CA2358013A1

CA2358013A1 - Compositions obtained from mangifera indica l.

Info

Publication number: CA2358013A1
Application number: CA002358013A
Authority: CA
Inventors: Alberto Julio Nunez Selles; Eleuterio Paez Betancourt; Daniel Amaro Gonzalez; Jhoany Acosta Esquijarosa; Juan Aguero Aguero; Raul Capote Hernandez; Maria Rosa Garciga Hernandez; Ivan Gaston Morales Lacarrere; Oscar Garcia Pulpeiro; Gabino Garrido Garrido; Gregorio Martinez Sanchez; Miguel Morales
Original assignee: Centro De Quimica Farmaceutica
Current assignee: Individual
Priority date: 1998-12-29
Filing date: 1999-12-29
Publication date: 2000-07-06
Also published as: PA8488701A1; AU2253100A; CU22846A1; CO5160343A1; WO2000038699A1

Abstract

The present invention relates essentially to the pharmaceutical, food and cosmetic industries and in particular to the preparation of formulations of active principles which are derived from the plant Mangifera indica L. amongst which are the polyphenols, the terpenoids, the steroids, the fatty acids and microelements which have anti-oxidating, anti-inflammatory, analgesic and antispasmodic properties thereby conferring to said formulations a high value as dietary supplements for the improvement of the quality of life of patients suffering from degenerative diseases, anti-aging treatment as well as for the consumption by healthy persons.

Description

27-02-2001 ~ - CU 009900007 y' CO~POi~ITIONB 08fiAINEb FRO Man~sra Indfca L.

Z'eshn,ical sector i The present invention relates essentially to pharmaceutical, food, y. and r, cosmetic industries, and particularly with a mi~cture of active principles obtained from the specie Mangifera indices L., which contains t polyphenols, terpenoids, steroids, fatty acids, and microelements.
That r, tx~.ixture gives useful properties to the compositions which are prepared i using it as active ingredient for food supplements to improve the quality ' io of life of patients with degcnrrativc diseases, the anti-aging treatment, ,.
and to improve the status of healthy persons as ucreli.

prior Art Although there is a documented ethno pharmacological knowledge for the use of different pasta of the mango tree (Mangifera tndica L.) as H , '; is leaves, roots, stem, stem bark, flowers, and fruits, in the treatment of i pathologies such as menorrhagia (Chopra, R. N. 1933, Indigenous Drugs of India. Their Medical and Economic Aspects. The Art Press, Calcutta, India , the scabies Sixi ( gh, Y. N. 1986. Traditional Medicine in Fiji: Some herbal folk cures used by Fiji Indians: J Ethno pharmacology 2o 15 1: 57-t38), the diarrhea (Darias S. ZT. and cola, 1989. Ncw i contribution to the ethna pharmacological study of the Canary Islands.

J Ethno pharmacology 25 1; 77 92; him Grarxid, A. 1989. Anti-infectious phytatherapy of the tree-Savannah, Senegal, Western Africa III: A

Review of the phytochemical substances and anti-microbial activity of zs 43 species. J Ethno pharmacology 25 3: 315-338; Ponce-Mecotela, M.
et al. 1994. In vitro anti-giardias:c activity of plant extracts. R.
Rev Invest Clip 45 5: 343-347; Muanza,~ D. N et al. 1994. Antibacterial and antifungal activity of nine medicines plants . from Zaire. Int J

Pharmacology 32 4: 337-345), the syphilis (Sirigh, Y. N. 1986.

30 Traditional Medicine in Fiji: Some herbal folk cures used by Fiji Indians:

J Ethno pharmacology 15 1: 57-88), the diabetes (Anjaneyulu and cols.

1989. Triterpenoids from MaruJfjera indices, Phytochemistry 28 5:

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.~27-02-2001 ~ - CU 009900007 14'77; Muanza, L7. N et sl. 1994. Antibaoterial and antifungal acaivity of .'.
nine medicines plants from Zaire. Int. J Fharmacolog. 32 4: 337-345), r.,;
the cutaneous infections (Him C?rrand, A. 1989. Anti-infectious phyt therapy of the tree-Savannah, Senegal, ~iJestern Africa III: A
i Review of the phytochemical substaxices and anti-microbial activity of 43 species_ J. Ethnopharmacol. 25 3: 315-338), the teeth pains (Hirn Grand, A. 1989. Anti-infectious phytotherapy of the tree-Savannah, Senegal, Western Africa III: A Review of the phytochernical substances and anti-microbial activity of 43 species. J. Ethnopharmacol. 25 3: 315-~0 338; Chhabra, S. C. and cabbage, 1987 Plants used in traditional medicine in Eastern Tanzania. 1. Pteridophytes and Angiosperms. J.

' Ethnopharmacol. 21 3: 253-277), and anemia {Muar~za, D, N. and cola.
1994, Antibacterial and antifungal activities of nine medicinal plants from Zaire. Int. J. Pharxnacol. 32 4: 337-345, it is not lo~own the use of 1 i formulations wherein a mixture of polyphenols, terpenoids, steroids, ~I
fatty acids, and microelements is used as active ingredient, as that one obtained starting from the stem bark of the mango tree.
The known reports about the ethno pharrnacolo;gical use of extracts obtained from parts of the mango tree (Mangifera indices L.) indicate i 2o different types of biological activities, such as insecticide, axitigiardiasic, antirnalaric, antipyretic and antispasmodic (Ponce-Macotela, M. and cola. 1994. In vitro antigiardinsic activity of plant extracts. R. Rev Invest Clip 46 5: 343-347; Awe, S. O. et al. 1998. Antiplasmodial and antipyretic screening of Mangifera indices. Phytotherapy Res. 12:437-zs 438; Kambu, K. and cots, 1990. Antispasmodic activity of extracts proceeding of plant antidiarrheie, Traditional preparations used in Kinshasa, wire. Ann. Pharm. Fr 48 4: 200-208), but there are not reports about controlled studies allowing the correlation of those findings with clinical results in humans.
30 C?n the other hard, the patent application No. WO 96/ 16fa32 A1 refers to the obtainment of cosmetic or pharmaceutical compositions using as AMENDED SHEET

