Download Development of Pharmaceutical Products from Medicinal Plants

Development of Pharmaceutical Products from Medicinal Plants (TWNSO)
1. Section One: General Information:
a.
Title of Innovative Experience: “Molecular- and Clinical-Based
Support for the Use Of Peruvian Natural Product Extract as an antiinflammatory agent”
b.
Keywords: Anti-inflammatory agents; Phyto-extracts; Cat’s claw;
Uncitolina; NF-kB; cytokines; Phyto-pharmaceuticals.
c.
Implementing Institution: Section of Immunology, Department of
Microbiology, Faculty of Sciences. Universidad Peruana Cayetano
Heredia. Address: Sectioin of Immunology, Department of
Microbiology, Faculty of Sciences. Universidad Peruana Cayetano
Heredia, Box 4314. Lima 100. Perù. Phone: (511) 319-0014; Fax:
(511)
319-0003,
E-mail:
[email protected],
Website:
www.upch.edu.pe/facien/microweb/inmuno/principal.htm
d.
Head of Institution : Oswaldo Zegarra, M.D., Rector Universidad
Peruana Cayetano Heredia. Phone : (511) 319-0000
e.
Person responsible : Jose L. Aguilar, M.D., Head of Section of
Immunology, Faculty of Sciences. Universidad Peruana Cayetano
Heredia.
f.
Other individuals involved: Biologist Percy Rojas, Associate
Investigator at the Section of Immunology. He has ended his Master
Degree studies on Immunology. He is responsible of animal
experiments with Cat´s claw; Biologist Adolfo Marcelo, Associate
Investigator at the Section of Immunology. He has ended his Master
Degree studies on Immunology. He is responsible of cellular and
molecular studies with Cat´s claw; Patricia Hurtado, M.D. Associate
Investigator at the Section of Immunology. She has ended his Master
Degree studies on Epidemiology. She participate in clinical
experiences with Cat´s claw; Fidel Barrantes, medical student. He
participated in clinical experiences with Cat´s claw.
g.
Implementation Period and Total Costs: It has been several different
studies developed during around 3 years. Total cost will be very
difficult to establish because it involved some participation of pregraduate and post-graduate students which spend time and costs for the
experiments. However, it may be projected to around US $25,000. It
has been contributions from governmental funds around US $2,000;
from private funds around US $15,000 and foreign funds US $3,000;
and own funds around US $5,000.
2. Section two: Summary of the Innovative Experience:
Chronic inflammatory diseases are a big public health problem all around the world,
worsened because there is an increasing percentage of older people in developed and
developing countries. Traditionally exists several natural products with attributed
anti-inflammatory properties, like Cat´s claw in Peru. We pay attention to an
alcoholic extract of Cat´s claw and developed studies in the cellular and molecular
bases to explain its properties and to develop clinical studies to demonstrated the
scientifically probed useful of this extract.
3. Section three: Background and Justification:
The augmenting amount of people with chronic inflammatory diseases need to be
pointed. In developed countries this is a currently programmed situation. However,
in developing countries this will turn on a critical issue because of the economical
crisis that reserve all resources for “productive issues”, but there is not resources for
older people. This situation will maintain a big part of the population outside of
pharmaceutical products. Also the develop of side effects on pharmaceutical
products chemically synthesized is a consideration. Then is critical to develop
therapeutic alternatives. Used the regional resources also will reduce the spend costs
to imported substrates.
Traditional information is useful, however, in a global word, scientifically proved
data is mandatory to support wide use of a natural product. This data must be hard
and provide molecular, cellular and clinical solid information to be accord with the
current concepts of evidence-based medicine.
4. Section four: Description:
We start this project with the wide investigation of the properties of Peruvian natural
products. We select Uncaria tomentosa (Cat´s claw) and decided to evaluate a
standardized hidro-alcoholic extract (Uncitolina®). First we evaluate the possible
toxicity of this extract in two models: animal lethal doses 50 (LD50), and in cell culture.
In both systems the extract demonstrated no toxicity. (Capcha R., Rojas PA, Aguilar JL. Acute
toxicity (LD50) for two standardized extracts of Uncaria tomentosa Willd DC and an extract of Lepidium
meyenii (Maca) rich in glucosinolatos. Published on Proceedings of the First International Congress FITO
2000, First Peruvian Congress of Medicinal Plants and Phytotherapy. American Phytotherapy Institute.
Lima, September 2000).
Then we evaluate the capability of the extract to induce anti-inflammatory effect on an
animal model (Carragenin induced paw edema), demonstrating good effect. Then we
developed molecular and cellular scientific experiments: inhibition of the production of
a pro-inflammatory cytokine: tumour necrosis factor alpha (TNF) in the air pouch
model in mice, inhibition of the activation of the transcription factor NF-kB in animal
Jurkat T cell culture, inhibition of the production of the inflammatory enzyme cyclooxigenase in cell culture (Aguilar JA, Rojas PA, Marcelo A, Plaza A, Bauer R, Reininger E, Klaas
CA, Merfort I. Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae).
Journal of Ethnopharmacology 2002;81(2):271-6), inhibition of the adhesion molecular ICAM-1
expression on human macrophages, experiments which corroborate the antiinflammatory effect and provide hard data of the molecular steps where this extract act.
