound from theleaves of Amaranthus spinosus is shown in Table – 2. It appears from the table that antiulcer activity increased with time of extraction. This was up to one hour. Extraction withethanol (50 % v/v) for thirty minutes as well as for one hour gave ulcer index 17.3 ± 1.1 and 10.9 ± 1.3 respectively. There was, however, no significant change in anti ulcer activity evenwhen extraction time was extended up to two hours. Anti ulcer activity after one and halfhours of extraction came 10.8 ± 1.5 and after two hours period of extraction it was 10.9 ±1.2 .
Table 2. Anti ulcer activity of extract of leaves of Amaranthus spinosus Linnaeus against
indomethacin induced gastric ulcers in albino rats : Effect of time on extraction process.
Ulcer index
(minutes)
Data shown in mean ± SEM (n = 10). Indomethacin inducedulcer index was 26.6 ± 1.3 *p<0.001 Table – 3 shows the effect of temperature on extraction process for isolation of anti gastriculcer compound from the of leaves of Amaranthus spinosus. Increase in temperature duringextraction elevated anti gastric ulcer activity. When extraction was done at 30º C ulcerindex came 18.3 ± 1.3, at 40º C it was 10.7 ± 1.0. After that, increase in temperature duringextraction had no effect on anti ulcer activity. Ulcer index came 10.7 ± 1.3 when temperaturewas increased to 50º C.
Table 3. Anti ulcer activity of extract of leaves of Amaranthus spinosus Linnaeus against
indomethacin induced gastric ulcers in albino rats : Effect of temperature on extraction process.
Ulcer index
Data shown in mean ± SEM (n =10).Indomethacin inducedulcer index was 26.6 ± 1.3, *p<0.001 DISCUSSION
Due of its frequency and worldwide distribution peptic ulcer continues to be a subject ofnumerous investigations, both experimental and clinico pathological. In this respect pepticulcer occupies a place secondary to carcinoma in the field of gastroenterology.
There are medicines to treat peptic ulcer (Tierrey et al. 1978). These drugs are broadlyclassified into two categories: 1. Those can decrease or counter acid – pepsin secretion viz. ranitidine, famotidine etc. (H - blockers), pirenzepine, telenzepine etc. (M1– blockers), omeprazole, lansaprazole, pantoprazole etc. (proton pump inhibitors) 2. Those can affect cytoprotection by virtue of their effects in mucosal defense factors like sucralfate, carbenoxolone etc. (Yeomans et al.1998) 238 Extraction of anti ulcer compound present from Amaranthus spinosus No doubt the above mentioned drugs have brought about remarkable changes in peptic ulcer therapy; the efficacy of these drugs is still debatable. Reports on clinicalevaluation of these drugs show that there are incidences of relapses and adverse effectsand danger of drug interactions during ulcer therapy. Hence, the search for an ideal anti –ulcer drug continues and has also been extended to medicinal plants / herbs in search fornew and novel molecules, which afford better protection and decrease the incidence ofrelapse.
Numerous medicinal plants, fruits etc. showed anti gastric ulcer activity. Sanyal et al. (1961) found that vegetable banana is efficacious not only for experimentally induced gastriculcers in albino rats, guinea pigs etc. but also for human being suffering from peptic ulcers.
Akah & Nwafor (1999) demonstrated anti gastric ulcer activity of the herb Cissampelosmucronata A. Rich. Likewise Shetty et al. (2000), Sairam et al. (2001), Maity et al. (1995,2003), Dharmani & Palit (2006) and Malairajan et al. (2008) confirmed anti gastric ulceractivities of Ginkgo biloba Linnaeus, Convolvulus pluricaulis Choisy, tea root extract,Vernonia lasiopus O. Hoffmann and Polyalthia longifolia (Sonnerat) Thwaites respectively.
We also reported anti gastric ulcer activities of few medicinal plants in different experimentalulcer models (Mitra1980, 1981, 1982, 1985, 2001; Mitra & Mitra 2005, 2008; Mitra et al.
2008, 2010, 2011).
Recently we observed anti ulcer activity of the leaves of Amaranthus spinosus against indomethacin induced gastric ulcer in albino rats [unpublished observation]. Weundertook isolation studies to get the active compound present in the leaves ofAmaranthus spinosus. Extraction process is a part of isolation work. Different solventsyield different extracts variable compositions (Zarnowski & Suzuki 2004). Therefore, asuitable extracting solvent should be selected for extraction of the active compound forits maximum activity (Wang & Weller 2006). We thus used distilled water, acetone,ethanol, methanol and petroleum ether separately as solvents for the extraction of theactive compound from leaves of Amaranthus spinosus. Results showed that ethanol(50 %, v/v) extract had maximum anti ulcer activity against indomethacin induced gastriculceration in albino rats. This was followed by acetone extract. Water extract had alsosome anti gastric ulcer activity but methanol and petroleum ether extracts had no antiulcer activities.
As extraction time is important to extract active compound in maximum amount, we allowed extraction time as half an hour, one hour, one and half hours as well as two hours inseparate experiments. It was found that one hour extraction time produced the extract fromleaves of Amaranthus spinosus had maximum anti gastric ulcer activity against indomethacininduced gastric ulcers in albino rats.
The extraction temperature is another important factor influencing the recovery of the bio reactive compound from the source (Wang & Weller 2006). In separate experimentswe thus conducted extraction at temperature stages like 30, 40 and 50 degree Celsius.
Results revealed that extraction of the leaves of Amaranthus spinosus at 40º C had moreanti gastric ulcer activity against indomethacin induced gastric ulcers in albino rats than theextraction temperature at 30º C. Further increase of extraction-temperature, however, hadno significant effect on the anti gastric ulcer activity against indomethacin induced gastriculcers in albino rats.