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27-02-2001 ~ - CU 009900007 ' 3 ., I
active ingredient "mangiferin or its derivatives in pure farm or in extract ;' of plantsn and the formulation of emulsions, aqueous or hydroaleoholic gels, creams, oils, aqueous or hydroalcohalic lotions, labial pencil, shampoo or hair conditioner, et~., for the photoprotection of the skin, the lips, and the hair. This compound, which is obtained essentially by means of extraction starting from the leaves of the species Aphloia and ,., i!Idangi ferina, was found to have activities such as anti-ultraviolet, anti-colagenase, anti-elastase, anti-free radicals, and anti-tyrosinase.
Disclasnre of the Iuveatioa io An objective of the present invention is the obtainment of a mixture of active ingredients such as polyphenols, terpenoids, steroids, fatty acids, I
' and microelements starting from the stem bark of the mango tree (Mangifera indicct L.), k' Another objective of the invention is the obtainmcnt of formulations ' is starting from the mixture of the active principles previously referred with important pharmacological, and dietary properties for oral, topic and parcnteral uses.
It is also another objective of the present invention the use of these formulations as food supplement, urith anti-oxidant, anti-inflammatory, 2o and analgesic properties, that increases and/or improves the quality of life indices in patients with degenerative illnesses, their use for anti-aging treatment, as well as it consumption for healthy people.
The formulations included in the present invention have a peculiar qualitative and quantitative composition of active ingredients, which ~s have been obtained from Mangiferc~ indices L., with differentiated properties, on the basis of the following facts:
A. Tr~ere is not known a formulation in the pharmaceutical and food !', brar~ch~s Cc~r the improvement of the quality of the life or as food ' supplement that is claimed in the present invention.
3o B. The formulations of the present invention are completely of natural origin, which combine components of pharmacological activity i AMENDED SHEET

27-02-2001 ~ - CU 009900007 r is r I
(polyphenols, terpenoids, and steroids) with others of dietary value (fatty acids and mieroelements~.
C. The mixture of components with phax~naCOlogical activity, and of dietary value, gives to the formulations a high antioxidant capacity that 'v' ~
S leads to the increase of the indexes used to evaluate the quality of the life and as food supplement as well, that differeratiates it substantially t from other similar products in the market.
For the deterrriinatiot~ of the mixture composition of the present invention, the following steps were carried out:
t o . Column Chromatogi aphy, I-Iigh Performance Thin Layer Chromatography, and further identification by spectroscopic y techniques (polyphenols), i ' ~ High Performance Liquid Chromatography and Capillary Gas Chromatography, coupled to mass spectrometry (terpenoids and ~s steroids), ~ High Resolution Cress Chromatography coupled to mass spectrometry (fatty acids), ~ Scanning Electronic Microscopy, Atomic Emission Spectral Analysis, I
and X-ray hluorescence Spectxontetry (m.icroelements).
i ' 2o The results of the qualitative and quantitative analysis of the mixture obtained, starting from the stem bark of mango (Mangifera indices L_), are sho~'vn in Table I.
I
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CA 02358013 2001-06-29 G'U o~9900007 s v .
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Com onent Coat~at ~~) 1. poi hrao~s ~o - 60 1. i Man 'ferirt 10 - 20 1.2 3-Pentade I henol 5 - 10 1.3 3-Oc 1 henol 5 - 10 1.4 Amentoflavone 10 - 20 2. Ttr eaoids 10 - 40 2 .1 M an i heronic acid 2.2 Beta-element - 5 - 10 2.3 AI ha- aiene 5 - 10 . 2.4 Aramandrene 5 - 10 i 2_5 Hinesol 1 - 5 2.5 C cloartanoles 1 - 5 2 . 7 Ledol i 2. $ Taraxero! 1 - 5-3. Steroids g - lg 3.1 G amma-sitosterol 2 _ g 3. 2 Be ta-sitosterol _ 1 - 5 3.3 Cam sterol 1 _ 5 3.4 Daucosterol 0.5 - 3 3.5 Multifluorenone 0.,5 _ g 4. Fatt ~cid~

' 4.1 Myristic 0.1 - 3.0 -..

