Isolation and Characterization of Phenolic Glycoside from the Bark of Symplocos Racemosa Roxb

A new phenolic glycoside, 3, 5 dihydroxy 2(hydroxyl methyl) 6-(3,4,5-trimethoxy phenoxy)tetrahydro-2H-pyran-4-yl, 4-hydroxy-3-methoxy benzoate have been isolated from the dried bark of Symplocos racemosa. The structure was identified by extensive spectral analysis, especially FT-IR, GCMS, H NMR and C NMR techniques. The method of isolation was simple, cost effective and efficient. The preliminary bioactivity of the compound was also evaluated. The ethanolic extract of Symplocos racemosa (EESR) was investigated for its anti-pyretic activity against brewer’s yeast induced pyrexia. The antipyretic effect of EESR (measured as % reduction in body temperature) was compared with paracetamol (100 mg/kg, orally). EESR in dose of 200 mg/kg caused significant decrease in body temperature of rats. This study has established the antipyretic activity of Symplocos racemosa and thus, justifies the ethnic uses of the plant.


Introduction
Symplocos racemosa roxb.(Fam.symplocaceae) is a widely used ayurvedic remedy for various ailments.It is also known as lodhra and is used in Indian System of Medicine (ISM) as single drug or in multicomponent preparations (viz.lodhrasava).It's bark is acrid, digestible, astringent to bowels.It is useful in treatment of fever, eye diseases, for spongy gums and bleeding.It cures 'Kapha', diseases of the blood, leprosy, dropsy and liver complaints 1,2 .It is also useful in abortions and miscarriages and for ulcers of vagina.Traditionally bark is given in menorrhagia and other uterine disorders.Unani medicine uses it as emmenogogue, aphrodisiac 3 .It is a potent remedy for inflammation and cleaning uterus.This is used to treat leucorrhea and menorrhagia 4 .It contains salireposide and benzoylsalireposide which are inhibitors 5 of phosphodiesterase I and have showed its depressant action on blood pressure and instestinal movements 6 .
Pyrexia or fever is caused as a secondary impact of infection, malignancy or other diseased states.It is the body's natural defense to create an environment where infectious agent or damaged tissue cannot survive 7 .Normally the infected or damaged tissue initiates the enhanced formation of pro-inflammatory mediator's (cytokines like interleukin 1β, α,β and TNF-α), which increase the synthesis of prostaglandin E2 (PGE2) near preoptic hypothalamus area and thereby triggering the hypothalamus to elevate the body temperature 8 .As the temperature regulatory system is governed by a nervous feedback mechanism, so when body temperature becomes very high, it dilate the blood vessels and increase sweating to reduce the temperature; but when the body temperature becomes very low hypothalamus protect the internal temperature by vasoconstriction.High fever often increases faster disease progression by increasing tissue catabolism, dehydration and existing complaints, as found in HIV 9 .Most of the antipyretic drugs inhibit COX-2 expression to reduce the elevated body temperature by inhibiting PGE-2 biosynthesis 10 .Moreover, these synthetic agents irreversibly inhibit COX-2 with high selectivity but are toxic to the hepatic cells, golmeruli, cortex of brain and heart muscles, whereas natural COX-2 inhibitors have lower selectivity with fewer side effects.A natural antipyretic agent with reduced or no toxicity is therefore, essential.As bark of symplocos racemosa is used in ailments that caused fever 11 , so it will be a cost effective alternative approach to study this plant for the development of an effective antipyretic agent.

Plant material
The plant Symplocos racemosa (Family: symplocaceae) was collected from Kolli Hills at Namakkal District, Tamilnadu, India.It was authenticated by Dr. V. Sathyanathan, Taxonomist, Epoch Pharma and Research Labs Pvt. Ltd.Chennai and its voucher specimens were deposited in the Herbarium for further reference.

Extraction and isolation of plant materials
After proper identification, the bark of Symplocos racemosa was dried under shade and then coarse powdered with a mechanical grinder.The coarse powder was stored in an airtight container for further use.The dried coarse powder material of bark (250 g) was extracted with ethanol (95%) for 72 h in soxhlet apparatus.The extract was made solvent free distillation process under reduced pressure and the resulting semisolid residue was vacuum dried using rotary flash evaporator.The yield of the ethanolic extract was 18.28% w/w and it was used for the isolation and study of antipyretic activity.

