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In vivo and in vitro anti-inflammatory potential of pentahydroxy-pregn-14-ol, 20-one-[Beta]-D-thevetopyranoside in rats.



Wattakaka volubilis Linn. (Stap.f.)

Polyhydroxy pregnane glycoside (PPG)

Carrageenan-induced inflammation

Cotton pellet granuloma

N-acetyl- [beta] -D-glucosaminidase




Polyhydroxy pregnane glycoside (PPG), a steroidal glycoside was isolated from Wattakaka volubilis Linn. (Stap.f.). PPG was evaluated for in vivo and in vitro anti-inflammatory activity using acute inflammation and chronic model of inflammation in rats and LPS-induced RAW 264.7 macrophage cells. PPG seemed to be responsible for the anti-inflammatory activity in the studied models. PPG at dose level of both 5 and 10 mg/kg significantly reduced the edema induced by the carrageenan in acute model of inflammation. It also showed significant anti-proliferative effect (dry pellet weight basis) in chronic model of inflammation. Cellular content of granuloma was measured by assaying activity of N-acetyl glucosaminidase (NAG) and total nucleic acid content. PPG at 5 and 10 mg/kg significantly suppressed the cellular infiltration measured by total nucleic acid content. In contrast. NAG activity decreased over a period of 10 clays resulting in inhibition of granuloma weight gain. PPG had a more effective response than the reference drug diclofenac sodium in both the models of inflammation. Wattakaka volubilis steroidal glycoside mixture (WVSM) and PPG (1-50[micro]M) significantly inhibited the COX-2 and iNOS enzymes resulting in low levels of [PGE.sub.2] and NO in LPS-induced RAW 264.7 macrophage cells. Hence the study supports the traditional use of Wattakaka volubilis and its constituent PPG in treatment of inflammatory disorders.

[c] 2013 Elsevier GmbH. All rights reserved.

Introduction Inflammation generally occurs in response to tissue injury and is associated with the release of different mediators like bradykinin, nitric oxide (NO), vasoactive amines (histamine. serotonin, adenosine), interleukin 1 (IL-1), tumor necrosis factor alpha (TNF-[alpha]) and eicosanoids (prostaglandins. thromboxanes, leukotrienes, lipoxins (Bennett and Brown 2003). The subcutaneous injection of carrageenan causes extravasations of plasma resulting in increased exudation of water and plasma proteins along with infiltration of neutrophils (Szolcsanyi et al. 1998). Lipopolysaccharide (LPS), derived from the cell wall of gram negative bacteria, activates multiple signaling pathways in macrophages and enhances production of inflammatory mediators (Chao et al. 2009). Macrophages produce pro-inflammatory response cytokines such as TNF-[alpha], IL- [beta] which activates NF-[kappa]B, leading to expression of inflammatory genes such as cyclooxygenase (COX) and inducible nitric oxide synthase (iNOS).

In Ayurveda. Wattakaka volubilis (L.f.) Stap.f. is extensively used to treat inflammation, piles, leucoderma, asthma, tumors, urinary discharge, etc. (Kirtikar and Basu 1935). Wattakaka volubilis (L.f.) Stap.f. is a large twining shrub having woody vines, widely distributed in India. Sri Lanka, Myanmar, Indonesia, Thailand and China. Present study was undertaken to investigate the anti-inflammatory property of Wattakaka volubilis and its constituents in rats and LPS-induced RAW 264.7 macrophage cells.

Materials and methods

Extraction and isolation of PPG

Wattakaka volubilis (L.f.) Stap.f. leaves were collected from Khanapur forest, Bidar, Karnataka. India during August-September 2009. Authentication was done by Y.N. Seetharam. Professor of Botany, Gulbarga University, Gulbarga (HGUG No. 83). 5g of the Wattakaka volubilis steroidal glycoside mixture (WVSM) was loaded on to the column and was eluted with the different proportions of hexane and chloroform. The fraction 3:7 obtained was subjected to determination of melting point and spectral analysis mainly ID and 2D NMR studies.