4. 2 Palmitic 35.0 - 45.0 h.3 Linoleic 15.0 - 35.0 ' 4.4 Oleic 20.0 - 40.0 _ 4.s Stearic o.1-1.0 4.6 Eicosatrienoic 1.0 - 3,0 5. Microelements 1 - 3 ", S.1 Potassium __ _ __ _ p.8 - 1.0 5.2 C~lCium _ _ 0,2 - 0.4 5.3 Ma ncsiuxn 0.1 - 0.2 S.~ Iron 0.1 - 0.2 5.5 Co er less than 0.01 S.6 Zi nc less than 0.01 5.'7 Selcniur~ 0.03 - 0.08 s That mixture was obtained from r the stem bark of the mango tree (Manr~ifera irzdiea L.), which once collected and canstrved under apprr~priate conditions, it is processed through a drying process until Table 1. Qualitative and quantitative composition of the mixture of polyphcnols, terpenoids, steroids, fe~tty acids and microelements used in the pharrna~ceutical formulations.
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~ 27-02-2001 ~ _ V Y V C~U '009900007 ' b .., ,i reaching a humidity between 4 and 12 °~o. Then it was decocted with a polar solvent such as uiater, or other that contained ar hydroxyl group with a Cdrbon chain between 0 and 5 atoms of lineal carbon or j , cyclic, selected from the group constituted by methanol, ethanol, dioxanc or i s cyclohexanol or mixtures thereof. lauring this process, the virgin _ ' vegetable matter was decocted at temperatures between 65 and 101 °C, and fur they the extract was dried by atomization to obtain a dark brown t' solid, which melts with decomposition between 216 and 2 i8 °C, and 1t-~
whose physic-chemical progenies are described izz Table II.
m Table rT. Physical-chemical properties of the mixture of polyphenols, terpenoids, steroids, fatty acids and microelements, which are used in the pharmaceutical formulations.
Property _ , Inde x of pots taaco Water content _ I _ goo Solubffi 1-3 lOmL NaOH 0.1 mol 1 _ _ ,i ph i Refraction index ~'_ 1.33 - 1.35 l.'.elative densi 0.9 - 1.1 em3 Solution of 1 ~r:;m in 10 mL of dietiued Water is It has been found that the mixture used as active ingredient in the formulations of the present invention possess antioxidant, anti~-inflammatory and analgesic activities, whose action provokes a significant increase of the parametnc indexes of the quality of life in imrnunodeFrcssed patients by degenerative illnesses, more specifically v 20 in patients with different types of cancer. It could be proven irt those patients that the genera3 state reached an average index of 3,5 on a parametric scale of 5, in time periods between 1 and 6 months of treatment with the formulations object of the present invention.
The appropriate daily dose of the mixture of polyphenols, terpenoids, i zs steroids, fatty acids, and xnieroelements to achieve the pharmacological a~tivi lies of this invention are between 10 and 100 mg/kg of body weight, using as the most appropriate administration routes, the oral one in form of tablets, coated tablets, pills, or flavored suspension, and AMENDED SHEET

_ ___.._ _.. .._ .. -___ ___ ______~~~.. ..~ ~.... .~v...rrz.vv._nia .

_ .a the topical one in form of cream, ointment, or lotion, although it can be , administered also using the rectal, vaginal or parenteral route . . , s.

It has been found that the mixture used as active ingredient in the formulations of the present invention possess antioxidant, anti-inflammatory and analgesic activities, whose action provokes s significant increase of the paxametric indexes of the uali of life in tY

irnmunodeprcssed patients by degenerative illnesses, more specifically in patients with different types of cancer. It could be proven in those patients that the general state reached an average index of 3,5 on a ~o paxarnetric scale of 5 in time periods between 1 end 6 months of treatment with the formulations object of the present irtventxon.

The appropriate daily dose of the mixture of polypheols, terpenoids, ' steroids, fatt3 acids, and rnicroelements to achieve the pharmacological i activities of this invention are between 10 and 100 mg/kg of bod y is weight, using as the most appropriate administration routes, the oral one in form of tablets, coated tablets, pills, or flavored suspension, and the topical one in form of cream, ointment, or lotion, although it can be ',I
administered also using the rectal, vaginal, or parenteral routes.
i The ~~ni tart' dose of the formulations for the oral administration ~9 ,.
20 contains preferably, as active ingredient, between 8,0 and 55,0 % in weighs of the mixture of polyphenols, terpenoids, steroids, fatty acids, and micraclements. That dose is achieved when mixing the active ' ingredient with different excipicnts in the pharmaceutical formulation, either as a ; ;lutinants, disintegrants, lubricants, flavors, or colorings.
2s The unitary dose of the formulations, for the administration using h topical route contains preferably, as active ingredient, between 1,0 and 4,0~'o in weight of the mixture of polyphenols, terpenoids, steroids, fatty acids, and microelernertts. This dose is achieved when mixing the active ingredi-rnt with different excipients, either as vehicles or stabilizants.
30 The fnrmularions that are included in the present invention are innocmous, according to the results obtained in the sub-chronic and AMENDED SHEET

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27-02-2001 ~ _ CU 009900007 v' chronic toxicity trials, which were carried out in rodents; they did not reveal irritating activity; genotoxic potential or clastogenic actfvity.
Collateral effects have not been detected in the patients treated with the 'I mixture oC polyphenois, terpenoids, steroids, fatty acids, and s microelements, objectives of the present invention.
The objects of the invention arc described in detail below, where I
' reference is made to the practical examples, which do riot limit ire any circumstance the scope of the present application.
l~a.mple 1 is 100 kilograms of vegetable raw material obtained from the stem bark of the m au ga tree (Martgifera vzdica L.) was the starting material to which is applied a solid-liquid extraction process by means of a water/ethanol mixture, 70 % (water). The extract was concentrated by heating (85 °G) and it was further dried by atomization mith air with an inlet ~5 temperature of 150°C and outlet of 80 °C, until a dark brown powder was obtained.
YI
~~'I1 13y this way, ?,5 Kg of the mixture of polyphenols, terpcnoids, steroids, fatty acids, and microelements was obtained as described previously.
The physic-chemical properties of the mixture were the followings: water 2o content: between 5 and 9 °r6; solubility: 1,2 g/ 10 onL NaOH, 0,1 mal/ L;
pH: b;.tween 5 and 7; relative density: 1,0 g/cm3. The purity of the mixture was 93 %.
!' Example a n 1 ton of vegetable raw matter obtained from the stem bark of the mangy ' 2s fret (~~Iangi~'ara indices L.) was processed as the previous example, giving 82 k~ of the mixture of polypheno~s, terpenoids, steroids, fatty acids, and microelements. The physic-chemical properties of the mixture were the fc~llwving: content of water: 3,8 °lo; solubility: 1,$ g/1t3 mL
NaOH, 0,1 molJL; pH: 5,9; relative density: 1,1 g/cm3. The purity of the 30 mi~rturc was 97 %. The qualitative and quantitative composition of the i mixtures obtained in the Exarrlplca 1 and 2 arc shown in Table III.
AMENDED SHEET

