Evaluation of Anti Arthritic Potential of Gloriosa superba ( L . ) Elite Germplasm Collected from Eastern Himalayas , India

Introduction: Gloriosa superba (L.) is a traditionally known medicinal plant for its potential antigout property. The species is rich source of colchicine alkaloid and is commercially exploit in the international market for the same. Method: In the present study, elite chemotype of G. superba was identified from natural population in Eastern Himalayas based on their colchicine content through HPTLC calibrated method. The selected elite chemotypes were further evaluated for in vitro anti-arthritic potential via inhibition of protein denaturation along with hydroxyl radical scavenging potential. Result: The HPTLC quantification data reveals that the content of colchicine varies from 0.044 to 0.184% having maximum content in NBG-128 from Jorhat, Assam. The results of bioassay reflect a potentiating anti-arthritic and hydroxyl radical scavenging with statistically insignificant difference within the elite germplasms. Conclusion: The presence of bioactive polyphenolics with significant hydroxyl radical scavenging will further suggest that inhibition of inflammatory mediator cells by extract is superimposed action of colchicine and other chemical inhibitors like polyphenolics. The study will aid in site specific exploration of high metabolite yielding chemotype(s) with validated pharmacological action for commercial cultivation to meet out the industrial demand of colchicine and herbal product development.


INTRODUCTION
Gloriosa superba L. (Colchicaceae) is a tuberous climbing herb found throughout tropical parts of India up to an altitude of 1800 m and also distributed in other tropical parts of the World.Indigenously Gloriosa is known as 'kalihari' in Hindi, ' Agnishikha' in Sanskrit and its trade name is "Glory lily". 1 The species exhibits various medicinal properties viz.analgesic, anti-inflammatory, antimicrobial, larvicidal, antipoxviral, antithrombotic, antitumor and, also used in treatment of snake bite, gout, respiratory disorders. 2,3,4loriosa superba is a potential source of phenethylisoquinoline category of alkaloids, among which colchicine is the major metabolite for which Gloriosa is industrially exploited from time long and hence became a threatened species.Other chemical constituents in tubers are lumicolchicine, 2-demethylcolchicine, 3-demethylcolchicine, N-formyl-N-deacetyl-colchicine, benzoic acid, salicylic acid, sterols, resinous substances, colchicoside, gloriosine, tannins and superbine.5,6 The chemotaxonomic studies of G. superba were scarcely documented.7,8,9 We had reported the variations of colchicine content in G. superba L., germplasms collected from Central India using OPLC technique.10 The use of colchicine in gout was well established by several biochemical and clinical studies, 11,12,13,14,15 however scientific validation of G. superba for its traditional claim in gouty arthritis is still very less.16 Thus, looking towards the commercial relevance of colchicine, the study was aimed to record the intra-specie variation in colchicine content of G. superba population from Eastern Himalayas.This was further extended to validate the pharmacological potential i.e. antiarthritic activity of elite germplasm selected from the population.In addition, antioxidant activity of elite germplasms was also established.This will aid in site specific exploration of high colchicine yielding chemotype(s) with validated pharmacological action to meet out the medicinal and commercial demands.

In vitro anti arthritic activity Inhibition of protein denaturation
Inhibition of protein denaturation by test extract was evaluated by the method with slight modification. 22,23450 μL of Bovine Serum Albumin (5% aq.Solution) was added to 50 μL of test solution (methanol extract, 0.05 mg/ml).The reaction mixture was kept at 37ºC for 30 min, followed by heating at 50ºC for 10 min.In the resulting solution 2.5 ml of phosphate buffer (pH 6.5) was added and allowed to cool down at room temperature, absorbance was than recorded at 660 nm.Acetyl salicylic acid was taken as a positive control.The experiment was carried out in triplicates and percent inhibition for protein denaturation was calculated using, % Inhibition=100−((A t -A c )/A t )*100).Where A t is the absorbance of the sample, Ac is the absorbance of the product control.

Statistical analysis
Results were reported as means ± standard deviation (S.D) of three replicates of the same extract.Data were subjected to one-way analysis of variance (ANOVA) to test the level of significance (XLSTAT, 2010, Microsoft Corporation, USA).The cluster analysis was done by PAST 2.15 for the selection of elite chemotypes and clustering of samples.