Male Swiss albino rats (Central Animal House, Luqman College of Pharmacy. Gulbarga) weighing 200-250g, were housed 6 per cage (320 cm x 180 cm x 160cm) under a normal 12h: 12h light/dark schedule with the lights on at 07:00 a.m. and had free access to tap water and food pellets. Ambient temperature and relative humidity were maintained at 22 [+ or -] 2 C and at 55 [+ or -] 5 C, respectively. They were allowed at least one week to adapt to the laboratory environment before experiments. All studies were carried out in accordance with the CPSCE and Institutional Animal Ethical Committee (IAEC-No-346), minimizing animal suffering.

Drug administration

Different groups of rats were used for drug treatment. Drugs were dissolved or dispersed in saline. All doses were expressed as mg/kg body weight of the respective drugs. Food, but not water, was withdrawn from the animals 1 h prior to drug administration. PPG (5 and 10 mg/kg), standard drug diclofenac (5 mg/kg) and saline (15 ml/kg) were administered by gastric gavage daily once at 11:00 a.m. to 12:00 p.m. to different groups of animals.

Carrageenan induced model of acute inflammation

The acute inflammatory response was measured using carrageenan (0.1 ml, 1%, w/v, in normal saline, Winter et al. 1962) wherein rats were divided into seven groups (n=6 per group). One hour prior to carrageenan injection, the control group (Group-I) received normal saline, Group-II was treated with diclofenac (5 mg/kg) and Group-III and -IV received PPG (5 mg/kg and 10 mg/kg body weight respectively) p.o. The paw volumes were measured immediately before and 1-24 h following carrageenan injection using a plethysmometer with slight modifications and the percentage inhibition was calculated.

Cotton pellet granuloma model of chronic inflammation

The anti-proliferative studies were carried out using cotton pellet granuloma in rats using the method of Swingle and Shideman (1972). Albino rats of either sex weighing 150-200g were used. (Group-I) received normal saline, Group-II was treated with diclofenac (5 mg/kg) and Group-III and -IV received PPG (5 mg/kg and 10 mg/kg body weight respectively) p.o. Two sterilized pellets of cotton weighing 35 [+ or -] 1 mg were implanted subcutaneously, one on each side along the flanks of the animal, under anesthesia and sterile conditions.

Sampling and extraction of granuloma

Rats were killed by asphyxiation in carbon dioxide. Cotton pellets and the accompanying granulomatous tissues were removed from the rats with a pair of forceps by reflecting the overlying skin and removing the granuloma together with a small amount of any capsular material present. One granuloma was placed in a glass petri dish, air dried at 60 C for 18 h. and weighed. The other granuloma was used for biochemical assays and was frozen in dry ice and kept frozen at -10 C until processed. The granuloma was placed in 9.0 ml of ice-cold 0.9% saline containing 0.1% (v/v) Triton X-100 and was then subjected to disintegration. The cotton and other solid material were pelleted by centrifugation at 1500 x g for 10 min, with two centrifugations usually necessary to remove all the cotton. NAG and nucleic acid content were determined directly from the supernatant fractions.

Determination of N-acetyl glucosaminidase (NAG) activity and nucleic acid levels

NAG activity was based on the hydrolysis of p-nitrophenyl-N-acetyl-D-glucosamide in a modified procedure. The supernatant was mixed with the substrate (2.24 mM p-nitrophenyl-N-acetyl-[beta]-D-glucosamide). The mixture was subjected to incubation at 37 C for 15 min. The reaction was arrested by addition of 0.2M glycine buffer (pH 10.4). The mixture was used to record the absorbance at 400nm in a spectrophotometer (Elico India, SL 117), where the p-nitrophenol liberated by the action of the enzyme on the substrate was measured. From the height of the tracing and extinction coefficient of the p-nitrophenol under the actual conditions of experiment, enzyme activity was calculated and is expressed as micromoles p-nitrophenol liberated per hour.