Animals
Male albino Wistar rats of body weight 150-250 g were obtained from the Sri Venkateshwara Enterprises, Bangalore and were maintained in J.K.K. Nataraja College of Pharmacy animal house.The animals were housed in well ventilated large spacious polypropylene cages and had 12 ± 1 h light and dark cycles throughout the experimental period.The animals received a balanced diet of commercially available pellet rat feed and water ad libitum.The Guidelines for Breeding and Experiments on Animals, 1998 defined by the Ministry of Social Justice and Empowerment of India were followed and the protocol was approved by the Institutional Animal Ethics Committee (887/ac/05//CPCSEA).

Acute toxicity studies
Ethanolic extract of Symplocos racemosa was studied for acute oral toxicity as per revised OECD (Organization for Economic Cooperation and Development) guidelines No. 423.The extract was devoid of any toxicity in rats when given in doses up to 2000 mg/kg by oral route.Hence, in our study 100 and 200 mg/kg doses of extract were dissolved in 0.1% Carboxy Methyl Cellulose (CMC) and used for the study 12 .

Screening for antipyretic activity
The antipyretic activity of EESR was evaluated using Brewer's yeast-induced pyrexia in rats.Fever was induced by administering 20 mL/kg of 20% aqueous suspension of Brewer's yeast in normal saline subcutaneously.The EESR (100 and 200 mg/kg, orally) was administered orally, paracetamol (100 mg/kg, orally) was used as reference drug and control group received distilled water.Rectal temperature was determined by thermal probe Eliab thermistor thermometer at 1, 2 and 3 hrs after test extract/reference drug administration 13 .

Statistical analysis
Data were recorded as mean ± SEM.The statistical significance of differences between groups was determined by analysis of variance (ANOVA), followed by Dunnett's test for multiple comparisons among groups.Differences of p<0.05 were considered statistically significant.The aromatic hydrogen (Ar H-2,6,2'', 6'', 5'') appeared as broad unresolved multiplets in the region between 6.22 -6.92.The hydroxyl groups appeared as broad peaks at 7.24 and 9.28 respectively. 13C NMR spectra showed resonance that was readily attributed to β-glucose and two phenolic esters.

Results and Discussion
The results of effect of EESR on yeast-induced pyrexia in rats are depicted in Table 2. EESR produced significant (P< 0.05) antipyretic effect in a dose dependent manner.Normal rats did not show any decrease in the body temperature on intraperitoneal administration EESR.The initial and final rectal temperature ( 0 C) for two groups of extract administered

Conclusion
Search for safe herbal remedies with potent antipyretic activity received momentum recently as the available antipyretic, such as paracetamol, aspirin, nimusulide etc. have toxic effect to the various organs of the body 14 .The acute toxicity result reveals that this plant might be considered as a broad non-toxic one.The antipyretic activity exhibited that the both doses of ethanol extract of barks possess a significant antipyretic effect in maintaining normal body temperature and reducing yeast induced fever in rats and their effect are comparable to that of standard antipyretic drug paracetamol.Such reduction of rectal temperature of tested animals by the both doses of EESR appears to be due to the presence of a single bioactive principles or mixture of compounds in them.The phytochemical analysis of the EESR showed the presence of carbohydrates, sterols, glycosides, alkaloids, phenols and saponins.The antipyretic activity observed can be attributed to the presence of steroids.Moreover, in many studies, glycosides, alkaloids have been reported to exhibit antipyretic effect [15][16][17] .It was also evident from the study that the antipyretic activity of EESR at 100 mg/kg body weight is almost similar to the standard paracetamol group and more than the 200 mg/kg body weight dose.The present study, therefore, supports the claims of traditional medicine practitioners as an antipyretic remedy.However, to know the exact mechanism of action of EESR further study with purified fractions/ bioactive compounds are warranted.
Antipyretic effect

Table 2 .
.85 ± 2.16 and 37.64 ± 2.15 (100 mg/kg); and 38.28 ± 1.14 and 37.15 ± 1.59 (200 mg/kg).Based on the result, it can be concluded that EESR produced antipyretic effect.Effect of EESR on Brewer's yeast induced pyrexia in rats Figure 1.Effect of EESR on body temperature of control and experimental group of rats