HPTLC quantification of colchicine
Seven germplasm of G. superba (tuber) were collected from the phytogeographical region of Eastern Himalayas of India, exhibiting the altitudinal variation from 94.48 to 1162.812 meter Table 1.Chemical profiling and method optimization for quantification of colchicine were carried out on 20 cm × 10 cm TLC aluminum precoated plates with 200 nm layer thickness of silica gel 60 F 254 (SD.Fine-Chem Ltd., Mumbai, India).Tracks (standard and sample) were applied as 6 mm bandwidth using Camag 100 μl sample syringes (Hamilton, Switzerland) with a Linomat 5 applicator (Camag, Switzerland) under a flow of N 2 gas.The linear ascending development was carried out with chloroform: acetone: diethylamine (5:4:1 v/v/v) as a mobile phase in a Camag glass twin trough chamber. 24fter development, the plates were air dried for 30 min, and scanning was performed using Camag TLC Scanner 3 at λ max of 350 nm in UV absorbance-reflectance mode operated by win CATS software (version 3.2.1)for the colchicines content.Quantification was performed using regression analysis of peak area versus concentration of standard marker in the range of 100-400 ng/band having statistically, acceptable regression coefficient (r 2 ) of 0.9987.The slope and intercept of linear line was 14.837 and 417.63 with statistically acceptable LOD and LOQ Table 2. Quantification data (% dry wt. of sample) reveal that the content of colchicine varies from 0.044 to 0.184 % Figure 1, maximum content was observed in NBG-128 from Jorhat, Assam.While minimum content of colchicine was observed in NBG-24 and NBG-25 from 6th mile, Kalimpong, Darjeeling and Kalimpong, West Bengal.Branch II was further bifurcated and separating the one germplasm, NBG-128 as elite chemotype having highest content of colchicine.Beside this, NBG-23 and NBG-24 having lowest content of metabolite was also selected with an approach that the pharmacological response may be due num plates 60 F 254 , (0.25 mm) were purchased from Merck (Darmstadt, Germany).

Plant material and extraction protocol
The tubers were collected during the month of September from North-Eastern Himalayan region covering Sikkim and the adjoining area of West Bengal.Five samples were collected from varied condition(s); specimens were authenticated and deposited in repository of CSIR-NBRI with individual voucher number.GPS information of the collected samples is recorded and summarized in Table 1.
The samples were washed with water, chopped, and shade dried.The dried tubers were coarsely powdered by passing through 40 mesh sieves (up to 500 mm), and the powdered sample (about 5 g) is defatted using petroleum ether by maceration method (cold) to remove the fatty materials/impurity.The defatted material was then macerated (cold method) with methanol (25 ml) for 24 h at room temperature (25°C ± 2°C) for 3 consecutive days.Extraction was repeated thrice, filtered (Whatman no.4), and the pooled filtrate was dried in a rotatory evaporator (Buchi, USA) under standard conditions of temperature (55°C ± 2°C) and pressure (40 mbar).The concentrated extract was finally lyophilized to solid residue (Labconco, USA).

HPTLC quantification of bioactive metabolite colchicine
The collected samples of G. superba were quantified for bioactive alkaloid(s), colchicine through HPTLC calibrated method as per ICH guidelines (2005). 17Quantified markers are expressed as % dry weight of crude samples.

In vitro anti-oxidant activity Estimation of total phenolics
Assay of total phenolic 18 (TPC) and total flavonoid 19 (TFC) content was quantified by spectroscopic method as per standard protocol based on regression curve of reference standard.Total phenolic and flavonoid content was expressed in mg/g of gallic acid equivalent (GAE) and quercetin equivalent (QE).

Hydroxyl radical scavenging activity (HRSA)
Hydroxyl radical scavenging activity of samples was determined according to the standard method 20 with slight modification.The reactive mixture, containing methanolic extract (0.2 mg/ml) in variable concentration (20 -100 µl) was incubated with 0.1 ml EDTA (0.1 mM), 0.01 ml of ferric chloride (10 mM), 0.36 ml of deoxy ribose (10 mM), 0.33 ml of phosphate buffer (pH 7.9, 50 mM) and 0.1 ml ascorbic acid (1 mM).The reaction was initiated by addition of 0.1 ml of H 2 O 2 (10 mM), followed by incubation at 37 0 C for 1h.An aliquot of 1.0 ml of the incubated mixture was mixed with 1.0 ml of 10 % trichloro acetic acid and 1.0 ml of 0.5 % thio barbituric acid (in 0.025 M NaOH) to develop the pink chromogen measured at 532 nm.The hydroxyl radical scavenging activity of the extract is reported as % inhibition of deoxy ribose degradation.