The nucleic acid content of the granuloma was determined flourimetrically. The method is specific for DNA and RNA and is based on original ethidium bromide reaction developed by Blackburn et al. (1972). Equal amounts of supernatant and ethidium bromide (20 [micro]g/ml) in calcium free Krebs medium (pH 7.4) were mixed and incubated for 10 min at room temperature. The incubated mixture was subjected to spectroflourimeter (Shimadzu RF 5301) and the relative fluorescence measured. A series of DNA standards were subjected to the same procedure and a standard curve (which is linear past 100 [micro]g/ml), the amount of DNA and RNA were determined and expressed as microgram of total nucleic acids per milliliter.

Cell lines and culture medium

Macrophage cell line RAW 264.7 was procured from National Centre for Cell Sciences (NCCS), Pune, India. It is a suspension culture and stock cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% inactivated fetal bovine serum (FBS), penicillin (100 IU/ml), streptomycin (100 [micro]g/ml) and ampho-tericin B (5 [micro]g/ml) in a humidified atmosphere of 5% C[O.sub.2] at 37 C until confluent. The confluent cell suspension was centrifuged at 2000 rpm for 10 min and the cell pellet was re-suspended in fresh medium. The stock cultures were grown in 25[cm.sup.2] culture flasks and all experiments were carried out in 96 micro titer plates (Tarsons India Pvt. Ltd., Kolkata, India).

Nitrite. [PGE.sub.2]. iNOS and COX-2 measurements

Macrophage cell line RAW 264.7 was cultured and cell viability assay was conducted. The cells were treated with different concentrations of Wattakaka volubilis steroidal glycoside mixture (WVSM) and PPG (1-50 [micro]M) for 24 h. The percentage of viable cells was calculated with respect to cells treated with 0.1% DMSO. Solvents used at these concentrations showed no toxicity on cell viability (>80%). DMSO at <0.1% presented 95% cell viability.

Cell treatment was conducted by seeding 2 x [10.sup.5] cells in a 6-well plate and allowed to grow for 48 h at 37 C in 5% C[O.sub.2]/95% air. After 48 h incubation, cells were treated with Wattakaka volubilis steroidal glycoside mixture (WVSM) and PPG (1-50 [micro]M) followed by 1 [micro]g/ml of lipopolysaccharide for 24h. After 24h treatment, the spent media was collected and analyzed for NO and [PGE.sub.2] Cell lysates were used to study the effect of PPG on the expressions of iNOS and COX-2 by using kits.

Statistical analysis

Results were calculated as mean [+ or -] SEM where appropriate, one-way ANOVA followed by post hoc Dunnet's t-test was employed. Values p <0.05 were considered significant, using Instat @ Graph Pad Software.


Polyhydroxy pregnane glycosides

The phytochemical analysis of the colorless crystals formed in the fraction 3:7 from Wattakaka volubilis glycoside mixture revealed the presence of steroid by answering the Libermann-Burchard test for triterpenoids. The purity of isolated compound was confirmed through HPLC and was found to be free from impurities. The molecular formula of the PPG was determined to be C21 H30 06 (allomethyllose-thevetose and tigloyl are not included in the formula) by MS-MS (positive mode found m/z 471.2713, calcd for [M+Na] 471.2723) and [.sup.13]C NMR spectral data (Fig. 1).

Carrageenan-induced edema

Both the doses of PPG (5 and 10 mg/kg) showed a significant increase in percentage reduction of paw volume when compared to control, with anti-edematogenic activity seen as early as 1/2 h of induction of inflammation (Table 1). Diclofenac sodium (5 mg/kg) exhibited a significant inhibition of edema at all the studied time intervals. Treatment with PPG at 10 mg/kg body weight showed anti-edematogenic activity superior over the diclofenac treated group.

Table 1

Effects of PPC isolated from Watakaka volubilis L.f. (Stap.f.)
on carrageenan-induced hind paw edema in rats.