~27-02-2001 ' _ _ _ . _ _ _ _ _ _ _ _ _ _ _ Table I1I, Qualitative and quantitative composition of the mixture of poly-phenols, terpenoids, steroids, fatty acids and rnicroelements obtained in examples one and two.
f'!ww~rwts 1OG1 .~ _ l..t Components _ .,_ 1,~ 1 ale ~

1. Poly henols 42.2 ~~ 46.8 2.Te naids 28.6 24.6 3. S keroids 13.9 18.4 4. ~'at acids 4.6 3.8 S.Microelements 3.6 3.4 TOTAL -- 92.9 97.0 j s Example Starting from the mixture of polyphenols, terpcnoids, steroids, fatty acids, and microelemcnts obtained as the Example 2, coated tablets of 300 rn~; of this mixture were manufactured, which contained the componcilLS shown in Table IV.

io Table IV. coated tablets.
Pharmaceutical formulation of the Y~

.
'; _ ._ . Component Contant (% ~

vlix of polyphenols, terpcnoids, ! I su:roids, 4$.g fatty acids and ' I . . microeIcmcnts . _ ATic:r, ocrystalline 21.5 cellulose horn st;urrh 18.6 _ ___ ~~ y~ ~

PolSrvin Ipyrrolidone .. 4.3 i . ...-_ .. ... Tic 3. 4 '' L___ Aerosil 200 silicon dioxide ~ p.5 I

M_a~nesium atearate , ~
5 p.

Hydrox~ ropylmethylcellulose 2.p _ _0.3 j .. _ ~e~n 80 Exarnplc 4 Start.in8 frarn terpcnoids, steroids, fatty Lhe mixture of polyphenols, acids, and microelemeots Example 2, the flavored obtained as the suspension was prepare i to 10 % m weight of this mixture, which a.
15 contain~:ct the componen rs shown in Table V.
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27-02-2001 . _ CU 009900007 to H.I
Table V. Pharmaceutical formulation of the flavored suspension.
Component __ Cont~at /%1 Mix. of polyphenols, terpenoids, -1 steroids, fatty acids and 10.00 i _ microclemcnts _ _. __._. . . .. Sorbitol 70% 19.61 Sodium benzoate . . M. _ 0.21 - . . , Sodium metabisulfite _ ___ 0.13 Sodium carbo mmh lccllul ose 0,46 I-_.._. C31 ccrin 3.27 - .. __ l Sodium sacrharine 0.06 ._ ..__ ~.__ ,.
_ ~ T~aspberry flavoring _ _ O.OI
I IJISOdium edetatr dih drag ~ . ~ ~ ~ ~~ - < U.OI
i ... _ Ethanol --""1.30 _ _ Deionizcd water 65.36 .._ ;.
Example 5 ! Starting from the polyrhenols, terperioids, steroids, fatty acids, and s microelements mixture c~:~tained as the Example 2, the hydrophilic cream was made to 2,4 °~~ in weight of this mixture, which contained the components shown in Tame Vl.
Table v). ~harmaceutic:~l formulation of hydrophile cream Co~poneut _ Content (°.6~
Mix cf polyphenols, terpenoids, ~!~roids, fatty a~~ds and 2,4 microelem a n is _ ._..~efiYl ~colyl 2.3 ' 1~'Coi:os~earaie ~;'.~.~cerin, 14.0 -_. .. . .
Meth 1 araben . , .. 0,2 Propylpar.~~hen . , _ _ a0.1 Polysorb_at_~ ti0 _ 1.7 . 1'ro rlene ~lycol __. _ . . . . 5.2 _ T'ttrilicd -ur~ter ; 74.2 .. . _ . _ &yrample ~6 Starting from the polyphcnols, terpcnoids, steroids, fatty acids, and microclcrnetlts mixture c~otained as in the Fxsmple 2, the lipophilic 0111tIn~1': C W na m:~d a to 2 , 4 °/n in weight of this mixture, which contained the compoxicncs shown in fable VII.
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~27-02-2001 ~ _ i~! 11 fl .i Table VII: Pharmaceutical formulation of ligophilic unguent.
_ .-._ _ Cotapo~teat Content % _.
Mix of polyphenols, terpenoids, '"' steroids, fatty acids and microelements ~i -_.~rarattin ~ f .O
.. ..
F~ample 7 Tablets, 300 rig of the active ingredient each, as described before (Example 3), were orally administered to a group of 38 patients affected k' by differ~::lt types of cancer, ar 3 mL of the flavored suspension, that contained 10 % in weighs of the mixture (Example 4). In the cases of ~lcerations in skin ttxe crc;am was applied to 2,4 % in weight (Example 3j or the oimment to 2,4 % in weight (Example 7) of this mixture on the to affected surface, according to the lesion type. The class~cation of the patients, according to the diagnosis, is shown in Table VIII.
Tahlc V)It. Classitica.tion of patients included in the study 'I I No. ! pia,~aosis i ~l~lt~mbes of -._ ~ _ i pationts _ -_ Mamma cancer, r 12 ' I Lung cancer ~ 5 '' ... .
y3 ' .. , _ ~ warier carcinoma 4 ~ Prostate cancer ! 5 l-1 ~ad~kin's, Lymphoma ~ 2..
- G Non-Hodgl~n's_ Lymphoma ' 2 - _ ~ . . ~ Stomach Cancer _ . ~ 2 _ CLitancous melanoma j _ 2 Carcinoma of the neck of the uterus ~ 2 . . I ~ _, Palate Carcinoma ~ 1 . _ 7 1 C: ~~ r ehellar astrOCYtOrila _ ~ ~.
I
? 2 ~~ Colon cancer ~ ~ 1 The uiz i r;~;. .- dose (300 m of the active ingredient] ~uvas administered orally ~ times ~ day and it was applied the cream or ointment so menu times c s it was neces5:~r:. The treatrb:ent lasted 6 months and the ! quality of iife was en;j'.uated by means of systems Of surveys internal :orally accepted ~,~r the measurement of the patient's general AMENDED SHEET