DPPH radical scavenging activity
The radical scavenging activity of extract was measured using DPPH˙ stable radical. 21As per standards, the reaction mixture contains methanol extract of sample (0.1 ml) and 2.9 ml of freshly prepared DPPH solution (6 x 10 -5 M).The mixture was then shaken vigorously before incubation for a period of 30 min in dark at 27 0 C. The decrease in absorbance at 517nm was recorded and the results were expressed in terms of concentration required for 50% inhibition of DPPH radical by test sample as IC 50 (mg/ml).Quercetin is used as positive control having similar treatment protocol as mentioned above.to synergistic effect and not exclusively due to colchicine content.Thus, three germplasm NBG-24, NBG-25 and NBG-128 are selected for in vitro anti-arthritic activity.

Total polyphenolic content
All the seven samples were quantatively analysed though spectrophotometric method for the estimation total phenolic (TPC) and flavonoid content (TFC) in extract and results reveal that there is significant difference within the samples.The total phenolic content in extract(s) was estimated against calibration curve of standard Gallic acid (0.1 mg/ml) at concentration range of 20 -100 µl using regression equation; y = 95.54x+ 0.384 and coefficient of 0.989.Whereas, total flavonoid content was calculated against calibration curve of quercetin (0.01 mg/ml) at variable dilutions of 0.2 -0.5 µl, using regression equation; y = 74.61x+ 0.058 and r 2 =0.998.The TPC ranges from 0.3 to 2.95%, maximum content was in NBG-25 (Kalimpong, W.B) and minimum was in NBG-52 (Itanagar, Arunanchal Pradesh).The TFC varies from 0.0074 to 0.0335%, maximum was in NBG-27 (Jorethang, West Sikkim) and minimum in NBG-24 from 6th mile Kalimpong, Darjeeling, West Bengal Figure 3.A cluster analysis Figure 4 was performed to segregate the population based on TPC and TFC for identification of elite germplasm, UPGMA dendogram was constructed and it was observed that the population was divided into two branches (I and II).Branch one was further divided to separate NBG-23, NBG-24 and NBG-52 into one group.The other arm (II) of dendogram contains four germplasm among which NBG-25 was distinctively separated out as elite germplasm with considerably higher content of both TPC and TFC.

Variability in anti-arthritic and antioxidant activity
The selected elite germplasm (based on colchicine and polyphenolic content) viz.NBG-24, NBG-25 and NBG-128 were evaluated for anti-arthritic potential.In addition to this, radical scavenging potential via DPPH radical scavenging and hydroxyl radical scavenging assay was also checked.The anti-arthritic potential of elite germplasm was analyzed by inhibition of protein denaturation of Bovine serum albumin, with increase in concentration of test extract the denaturation of albumin protein decreases characteristically.At a concentration range of 0.001 to 0.005 mg/ml, the inhibition (%) in NBG-24, NBG-25 and NBG-128 varies from 25.81 to 67.66 %, 23.21 to 60.18 % and 28.33 to 64.46 % respectively with IC 50 at 0.0031, 0.004, 0.0032 mg/ml respectively.Pure colchicine under the identical in vitro experimental condition exhibit an IC 50 at 0.0048 mg/ml, at a concentration range of 0.001 to 0.005 mg/ml.Aspirin, the positive control of study exhibit the maximum inhibition of 74.64 % at concentration of 200 µg/ml with regression coefficient of 0.989.The elite germplasm was found to have promising radical (Hydroxyl) scavenging activity when tested under standardized protocol.The DPPH radical scavenging assay was performed under the mentioned protocol at stock concentration of 2 mg/ml, the IC 50 of elite germplasm NBG-24, NBG-25 and NBG-128 was at 2.18, 1.62 and 0.531 mg/ml respectively.The inhibition (%) of DPPH free radical by test extract of elite germplasms varies from 1.77 to 8.6 %, 4.08 to 20.18 % and 10.53 to 44.52 % for NBG-24, NBG-25 and NBG-128 respectively.The calibration curve of elite sample was documented in supplementary Figure 1.The Inhibition (%) of quercetin ranges from 12.77 to 72.43 % at a concentration range of 0.002 to 0.026 mg/ml.IC 50 of quercetin was recorded at 5.95 ± 0.115 µg/ml.In the other assay, the hydroxyl radical scavenging activity of elite germplasm were evaluated at stock concentration of 0.05 mg/ml, exhibit a statistically significant regression coefficient Supplementary Figure 2. The inhibition (%) of hydroxyl radical in elite sample varies from 25.81 to 67.66%, 23.21 to 60.18 % and 28.33 to 64.46 % for NBG-24, NBG-25 and NBG-128 having IC 50 at 0.0029, 0.0048 and 0.003 mg/ml respectively.The scavenging activity of quercetin standard (positive control) at a concentration range of 20 -100 μg/ml varies from 10.03 to 82.15% with an IC 50 value of 41.05 μg/ml.