             Mean                                    %
              paw                           Inhibition
            in mm

Treatment   1/2 h    1 h       2 h     4 h       1/2 h   l h   2 h   4 h

Control     0.365  0.342   0.338[+   0 326           -     -     -     -
            [+ or  [+ or     or -]   [+ or
               -]     -]      0.23      -]
             0.04   0.21              0.21

Diclofenac  0.225  0.210  0.200 [+   0 150        35.2  38.1  46.9  50.6
(5mg/kg)    [+ or  [+ or     or -]   [+ or                             *
               -]     -]      0.07      -]
             0.01   0.02              0.21

PPC         0.250  0.205  0.182 [+  0 .160        28.4  40.3  45.6  48.1
(5mg/kg)    [+ or  [+ or     or -]   [+ or
               -]     -]      0.06      -]
             0.09   0.09              0.40

PPG         0.220  0.195  0.175 [+   0.151        34.5  43.8  48.8  51.2
(10mg/kg)   [+ or  [+ or     or -]   [+ or                             *
               -]     -]      0.01      -]
             0.08   0.03              0.03

PPG, polyhydroxy pregnane glycoside. Values are mean [+ or -] SEM.
* p < 0.01

Cotton pellet granuloma

A significant anti-proliferative effect was seen in the PPG treated groups that were far superior to diclofenac sodium used as a standard reference drug at a dose of 5 mg/kg (Table 2). The mean weight of the dried pellets implanted under the skin in the rats of control group was found to be increased when compared to the diclofenac sodium (5 mg/kg).

Table 2

Effect of PPC isolated from Watakaka volubilis
L.f. (Stap.f.) on cotton pellet granulorna in rats.

Treatment           Net dry pellet  Anti-proliferative
                       weighi (mg)              effect

Control         38.2 [+ or -] 3.12                  --

Diclofenac      20.8 [+ or -] 2.45               45.89
(5 mg/kg)

PPG (5mg/kg)    16.7 [+ or -] 1.54               55.12

PPG (10mg/kg)  13.78 [+ or -] 1.25               62.74

PPG, polyhydroxy pregnane glycoside. Values are mean
[+ or -] SEM.
* p < O.O1

Levels of NAG activity and nucleic acid levels

NAG and nucleic acid levels of the granuloma were affected by treatment with diclofenac sodium (5 mg/kg) and PPG (5 mg and 10 mg/kg) (Fig. 2A and B). The effect of PPG at 10 mg/kg showed a significant lowering of NAG and nucleic acid levels. whereas the PPG at 5 mg/kg and the diclofenac sodium at 5 mg/kg showed a similar effect in lowering the NAG and nucleic acid levels resulting in effective inhibition of the development of granuloma.

Nitrite. PGE2, iNOS and COX-2 measurements in LPS-induced RAW 264.7 cells

Macrophages showed a survival rate of >90% when incubated with WVSM and PPG at concentration [less than or equal to] 50[micro]M. WVSM had no effect of NO production and iNOS activity at 1 and 10[micro]M whereas 25 and 50 [micro]M showed a reduction in NO levels and inhibition of iNOS activity (Fig. 3A and B). But PPG produced a dose-dependent manner reduction of NO production and inhibition of iNOS activity. Moreover WVSM and PPG produced a dose dependent manner inhibition of COX-2 resulting in reduction in PGE2 production (Fig. 3C and D).


Present investigation illustrates an in vivo and in vitro anti-inflammatory activity of steroidal glycoside, PPG isolated from Wattakaka volubilis. PPG seemed to inhibit both the phases of carrageenan induced edema, since the inhibitory response was seen from 1/2 h of irritant injection till the 4th hour, indicating that PPG is inhibiting the release of inflammation mediators. PPG which has been isolated in our studies seems to be the structural analog of the steroids and glucocorticoids. Oleanane-type triterpenoids have been shown to be reducing NO (nitrous oxide), PGE2 (prostaglandin E2), and TNF-[alpha]production by inhibiting COX-2 (cyclooxygenase 2) and iNOS (inducible nitric oxide synthatase) activity and down-regulating nuclear factor (NF)-[kappa]B. Possibly PPG is exhibiting the anti-inflammatory effect by inhibiting COX-2, iNOS, down-regulating (NF)-[kappa]B, thus reducing NO, PEG2. Diclofenac also seems to act by inhibiting both phases of carrageenan-induced edema. But PPG (10 mg/kg) seems to be more potent anti-inflammatory compound than the diclofenac sodium (5 mg/kg). The present results indicate a significant anti-inflammatory activity of PPG isolated from Wattakaka volubilis in cotton pellet granuloma and found to be effective in chronic inflammatory conditions, which reflects its efficacy in inhibiting the increase in the number of fibroblast and synthesis of collagen and mucopolysaccharides during granuloma tissue formation as reported by Recio et al. (1995).