Also clinically we demonstrated security of the extract on humans on a phase I clinical
study (Rojas PA, Aguilar JL, Timoteo OS, Capcha R, Marcelo A, Plaza A, Merfort I, Bauer R. Effect of
an extract of Uncaria tomentosa (Uña de gato) upon the production of the Tumoral Necrosis Factor - 
in a model in vivo and its action as an anti-inflammatory agent. Published at Proceedings of the VIth
International Congress of Immunology. La Hababa, Cuba 2002. It received one of the awards of OPSWellcome Trust 2002), and demonstrated a clinical response of patients with acute
inflammatory process when use pharmacological doses do the extract, with no side
effects (Aguilar JL, Barrantes F. Open Clinical Study of the Anti-inflammatory Effect of Uncitolina in
articular diseases. Published at the Proceedings of the Ist International Congress of Immunology. Lima,
August 2001.
5. Section five: Partnerships:
The community has been involved in clinical studies with a previous informed
consentient. Also community will be improved and stimulate to produce a product with
scientific support. Academic part has been involved by the UPCH. Private has
supported in part this investigations.
6. Section Six: Replicability:
This methodology may easily be reproduced and applied to other natural products from
different areas. The idea is maintain local resources to avoid spend on imported
products. Also an interchange between developing countries may be planned.
7. Section Seven: Impact:
Right now there is more scientific hard data to support the incorporation of this product
in the pharmaceutical strategies provided to medical doctors and general population. It
need to be quantified, to offer this alternative to local pharmaceutical corporations, to
introduce natural product extracts on its plans. The sustainability will depend on the
introduction (marketing) in the medical programs not only in my country but all around
the region.
8. Section Eight: Lessons Learnt:
The main obstacle to the develop of this project was the absence of economical supports
to develop the studies. Finally this was obtained slowly, with great enforce. Another
obstacle was the difficulty to introduce a natural product extract in health professionals,
which prefer synthetic marketed products.
9. Section Nine: Future Plans:
Now, a great enforce is been developed to obtain economical support to develop a
clinical study phase III, with a big number of patients to demonstrate again clinically the
benefits of the product.
Also, it need to continue basic investigation to show the mechanisms by which work
this plant, not only as an anti-inflammatory agent, but as a therapy for cancerous cells.
World wide institutions or corporations are welcome to participate in the final
developing of this product.
10. Section ten: Tables and graphics:
Table 1. Anti-inflammatory effect of an hydro-alcoholic and an aqueous cat’s claw
extract in mice’s paw oedema after 4 hours.
Anti-Inflammatory Activity (%)
Dose
(mg/kg)
n
7
8
50
8
35,3
21,9
100
8
37,3
23,8
200
8
43,0
33,0
500
8
51,7
26,7
Hydro-alcoholic extract
Aqueous extract
Indomethacin
37,8
Table 2. Inhibition of cyclooxygenase-1 and -2 by an hydro-alcoholic and an
aqueous cat’s claw extract at a concentration of 50 g/mL (standard deviation in
brackets)
Extract
Cyclooxygenase-1
Cyclooxygenase-2
Hydro-alcoholic
7,8 (  10,4)
21,7 (  3,1)
Aqueous
32,7 (  6,6)
12,2 (  6,5)
Table 3. Variation on functional class on patients treated with Uncitolina.
Time of evaluation
Functional
Class
Begining
3 weeks
6 weeks
III
70,5 %
40,0 %
16,6 %
II
29,5 %
35,0 %
33,3 %
I
0
25,0 %
50,1 %
Figure 1
HPLC analysis of oxindole alkaloids in hydro-alcoholic extract
Figure 2
The effect of the hydroalcoholic (A) and the aqueous (B) Cat's claw bark extracts on
NF-kB DNA binding.
Extract A
µg/mL Extract:
TNF
_
1
Extract B
50 100 200 500
50 100 200 500
+
+
+
+
+
+
+
+
2
3
4
5
6
7
8
9
+
10
Lane 1 shows unstimulated Jurkat cells, in lane 2 cells were treated with 200
U/mL TNF- alone. In the other lanes (3-10) cells were pretreated for 1 h with
various concentrations of the respective bark extract and subsequently
stimulated with TNF- for 1 h. Equal amounts of protein from cell extracts were
analysed for NF-B activity by EMSA. The filled arrowhead () indicates the
position of NF-B DNA complexes. The open circle () denotes to non-specific
activity binding to the probe and the open arrowhead ( ) shows unbound
oligonucleotide.
Figure 3. Inhibition of the Amount of TNFalpha in the air pouch model using
Cat´s claw extract.
*
*
4000
TNF -  ( pg/ml )
3500
3000
2500
*
2000
*
1500
*
1000
500
*
*
0
PBS
Carragenina
UG (100)
UG (250)
Pentoxifyllina
UG (100) Pentoxifyllina
+ carragenina
Tratamiento
Figure 4
8.00
7.00
6.00
VAS on rest
VAS
5.00
4.00
VAS during
movement
3.00
2.00
1.00
0.00
Bening
3 weeks
6 weeks
Time
Evolution of the visual scale analogue in patients dealt with Uncitolina