DISCUSSION
It is well evident that the use of Gloriosa superba in gout is due to action of colchicine on several biochemical reactions, majorly on inflammatory mediators.The effect is multifacet and involves at molecular level under lying the cascade of inflammatory action.Specifically, it is attributed due to various proposed actions as disruption of microtubule in neutrophils and inhibiting their migration towards the chemotactic factors, alteration in distribution of adhesion molecules on the surface of both neutrophils and endothelial cells, which significantly inhibit the interaction between WBC and endothelial cells, inhibition of monosodium urate crystal activation of the NLRP3 inflammasome, inhibit the release of IL-1β and suppression of gene expression. 12,13,14,15This however is dependent upon the dose of colchicine and duration of treatment; the effect is at both molecular and genetic level. 25The competitive binding of colchicine to serum albumin was well established 26 and thus supports the selection of appropriate method in study to check the existing variability in targeted pharmaco logical activity.Quantification data reveals significant variation in the colchicine content of collected samples, among which NBG-128 was statistically selected as the elite chemotype with high colchicine content.However pharmacological response of anti arthritic activity was not significantly varied in selected three elite chemotypes viz.NBG-24, NBG-25 and NBG-128.Moreover, the elite germplasm(s) exhibit more prominent anti-arthritic activity than pure colchicine and thus we presumed that this may be due to synergistic action of other unknown metabolites along with colchicine in extract for such potentiating response towards the inhibition of pro-  tein denaturation.The results of radical scavenging assays were variable but pronounced in elite germplasm.Thus, there may be a possibility that the inhibition of inflammatory mediators will be more accentuated when whole extract of G. superba is used in the form of herbal medi cine/formulation than pure colchicine, especially in cases of long term treatment considering the toxicity factor of species.As in such cases anti inflammatory action of colchicine is super imposed with the anti oxidant action of other polyphenolics present in extract.

CONCLUSION
Seven germplasm were collected from different location of Eastern Himalayas up to an altitude of 1162.812meters.A calibrated HPTLC method was used to quantify colchicine in collected samples, ranging from 0.044 to 0.184% having maximum content in NBG-128 from Jorhat, Assam.While minimum content of colchicine of observed in NBG-24 and NBG-25 from 6th mile, Darjeeling, W.B. and Kalimpong, W.B. Germplasm from Jorhat, Assam (NBG-128) was statistically screened as elite chemotype with highest colchicine content.Three germplasm viz.NBG-24, NBG-25 and NBG-128 with highest and lowest colchicine content was pharmacologically validated.The test extracts reflect potential anti arthritic activity with promising hydroxyl radical scavenging activity in addition with no statistically significant difference within the selected germplasm.The results, thus suggested that the effect of Gloriosa superba whole extract on curbing the inflammation causing cells is more pronounced than pure colchicine and which may due to superimposed action of colchicine and, other chemical inhibitors like polyphenolics.Thus, it will aid in site specific exploration and commercial cultivation of high metabolite yielding chemotype(s) with validated pharmacological action to meet out the medicinal and commercial demands of colchicine.This will also promote the use of species in herbal product development as an adjuvant therapy in acute gouty flare for inhibition of inflammation.
Hierarchical cluster analysis was performed to group the samples on basis of colchicine content and altitude in seven samples (Hammer et al. 2001) as other biotic and abiotic factors are considered salient within the same phytogeography of Eastern Himalayas.A UPGMA dendrogram Figure 2 reveals that the whole population was divided into two branches, clustering NBG-23, NBG-24 and NBG-25 into one branch (I), whereas NBG-26, NBG-27, NBG-52 and NBG-128 into other one (II).

Figure 1 :
Figure 1: HPTLC quantification of colchicine in G. superba samples collected from Eastern Himalayas.

Figure 2 :
Figure 2: UPGMA dendogram of G. superba germplasm collected from Eastern Himalayas on basis of Colchicine content and altitude for identification of elite chemotypes.

Figure 4 :
Figure 4: UPGMA dendogram of G. superba germplasm collected from Eastern Himalayas on basis of TPC and TFC.