This investigation also reports the in vitro anti-inflammatory activity in LPS-induced RAW 264.7 macrophage cells. Excessive production of NO, especially in macrophages, can lead to cytotoxicity, inflammation, carcinogenicity, and autoimmune disorders. Therefore, modulation of NO and iNOS can prevent inflammatory diseases. In this study. PPG was found to significantly suppress LPS-induced NO production in RAW 264.7 cells, due to its ability to inhibit the expression of iNOS. Inhibition of iNOS/NO signaling pathway is one strategy for anti-inflammatory drug development. PPG inhibited the inducible enzymes, iNOS and COX-2, suggesting that saponins may have additional chemopreventive potentials. It has been reported that saponins suppress the expression of COX-2 and Inos, resulting in a marked lowering of [PGE.sub.2] levels (Kim et al. 2006).

In conclusion PPG isolated from Wattokoka volubilis possesses anti-edematogenic activities, the anti-inflammatory potency of the PPG exceeded/equaled the reference drug diclofenac sodium, thus it can be developed further for long term symptomatic treatment involving inflammation.

* Corresponding author. Tel.: +919341806110; fax: +91 8472263202.

E-mail address: (P.L. Swamy).


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Chao. W.W., Kuo, Y.H., Li, W.C., Lin. B.F., 2009. The production of nitric oxide and prostaglandin E2 in peritoneal macrophages is inhibited by Andrographis paniculata. Angelica sinensis and Morns alba ethyl acetate fractions. Journal of Ethnopharmacology 122, 68-75.

Kim, J.Y., Hwang. Y.P., Kim. D.H., Han. E.H., Chung. Y.C., Roh, S.H., 2006. Inhibitory effect of the saponins derived from roots of Platycodon grandiflorum on carrageenan-induced inflammation. Bioscience. Biotechnology, and Biochemistry 70, 858-864.

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Recio, M.C., Giner, R.M., Manez. S., Rios, J.L., 1995. Structural requirement for the anti-inflammatory activity of natural triterpenoids. Planta Medica 61. 182-185.

Swingle, K.F., Shideman, F.E., 1972. Phases of the inflammatory response to subcutaneous implantation of a cotton pellet and their modification by certain anti-inflammatory agents. Journal of Pharmacology and Experimental Therapeutics 183, 226-236.

Szolcsanyi. J., Helyes, Z., Oroszi, G., Nemeth, J., Pinter. E., 1998. Release of somatostatin and its role in the mediation of the anti-inflammatory effect induced by antidromic stimulation of sensory fibers of rat sciatic nerve. British Journal of Pharmacology 123, 936-942.

Winter, C.A., Risley, E.A., Nuss, G.W., 1962. Carrageenan induced edema in hind paw of rats as assay for anti-inflammatory drugs. Experimental Biology and Medicine 111, 544-547.

Ram S. Jadhav (a), Md. Liyakat Ahmed (b), Paramjyoti L. Swamy * (a), Syed Sanaullahh (b)

(a) Department of Biochemistry, Gulbarga University. Gulbarga, Karnataka. India

(b) Department of Pharmacology, Luqman College of Pharmacy, Gulbarga. Karnataka, India
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Title Annotation:Short communication
Author:Jadhav, Ram S.; Ahmed, Md. Liyakat; Swamy, Paramjyoti L.; Sanaullah, Syed
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9INDI
Date:Jun 15, 2013
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