27-02-2001 ~ _ CU 009900007 ~'~.
state. All the patients were interviewed before beginning the treatment and 6 months after the tr~~atmcnt was started. The integral evaluation . was quantified according to the formula 5X1 + 5X2 + 3X3 + 2X4 + 4X5 + 2X6 + 2X7 = P, where Xi is the question, which was further multiplied s for a factor to the specialists' approach and P is the patient's result in the integral evaluation. According to the value of P the patient's state was classified according to the scales shown in Table IX.
Table IX. Ir~~aluation scale of patients Evaluat3oa - "' Value of P
Good 22 a P ~ 44 _ .-. ~ . .. _ Fair , 40 ~ P ~ 60 - ._ .
i ~ad.~ _.-_.._.. . , ...._ 6a ~ P ~ 100 ' to The results were evaluated by mesas of the nonparametric test of ~i !.' Witco~:o:z for a statistical significance p ~c 0,01. The results obtained in !a the improvement of the general state of the patients is shown in Table X
Table X. Integral evaluation of patients before treatment and after 6 months treatment, ___ _ ~~~~$ b$~,4r~ ~"t~ ~t~.~ s ' starting Evaluation aioathr o!' >lCoaluattoa No.~ _ treatment t~eat~ent 1.1 28 Good 22 Good _ 32 Good 6 Good 1.3 36 Good 22 Good _ _ 1 .~. 47 Fir 22 Q,ood ' 1:5 ~ 47 Fair 2B Good . 51 . Fair 30 Good l.f~

Fair 22 Good ' o.~,~ ' S4 Fair ~ 26 Good ' 1. ~ . 54 Fair 48 Fair 1.1 c) 57 Fair 30 Good 1.11 , Had 35 G
66 d "Bas ~ 42 oo Fair 2 .13 - 24 Good 22 Good 35 Good 26 Good '' 1 ? 42 Fair 26 prood . _ 42 Fair 28 Good 3.1 a _._ 2.17 8~ ~ad 48 F~

=1 ~'~ .. 51 ~ Fair I _ _ _ 4$ _~ F~
~

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_ ._. __ . _ _ .. - _- _- .__.._ .._ ~-~.=a-~~.~.~~ r-ra c~c~ Lu~~y..*da.kits 27-02-2001 . _ n~ ; 13 3 . z 9 5~ F~, 26 Goon 3 . 2 0 68 gad 44 F~, 3 _ ? 1 ' 79 Bad 26 Good 3.22 I 81 Bad 2b Good 4 . ? 3 Fair 58 p~~r ~ 53 4.24 i Bad 44 F~.

4.?5 I Had 28 Good 5: 25 Good 22 ~ G
36 d __ Good 3~0 oo ~ w Good ;
- -. 2 7 30 Good 22 Good F~.:?9 52 Fair i 3U Good i .30 38 Good 28 G
d .. 61 Bad 2f oo 7 , ~~ Good ' ~ . 3? 66 Fair 22 Good 8 33 80 Bad 36 Good ~~. 34 32 Good 22 Good ~

46 Fair _ F
42 i a I fl. ;c~ 58 Fair 28 x Good I 1. 3 74 $ad 48 Fair i .

1 ? .:, 36 C ood 22 Good ~~

'The f:r:;t number corresponds to the clnssificatieri in Table Vlll and the second num!:c:r is the consecutive seqt~nce.
At t!m beginning of the treatment, 50,0 % of the patients classified as FA~I: in its general state and 28,9 % as BAD. After 6 months of 5 tre:o. ~nc~nt, 23,7% of the patients only classified as FAIR and none as f Brll~. 60,5°/u of the patients included in the study (23j had a s~.~hst:ir.tial improvement in their general state. The statistical analysis 01 tl, c results, not only evaluating the change of the patient's ~lasaiC~eation, but also the variations of the points, on the basis of ' 1o paracnctric imdexes, it was indicated that the improvement of the pa::c~tit's general state with the used formulations was 64,7 %.
Example 8 In the same grnup of patient as the Example 7, inside the study dcscwibud prc;viously and with a similar methodology, the depression 1s irid~~~ was evaluated before beginning the treatment and after b months of trratrncnt. The depression index ~~as evaluated as LARGE, SMALL or I~URMAL according to the scale shown in Table XI.
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_.. - CA 02358013 2001-06-29 _ _ _ _ _._., .... _"...,..."
~27-02-2001 ~ _ CU 009900007 .r.