We are now interested to see the effect of mixture of solvents on the process of extraction. Works in this direction are in progress.
LITERATURE CITED
Akah, P.A. & Nwafor, S.V. 1999. Studies on anti – ulcer properties of Cassampelos mucronata leaf extract. Indian J. Exp. Biol. 37: 936 – 938.
Chopra, R.N. & Chopra, I.C. 1958. Indigenous drugs of India, U.N. Dhar & Sons Private Limted, Kolkata, Page : 668.
Das, A.P.; Ghosh, C.; Sarkar, A. & Choudhury, D. 2010. Hundred Medicinal Plants from North Bengal. University of North Bengal, Siliguri.
Dharmani, P. & Palit, G. 2006. Exploring Indian medicinal plants for anti ulcer activity. Indian J. Pharmacol. 38 : 95 – 99.
Gurung, B. 2002. The medicinal plants of Sikkim Himalaya, Gangtok, Sikkkim, P. 271.
Maity, S.; Vedasiromoni, J.R. & Ganguly, D. K. 1995. Anti – ulcer effect of the hot water extract of black tea (Camellia sinensis). J. Ethanopharmacol. 46: 167 – 174.
Maity, S.; Chaudhuri, T.; Vedasiromoni, J. R. & Ganguly, D. K. 2003. Cytoprotection mediated anti ulcer effect of tea root extract. Indian J. Pharmacol. 35: 213 – 219.
Malairajan P.; Gopalakrishnan, G.; Narasimhan S. & Jessi, K.V.K. 2008. Evaluation of anti – ulcer activity of Polyalthia longifolia (Sonn.) Thwaites in experimental animals.
Indian J. Pharmacol., 40: 126 – 131.
Mitra, P.K.1980. Anti gastric ulcer effect of vegetable banana. Am. J. Herbs.1: 11 – 12.
Mitra, P.K.1981. Influence of a banana supplemented diet on restraint stress in rats. Br. J. Exp. Gastroenterol. 2: 27 – 30.
Mitra, P.K.1982. Effect of Piper longum Linn. on aspirin induced gastric ulcer in albino rats. Bul. J. Herb Res.1: 61 – 64.
Mitra, P.K. 1985. Effect of Emblica officinalis Linn on swimming stress induced gastric ulcer in mice. Arc. Nat. Prod, 1: 81 – 86.
Mitra, P.K. 2001. In search of an anti ulcerogenic herbal preparation. Trans. Zool. Soc. Mitra, P. & Mitra P.K. 2005. Biochemical studies of the anti ulcerogenic activity of Nirmali (Strychnos potatorum Linn) in restraint induced gastric ulcers in rats. Trans. Zool.
Soc. East. India. 9: 39 – 42.
Mitra, P. & Mitra, P.K. 2008. Use of Astilbe rivularis Buch.– Ham. ex D. Don as anti – peptic ulcer agent. Pleione 2(1):74 – 76.
Mitra, P.K.; Mitra, P. & Debnath, P.K. 2008. Screening the efficacy of some East Himalayan medicinal plants against ethanol induced gastric ulcer in Albino rats. Pleione 2(2): 233– 238.
Mitra, P.K.; Mitra, P; Das, A.P.; Ghosh, C.; Sarkar, A. & Chowdhury, D. 2010. Screening the efficacy of some East Himalayan medicinal plants on ethanol induced gastriculcer in Albino rats. Pleione 4(1): 69 – 75.
Mitra, P.K.; Maitra, T.; Mitra, P.; Paul, B.; Ghosh, D.; Guria, M.; Chowdhury, D. & Das, A.P. 2011. Antiulcer activity of an isolated compound (MK-1) from Murraya koenigii(Linnaeus) Sprengel leaf in rats. . Pleione 5(1): 49 – 55.
Parmar, N.S. & Desai, J. K. 1993. A review of the current methodology for the evaluation of gastric and duodenal antiulcer agents. Indian J Phrmacol. 25: 120 – 135.
240 Extraction of anti ulcer compound present from Amaranthus spinosusSairam, K.; Rao Ch, V. & Goel, R. K. 2001. Effect of Convolvulus pluricaulis Chois on gastric ulceration and secretion in rats. Indian J Exp. Biol. 39: 137 – 142.
Sanyal, A.K. ; Das, P.K.; Sinha, S. & Sinha, Y.K. 1961. Banana and gastric secretion. J. Pharm. Pharmacol. 13: 318 – 319.
Shetty,R.; Vijay Kumar; Naidu, M.U.R. & Ratnakumar, K.S. 2000. Effect of Ginkgo biloba extract in ethanol induced gastric mucosal lesions in rats. Indian J. Pharmacol. 32:313 – 317.
Szelenyi, I. & Thiemer, K. 1978. Distension ulcer as a model for testing of drugs for ulcerogenic side effects. Arch. Toxicol. 41: 99 – 105.
Tierrey, L.M.; Mephee S.J. & Papadakis, M.A. 1978 : In “Current Medical Diagnosis & Treatment”, Pub. Mc Craw Hill, New York.
Wang, L. & Weller, C.L. 2006. Recent advances in extraction of nutraceuticals from plants.
Trends in Food Science and Technology, 17: 300 – 312.
Yeomans N. et al., 1998. Sucralfate and carbenoxolone as mucosal defense factors. N. Eng. J. Med., 338: 719 – 723.
Zarnowski, R. & Suzuki, Y. 2004. Expedient soxlet extraction of resorcinolic lipids from wheat grains. J. Food Comp. Anlys. 17: 649 – 664.

Source: http://www.ehsst.org/26%20PK%20Mitra_Final.pdf