1. NOVEL APPROACHES FOR AN ANCIENT DISEASE
Clinical Efficacy of Herbal Coded Formulation Gouticin for treatment of Gouty Arthritis and in vitro xanthine oxidase inhibition
Third International Conference on Functional Food and Nutraceuticals December 4-5, 2013 Faisalabad
Khan Usmanghani
Jinnah University for Women
Faculty of Eastern Medicine, HAMDARD UNIVERSITY
Consultant, Herbion Pakistan (Pvt.) Limited, Karachi, Pakistan

2. Hyperuricemia and Gout
The condition of hyperuricemia is indicative of a high level of uric acid in the blood (>7mg/dl for men, >6mg/dl for women).
Human beings have higher levels of uric acid (hyperuricemia) because of a deficiency of the hepatic enzyme, uricase, and a lower fractional excretion of uric acid.
Gout is defined as a arthritis resulting from the deposition of sodium urate crystals in one or more joints.
The Starburst Galaxy M82 shows
a frenzy of star formation in its central
regions and jets of gas perpendicular
to the galaxy. Both activities
are believed to have been initiated
by passage of the galaxy near its
neighbor, M81. (Courtesy Mark
Westmoquette, University College
London; Jay Gallagher, UW-Madison;
Linda Smith, University College London;
WIYN/NSF; NASA/ESA)

3. CAUSES OF HYPERURICEMIA
Accelerated generation of uric acid through purine metabolism
Impaired excretion in the kidney
High levels of fructose in the diet
Consumption of purine-rich diets.
Ingestion of high protein and fat
Starvation.
Tumor lysis syndrome
Lesch-Nyhan syndrome
High intake of alcohol.

5. CLINICAL TRIALS
TEST DRUG GOUTICIN
AND
CONTROL DRUG
ALLOPURINOL

6. METHODOLOGY
Case control, direct unicentre prospective randomized authentic allopathic, controlled two arms parallel group clinical trial.
Test group (Herbal): Apium graveolens , Colchicum autumnale, Withania somnifera, Smilax chinensis, Tribulus terretris, Zingiber officinale
Control Group (Allopathic) Allopurinol

7. XANTHIN OXIDASE INHIBITION ASSAY
Xanthine oxidase activity determined by measuring the formation of uric acid from xanthine.
Plant extract at 1 mg/mL will be prepared in 50 mM phosphate buffer solution (pH 7.0). Two millilitres of the sample will be mixed with solution containing xanthine oxidase (2 mL, 0.4 U/mL) and xanthine (100 μM).
After incubating at room temperature (24 oC) for 3 minutes, uric acid production will be determined by measuring the absorbance at 295 nm.
The blank used will be buffer and the control containing xanthine and xanthine oxidase. The inhibition percentage of xanthine oxidase activity will be calculated according to the formula = (Acontrol –Asample) / Acontrol x 100%( Geethaa S et al, 2009).

8. Common sites of gouty arthritis lesions

9. Swollen ankle and first etatarsophalangeal joints during an acute attack

11. Pain on Movement of joints
Objectives
Joint Pain
Tenderness of Joints
Swelling of Joints
Redness of Joints
Stifness of Joints
Pain on Movement of joints
Tophus Formation

12. Diagnosis
Arthrocentesis
Serum Uric Acid
Joint X-ray
Urine D/R
CBC

13. Objectives In vitro xanthine oxidase inhibition Clinical Trial
To investigate the in vitro xanthine oxidase inhibitory activity of the ingredients(Apium gravelens, Colchicum autumnale, Smilax chinensis, Tribulus terrestris, Withania somnifera, Zingiber officinale) of Gouticin
Clinical Trial
To investigate the efficacy of Gouticin (test group) in comparison with allopurinol (control group) for the treatment of gouty arthritis

14. Literature review of plants Zingiber officinale (Sondh)
TEST DRUG GOUTICIN COMPONENTS
1. Zingiber officinale: gouty arthritis, prevents acute gouty attack.
Withania somnifera: treat rheumatism, xanthin inhibitor, immune function
Apium greveolens: reduce serum uric acid, xanthin inhibitor, reduce serum uric acid
4. Smilax chinensis: antihyperurecemic, xanthin oxidase inhibition
Colchicum autumnale: acute gouty arthritis, prevent acute gouty flares
6. Tribulus teresteris: excretion of uric acid in urine/blood, xanthin oxidase inhibition
Literature review of plants Zingiber officinale (Sondh)