papsess>tou ittd~~ Valuo of P

22<P<40 Small 4Q < P < 60 ' Lar a 60 < P ~c 100 The results before the treatment and after 6 months of treatment are s sho«m in Table XII

Depression Before Percentage Afters 6 Peroenta~go index stastiag ~to~ths _. _ treatment ~a~a~t l~~ormal 11 28.9 26 68.4 --l.caser 9 23.7 11 28.9 i Greater 18 47.x+
1 2.6 i 44,7 % of tho patients treated with the referred formulations had a i r to rc:m~:~ kuulc decrease of the depression index. The statistical analysis of . the results, not only evaluating the change of the patient's clas~:iic:mion, but also the variations by the points scale, on the basis of parnm~°tric indexes, indic;xted that the improvement of the state of the patient's depression with the used formulations was of 59,1 %.
15 Example 9 Table XI. Scale of patient wa~uation 'fable X1T. Evaluation of the depression index of patients treated with the pharmaceutical formulations, Tablets, 300 mg of the active ingredient each as described btfdre (Cxl:nplc 3), were orally administered to a group of 122 patients affected by different types of cancer, or 3 mL of the flavored suspension that contained 10 °/u in weight of this rnixt-urc (F.xamplc 4). In the casts zo of ulcerZtions in skin the cream was applied to 2,4 % in weight (l~;x~.~ihi~ 5) nr the ointment to 2,4 % in weight (Example 7} of this . mixturr. can the affected surface, according to the lesion type. The c!;m~v;li~~uti~n of the patients, according to the diagnosis, is shown in t [I Tsl~'.~: Xllt.
' as a AMENDED SHEET

ma .:.voo~r~tvJ . trGV
CA 02358013 2001-06-29 rv . f'U 009900007 ' 27-02-2001 . _ h~~,l Table X111. Classification of patients included in the study No Dia~ao:ia Numba~ of patiettta .-1 Adenocarcinomas 53 Carcinomas 3 $

L homas 15 .. 4 Astro ors , Leukemias ~ 4 Lather s of neo lasm g Total ~ 122 The chit dose (300 rtig~ was orally administered 4 times a day and the . . cream or ointment was applied similarly when it was necessary. The treatment lasted between 1 and 12 months according to then patient ' initial state. The general state was evaluated according to imcrnationally recognized surveys for the measurement of patient . general state, hematological, biochemical, and clinical analyses. All ni analyses were done before and after treatment. The results of the 1o evahiation of these patients after the treatment with the formulations, on parametric scale, are shown in Table XiV.

I
Ta'~~ XIV. the end of the study Criteria for evaluating patients at State of patient p~e~~ sod d _ Cured Great im rovemer_t :~ c~tab:c im rovcmc:nt 3 ._ Little improvement 2 Nn response to treatment 1 Thr ncsults of the evaluation of those patients after treatment are shown t5 in T..;hlc XV.
n~
if i AMENDED SHEET

27-02-2001 ~ _ __ "~i i v Tab~e XV. Etfects on the general state of well being of patients with cancer after treatment.

i Type of No. of Hieaa Hoat results a aeo lasm tieata ~ evaluation .

Adcnocarcinomas 53 3,~. Colon, 1-eCtosl~moid, ., ov Carcinomas 38 3.5 Stomach, Prostate L,~mphomas 15 3.5 :'~strc~c to:nas ~ 3.7 _ Leukcmias 4 2.8 L m h oid adenome, C~ her 9 3,0 hYPoPhYsial, eperdinome, Wilrns T ' otal 1 3.5 _.. ?2 r, 98 , ~ of ~luc r~aticnts after the treatment, with duration between 1 and ' S nlUlltl':s, had a remarkable improvement in the evaluated indexes.

Example 10 The capacity of the active ingredient used was evaluated in the form a !: ~ i a ply S f<ar the stimt:lation of the production of nitric oxide, as an ~y imh~~rmnt m:~ssenger involved in such immunologieal events as the 'I
' to diffwem i i;~tW n of lymphocytes T, the macrophage activation, acrd the mechanisms of anti-tumoral defense. The effect of the active ingredient was Wudied thxough the possible stimulation of the inducible oxide nitrio- vnthase (iNOS) and its Comparison with the challenge for LPS
and tl:c effect induced by dexamethasone. To the anirn.als employed ,i ~ 5 (~l;': ~ ca r rats, males between 2 SD and 175 g of weight) the food vtras rctir~cf 16 hours before the treatment. Dexamethasone was ad n:in i ~; cored 1 hour before the administration of LPS and 30 minutes ai'zcr tlm~ administration of the active ing-edient to dose between 10 and 20(~ ~r_:; kg ol' hody weight. The animals were sacrificed after 18 hours 20 of rr~-;.~ ~ m~elW , Lhe blood was extracted to obtain the serums and it was drm~n:i::cd the concentration of nitrates and nitrites. The results of the Btucl,y :ir_~ shown in Table X'VI.
r ~i AMENDED SHEET

~ CA 02358013 2001-06-29 - CU 009900007 ~~27-02-2001 . _ j.:

i Treatmeat Nu~nxbar of auituatls~ Coaceatrstic,a of aitsic oxide . . Control rou 10 30 t g LPS 9 95 t 6 De:xarnethasone i0 33 f 3 Active in 7r. + LPS 10 7~ t b Active in r. ?00 m ! 10 ~ 400 t 8'~
k Active in r. 1.00 10 _ __ mg/k~ .~ ~ 409 t 4*

Actw in ~r :i0 m k IO 36 t 3 Activ c ingr.