15. CONTROL DRUG ALLPPURINOL
This is the only drug to affect uric acid concentrations at the production end.
Mechanism of allopurinol is to inhibit xanthine oxidase, thus decreasing production of uric acid.
But continuous use drug causes many adverse side effects like fever and rashes, leukocytosis, eosinopholia, vasculitis
aseptic menengitis,nephritis and renal dysfunctions.
Standard dose is 300 mg. Maximum dose of Allopurinol is 800 mg. Once daily(Govani et al., 2010).

16. Ethical issues and clinical trial approval
Methodology
Ethical issues and clinical trial approval
Study was conducted under the rules of Ethical Committee (EC) of Shifa-Ul-Mulk Memorial Hospital, Faculty of Eastern Medicine, Hamdard University Karachi, Pakistan.
Study design and protocols were presented to the board members of Ethical Committee (EC) and Board of Advance Studies and Research (BASR) for their clearance and permission before the start of clinical trial.
Clinical evaluation proforma was filled up before starting the treatment and was consequently updated during the course of the treatment.
In in vitro study, those plants were investigated that were ingredients of my herbal coded formulation Gouticin

17. Inclusion and exclusion criteria
Inclusion criteria
The patients suffering from gouty arthritis with serum uric acid >8 mg/dL
Male and female patients between 20 to 80 years of age.
All socio-econoic classes including lower middle and higher.
Patients having no obvious pathological finding on routine examination
Patients living in Karachi or its outskirt or suburban areas
Exclusion criteria
Uncontrolled hypertension, diabetes and kidney stone .
Cancer chemotherapy
Patients outside Karachi because of inherent difficulty in follow up.
Dialysis
Patient having renal disease

18. Design and Settings Clinical Trial:
Design
Case control direct, multicentre, prospective, randomized authentic allopathic controlled, two arm parallel group clinical trial was the target design
Setting
This case controlled examination based clinical study was conducted at Shifa-ul-Mulk Memorial Hospital for Eastern Medicine and Momina Clinic Korangi Crossing Karachi and experimental work was carried out at the PCSIR Labs Complex Karachi

19. Subjects and Interventions (Clinical Trial)
Patients of both genders male and female with clinical diagnosis of gouty arthritis included for the study.
Sample sizes for Gouticin contains100 and Allopurinol 100 subjects were registered and treated. Mean age male patients and mean age female was were male and 56 were female
Study was conducted between
Study period was 18 weeks with a window for the follow up visit of 6 weeks accounting for variable duration of gout treatment.

20. Methodology (Vitro Study)
Plant material
Apium graveolens, Colchicum autumnale, Withania somnifera, Smilax chinensis, Tribulus terretris and Zingiber officinale.
Plant Extraction
All plants were extracted with ethanol, 200 g dry weight macerated at room temperature for 15 days with solvent and filtered.
The solvent removed by rotary evaporation under reduced pressure and the resulting extracts tested for inhibition of xanthine oxidase.

21. Design and Methods
Xanthine oxidase inhibitory of the Gouticin and its ingredients (Apium graveolens, Colchicum autumanle, Smilax chinensis, Tribulus terrestris, Withania somnifera and Zingiber officinale was assayed spectrophotometrically at 200, 100, 50, 25 μg/ml concentrations.
Degree of enzyme inhibition was determined by measuring the increase in absorbance at 295 nm.

22. Outcome Measures .
In clinical trials, primary measure was to determine the efficacy parameter to decrease uric acid level in patients with gouty arthritis. Secondary measure was to investigate the efficacy of test drug on sign and symptoms of gouty arthritis
In in vitro study, outcome measure was to examine the xanthine oxidase inhibitory activity of ethanol extract of Gouticin and its ingredients.