~ I p ~ 33 f 2 mg/ka ~ Si~,nit~c:ant d.iff~rencc for p ~ 0.01 The st.ucly durrzonstrated that the active ingredient that is used in the 5 formul;.aions of chc invention stimulated the production of nitric oxide ' to close above 100 mg/kg of body weight, which demonstrates its effect on the immm:ological mediators related to the answer of that en2yme.
Example 11 i The ancil5esic effect of the active ingredient employed was evaluated in ~o the foru~ulations by means of the analgesia rehearsals by formol and acetic ;lcid, respectively, in mice. Mice Balb C of b weeks of age and body w-~:ight between 20 and 23 grams were used. Indomethacine mss used as reference standard and a group negative control. The analgesic efCc~c:l, rneasurcd by the reduction of the number of having licked, was 1s otU~inccl St'clI'tlIl~ from a dose of the active ingredient of S mg/kg of body wc~ i art C.
Table X"I. Ettect of the active ingredient used in the pharmaceutical formulations, on the production of nitric oycide in rate.
Example 12 The an ~ i-inCl;:,mmatory activity of the active ingredient employed was e1','1 J Lld L~: Cl in the formulations by means of the rehearsal of the s inclU~tl;ltl Of the plantar edema by carragenanc in Pirbright guinca~-pigs n of 4 wc~la of cge and body weight befiuveen 200 and 300 grams. Animal fecci ~v:~s rrtircd 24 hours before the rehearsal and the edema was in~luc~cl by subcutaneous injection of lambda carragenane in the aponeurvl plant. The inflarnmatian was measured by displacement of AMENDED SHEET

CA 02358013 2001-06-29 ' . ." vV _ .
~, 27-02-2001 . - C U ,009900007 the volume of li uid. The active in edicnt was 4 gr given orally 1 hour before the carragenane injection. Sodium naproxene was used as reference and the results were compared also with those of other natural products extracts. The results arc sshown in Fable XVII.
S Table XVII. Anri-inflammatory effect of the active ingredient used in the pharmaceutical formulations.
w Composition ~' Lntlammat3oaAntiiafiammatory Doss 11T
i Indnoed ~~Y'o~A~tiv~lty ~",~fO~ (~.g/~~

Sc>c? ium 17.1 t 0.8 54.6 t 0.6 _ 4 15 na roxen Active 18.7 t 3.6 45.0 ~ 0.8 1000 I1 in~:r~:dient 8oldoin 19.0 t ~.g 50,p ~ 0.9 40 36 "i Tr_evoa trinervis- _57.0 t 0.7 800 _ i 1;. ( ~l:.sebachii- 58.9 t 0.8 - _ O. Er~throrhiza- i,. ~ ~ 57.8 t 0.5 _ _ ~

The rc~;uas c'.emonstrated that the mixture of active ingredients that is used .n the formulations presented anti~inflamnzatory activity at a dose of 1 OUc.! and/ kg of body weight, iri a similar way to the activity of the so-liv.cn naprc:~;ene (4 rng/kg) and the boldine extract (40 mg/kg).
ri »aramplc 13 The amt io~,.idmt activity of the active ingredient used in the formulations was ~v;.Uuat~d ire uitm as described by means of the evaluation of its canczcitu of spontaneous oxidation, inhibition of phospholipid peroxidntion i.~ rat brain homogenate and the system bleomicine-iron, re~pcc:i~~~?y, a«d scavenging of hydroxyl radical and hipochlorous acid.
The et~:tive ingredient demonstrated to have a protective effect on en2vmmic Phc~spholipxd per oxidation in rat brain homogenate. This zo protecric.m dr.monstrated to be dose-related and the protective half dose was clcu.:rmined as 0,01$ % (p/v) as it is shown in Table XiX.
f, AMENDED SHEET

~27-02-2001 . _ TABLE XIX. Inhibition of spontaneous self-oxidation in phospholipids in rat's brains, using the active W gredient of the pharmaceutical formulation.
_ _ _ Concentration of aottv~e Absosbeaace ~ Inhibition of ingrCdiGnt °ib W ~ paso~did~aatioa - 0.955 - __ j 0.025 _ 0.191 80 _~_ 0.020 - ,. 0.295 _ 69 , U. U 17 _ 0. 544 33 _i _ O. U 13 ~ y~I 0.811 __ 15 0.005 ., 0.878 . 8 _ O~OO 1 0.956 ....
In the system bleomicine-iron the same effect was observed as described previously, but with a effective concentration slightly higher (0,0?Ei '; ~~), as it is shown in Table XX. Hoth inhibition concentrations (0,018 and G,~7?fi %) are higher than the necessary one to only inhibit the hr<.~c~~ss c;i~'.zalf filled peroxidation for enzymes.
to TAB LL XX, hreverition of peroxidation of phospholipids in the t"
blec~rnycirie-iron system, using the active ir~.gredient of the ph7rmaceutical formulations.
Concentration of the active Abaorbaaca - . Ix~.hibitioa of ingredient-(% W/v~ (635 nnnl Pasr,~_rtdatiost (%
- .. O.gg5 ~ _ -0.025 ~ _ 0.402 59 _ 0_020 o,b3o _ 36 _ ~:y.Q 17 ~ 0.734 . .. .. 25 ! Ct . 013 0.843 14 _.__. _ .
U.UUS o.905 s The scv mc~n~ ~ : : :; e~po.eity for hydroxyl radical was notably highh and it i s was ro a nd chut the concentration (w/v) of the mixture of polyphenols, ' tcrpcm~icis, :;LCroids, fatty acids, and microelements exhibited a rsduclion cf ~0 '% of generated hydroxyl radical was 0,011 %. It i dcrno~:stratecl the high antioxidant activity of the invention object in Eton; of one r.~f the reactive oxygen radicals that more frequently is I
2o assc~ci~rad m~i,.h processes of deterioration of the human organisrci.
i AMENDED SHEET

. _ _ ._ ____ _ . _ . _.._. ._ __ __ _..__ __.._ .t _ CA 02358013 2001-06-29 CU o0~900007 4' 27-02-2001 . _ _ 20 The scavenging capacity for hypoehlorous acid was also notable, since the cosicentration (w/v) that reduced 5D % of its concentration was detected as 0,lJ4 "/o. It dernosistrated the high antioxidant activity of the invention object by means of the protection of the alpha-one antiprotciriase, which. is degraded by the hypochlorous acid.
' The antioxidant properties described in the Example 14 for the polyphe~nols, terpenoids, steroids, fatty acids, and microelements mixture invention ob'ect have not been described reviousl for a P Y ~Y
other natural product at so low concentration levels, which ~ o demonstrates their effectiveness in the chemo-prevention of biological events related to aging and degenerative pathologies that cause a consiclcrnble stress in the human organism, as it was demonstrated in cancer haracn is according to the Examples ?, 8, and 9. These C.
antioxidant properties, in turn, are linked to physiological processes t5 rclatccl to inflammation and pain, as described in the Ex$mples 10, 11, 12, m::l 13, which allows to correlate the anti-inflammatory and analgesic:, a:, observed to the high capacity of lipid peroxidation inl~ihi~ic~~i, spontaneous self oxidation, the scavenging capacity of hyc)row~1 ricdical and hypochlorous, which confers to the object of z0 invention dis~inctivc characteristics, for their high effectiveness, not rehor~c~cl previously for another existent product in thc_ market AMENDED SHEET

Claims

21

1. Mixture of polyphenols, terpenoids, steroids, fatty acids, and microelements obtained from the stem bark of Mangifera indica L
wherein its qualitative and quantitative composition is:
Component Content (%) 1, Polyphenols 20 - 60 1.1 Mangiferin 10 - 20 1.2 3-Pentadecylphenol 5 - 10 1.3 3-Octylphenol 5 - 10 1.4 Amentoflavone 10 - 20 2. Terpenoids 10 - 40 2.1 Mangiferonic acid 15 - 30 2.2 Beta-element 5 - 10 2.3 Alpha-guaiene 5 - 10 2.4 Aromandrene 5 - 10 2.5 Hinesol 1 - 5 2.6 Cycloartanols 1 - 5 2.7 Ledol 1 -5 2.8 Taraxerol 1- 5 3. Steroids 5 - 15 3.1 Gamma-sitosterol 2 - 8 3.2 Beta-sitosterol 1 - 5 3.3 Campsterol 1 - 5 3.4 Daucosterol 0.5 - 3 3.5 Multifluorenone 0.5 - 3 4. Fatty acids 1 - 5 4.1 Myristic 0.1 - 3.0 4.2 Palmitic 35.0 - 45.0 4.3 Linoleic 15.0 - 35.0 4.4 Oleic 20.0 - 40.0 4.5 Stearic 0.1 - 1.0 4.6 Eicosatrienoic 1.0 - 3.0 5. Microelements 1 - 3 5.1 Potassium. 0.8 - 1.0 5.2 Calcium 0.2 - 0.4 5.3 Magnesium 0.1 - 0.2 5.4 Iron 0.1 - 0.2 5.5 Copper Less than 0.01 5.6 Zinc Less than 0.01 5.7 Selenium 0.03 - 0.08

2. Composition containing as active principle the mixture according to claim 1 and an appropriated excipient.

3. A pharmaceutical composition in the form of a coated tablet containing from 45 to 55 % of the mixture according to claim 1 together with fillers, agglutinants, disintegrants, lubricants, vehicles, and other pharmaceutically acceptable excipients.

4. A pharmaceutical composition in the form of a flavored suspension containing from 8,0 to 12,0 % of the mixture according to claim 1, together with vehicles, stabilizers, and other pharmaceutically acceptable excipients.

5. A pharmaceutical composition in the form of a hydrophilic cream containing from the 1 to 4 % of the mixture according to claim 1 together with fillers, vehicles, stabilizers and other pharmaceutically acceptable excipients.

6. A pharmaceutical composition in the form of a lipophilic ointment containing from the 1 to 4 % of the mixture according to claim 1, together with fillers, vehicles, stabilizers and other pharmaceutically acceptable excipients.

7. The use of the mixture according to claim 1, for the manufacture of a medication for the treatment of immunodepressed patients and degenerative illnesses, which is used to a daily dose between 1200 and 2000 mg for oral and/or topical route.

8.- Use according to claim 7 wherein the immunodepressed patient is a patient with Acquired Immunodeficiency Syndrome.

9.- Use according to claim 8 wherein the degenerative illness is cancer.

10.- The use of the mixture according to claim 1 for the manufacture of an anti-oxidant medication.

11.- The use of the mixture according to claim 1 for the manufacture of an anti-inflammatory medication.

12.- The use of the mixture according to claim 1 for the manufacture of an analgesic medication.

13. The use of the mixture according to claim 1 for the production of a food supplement, which is used to a daily dose between 900 and 1500 mg for oral or parenteral route.