23. Results Table 1: Mean Serum Uric Acid Level
Treatment Group
Base line
1st Follow up
2nd Follow up
3rd Folllow up
Test Group
10.12
8.57
6.41
4.62
Control Group
10.01
7.81
6.33
5.21

24. Table 2: Improvement in Lowering of Serum Uric Acid Level
Treatment Group
Percentage of patients treated at 1st follow up
pValue
Percentage of patients treated at 2nd follow up
P value
Percentage of patients treated at 3rd follow up
p value
Test Group 42
0.05 72
0.04 91
0.016
Control Group 30 61 78

25. Table 3: Improvement in Sign and Symptoms in Test and Control Group
Test (Gouticin)
Control
(Allopurinol
p value
Joint pain
70/80(87.5%)
55/74 (74.32%)
0.02
Tenderness of Joint
82/87(94.25%)
70/84(83.33%)
0.01
Swelling of joints
68/75(90.66%)
54/72(75%)
Redness of Joints
70/78(89.74%)
58/74(78.37%)
0.04
Stiffness of Joints
69/77(89.61% )
57/73(78.08%)
Pain on movement of joints
89/94(94%)
79/92(85.86%)
0.03

26. Table 4: Percentage of Enzyme Inhibition by Different Medicinal Plants
S. No
Plant Extract
Code
200 µg/ml
Percent
inhibition
100 µg/ml
50 µg/ml
25 µg/ml 1
Apium graveolens
A.G 69 54 31 11 2
Colchicum autumnale
C.A 79 61 42 30 3
Gouticin
G.C 96 88 40 4
Smilax chinensis
S.C 74 52 36 18 5
Tribulus terrestrus
T.T 91 78 49 6
Withania somnifera
W.S 76 48 26 7
Zingiber officinale
Z.O 92 80 51 8
Allopurinol
A.P 93 86 70 41

27. Graph 1: Percentage of Enzyme Inhibition by Different Medicinal Plants.

28. Table 4: IC 50 values of all plant Extracts with Allopurinol
S. No
Plant Extract
Code
IC50 μg/ml. 1
Apium graveolens
A.G
180 2
Colchicum autumnale
C.A 42 3
Gouticin
G.C
17.3 4
Smilax chinensis
S.C
121 5
Tribulus terrestris
T.T
19.8 6
Withania somnifera
W.S 95 7
Zingiber officinale
Z.O
23.6 8
Allopurinol
A.P
6.1

29. Graph 2: IC 50 Values Plant Extracts with Allopurinol

30. Conclusion
All the samples were found to have inhibitory activity greater than 50% at 100 μg/ml.
Gouticin and allopurinol exhibited the highest activity with an inhibition of 88 and 86% respectively. Gouticin has IC50 values (17.3 μg/ml).
The IC50 value of allopurinol used as the standard was 6.1 μg/ml. Gouticin (500 mg twice daily ) was found more effective than the allopurinol (300mg once daily) in the treatment of gouty arthritis.
Gouticin has proved effective in gouty arthritis. There was no untoward manifestation associated with the use of this medication except diarrhea and this is found a good acceptability by all treated patients.

31. References
Doha, Sahar(2008). Evaluation of antigout activity of some plant food extracts. Polish Journal of food and nutritional Science. 58(3)
Chen L, Yin H, Lan Z, Ma S, Zhang C, Yang Z, Li P, Lin B(2011). Anti-hyperuricemic and nephroprotective effects of Smilax china L. J Ethnopharmacol;135(2):
Govani SM, Higgins PD (2010). Combination of thiopurines and allopurinol: adverse events and clinical benefit in Inflammatory bowel disease. J Crohn colitis:4(4):
Miean KH, Mohamed S(2001). Flavonoid (myricetin, quercetin, kaempferol, luteolin, and apigenin) content of edible tropical plants. J Agric Food Chem.;49(6):
Hippocrates(1886): The Genuine Works of Hippocrates. Volume I and II. Edited by Adams F. New York: Woo

32. AN ICON FROM FAISALABAD Thank You For Participation
FIGURE 16-4 Variety in Spiral Arms
The differences in spiral
galaxies suggest that at least two mechanisms create spiral
arms. (a) This flocculent spiral galaxy has fuzzy, poorly defined
spiral arms. (b) This grand-design spiral galaxy has well-defined
spiral arms. (a: NASA; b: Gemini Observatory/AURA)
AN ICON FROM FAISALABAD
Thank You For Participation